structural(studiesof(mousecsf21(mcsf21)(and itscomplex ... · acknowledgments looking back, my...

Structural studies of mouse CSF-‐1 (mCSF-‐1) and its complex with the viral oncoprotein BARF1

Anaïs BEKAERT

Master’s dissertation submitted to obtain the degree of Master of Biochemistry and Biotechnology Major Biochemistry and Structural biology

Academic year 2010-‐2011

Promoter: Prof. Dr. Savvas Savvides Scientific supervisors: Drs. Jan Felix, Drs. Jonathan Elegheert Department of Biochemistry and Microbiology Laboratory for Protein Biochemistry & Biomolecular Engineering

Acknowledgments

Looking back, my first trip to the synchrotron in Hamburg last year during the first Master was more of a pleasure matter. At the time however, it seemed like a peak into the mystic world of biomolecular crystallography, where people could spend hours upon hours staring at screens depicting dotted canvasses. And just one year later, I think it is safe to say I have indeed experienced the synchrotron life. Because you haven’t been to a synchrotron until you get a 24h shift! Twice!

Although the amount of mental material to be processed there seemed overwhelming, I did learn a tremendous amount in a very short period of time. Where crystallography used to be a vague subject, some of its details have been revealed to me bit by bit over this last year.

And so, after 5 years of studying in the biochemistry field, which were sometimes laced with trials and tribulations, it’s time to thank some people.

Thanks to all my supervisors: Prof. Dr. Savvides for giving me the opportunity to do this thesis in the wonderful world of structural biology. Drs. Jonathan Elegheert for his seemingly endless wisdom of all things cytokine. Drs. Jan Felix for taking the time to help me out even during his busy schedule. And thanks to Drs. Nathalie Bracke, for being a great supervisor during the first semester.

I would also like to thank the reading commission for reading this thesis.

Thanks to all the other people at the L-Probe lab for always being helpful and for the fun trips to the synchrotrons and spicy food restaurants.

Thanks to my colleagues aka other thesis students at L-Probe for the moral support, you all made the coffee breaks a little more fun.

Thanks to my parents, my brother and other family members, who may not have a clue about what I’m studying but who support me nonetheless. Thanks to my friends. And many thanks to Basil, for keeping me motivated.

Anaïs Bekaert

And now for something completely different.

Table of contents Abbreviations i

List of Figures ii

Abstract v

Samenvatting vi

PART 1: INTRODUCTION ............................................................................................................................. 1 CHAPTER 1: INTRODUCTION TO THE CYTOKINES ......................................................................................................... 2 1.1 Hematopoiesis .......................................................................................................................................................... 2 1.2 The cytokines superfamily ................................................................................................................................... 2

CHAPTER 2: THE FOUR Α-‐HELICAL BUNDLE CYTOKINES ........................................................................................... 4 2.1 FLT3L ............................................................................................................................................................................ 5 2.2 SCF ................................................................................................................................................................................. 7 2.3 CSF-‐1 ............................................................................................................................................................................. 7

CHAPTER 3: RECEPTORS FOR THE FOUR Α-‐HELICAL BUNDLE CYTOKINES .......................................................... 10 3.1 The tyrosine kinase receptors ......................................................................................................................... 10

3.1.1 FLT3 ..................................................................................................................................................................................................... 11 3.1.2 C-‐KIT .................................................................................................................................................................................................... 12 3.1.3 CSF-‐1R ................................................................................................................................................................................................. 13

3.2 The Epstein-‐Barr virus ....................................................................................................................................... 14 3.2.1 Pathology ........................................................................................................................................................................................... 14 3.2.2 BARF1: a new receptor for CSF-‐1 ............................................................................................................................................ 15

CHAPTER 4: CYTOKINE LIGAND-‐RECEPTOR INTERACTION ...................................................................................... 17 4.1 Rationale .................................................................................................................................................................. 17 4.2 Conformational changes ................................................................................................................................... 17

4.2.1 FLT3 and FLT3L .............................................................................................................................................................................. 17 4.2.2 C-‐Kit and SCF .................................................................................................................................................................................... 19 4.2.3 CSF-‐1R and CSF-‐1 ........................................................................................................................................................................... 19

CHAPTER 5: X-‐RAY CRYSTALLOGRAPHY ...................................................................................................................... 21 5.1 Introduction ............................................................................................................................................................ 21 5.2 Lattices, unit cells and spacegroups ............................................................................................................. 21 5.3 From diffraction to electron density ............................................................................................................ 23

PART 2: GOAL ................................................................................................................................................ 25 PART 3: MATERIALS AND METHODS ..................................................................................................... 27 CHAPTER 6: OBTAINING THE CONSTRUCT AND OVEREXPRESSION IN E. COLI ...................................................... 28 6.1 Introduction ............................................................................................................................................................ 28 6.2 Methods .................................................................................................................................................................... 29

CHAPTER 7: REFOLDING AND PURIFICATION OF MCSF-‐1 ....................................................................................... 30 7.1 Introduction ............................................................................................................................................................ 30 7.2 Methods .................................................................................................................................................................... 31

CHAPTER 8: CRYSTALLIZATION AND HANDLING CRYSTALS OF MCSF-‐1 ............................................................... 33 8.1 Introduction ............................................................................................................................................................ 33 8.2 Methods .................................................................................................................................................................... 33

CHAPTER 9: DATA COLLECTION AND SOLVING THE STRUCTURE OF MCSF-‐1 ...................................................... 35 9.1 Introduction ............................................................................................................................................................ 35 9.2 Methods .................................................................................................................................................................... 38

PART 4: RESULTS ......................................................................................................................................... 40 CHAPTER 10: OVEREXPRESSION OF RECOMBINANT M-‐CSF1 IN E. COLI, REFOLDING AND PURIFICATION .... 41 10.1 Cloning of the construct and overexpression ........................................................................................ 41

10.1.1 Nickel affinity purification under denaturing conditions .......................................................................................... 42 10.1.2 Rapid dilution and dialysis ...................................................................................................................................................... 42

10.2 Purification of mCSF-‐1 .................................................................................................................................... 42 10.2.1 Affinity chromatography on nickel sepharose ................................................................................................................ 43 10.2.2 Gel filtration ................................................................................................................................................................................... 43 10.2.3 Thrombin cleavage of the His-‐tag and Source 30Q ion-‐exchange chromatography ...................................... 44

CHAPTER 11: CRYSTALLIZATION OF MCSF-‐1 ............................................................................................................ 46 11.1 Crystallization trials ......................................................................................................................................... 46 11.2 Additive screens .................................................................................................................................................. 47 11.3 Hanging drop crystallization ....................................................................................................................... 47

CHAPTER 12: STRUCTURE DETERMINATION OF MCSF-‐1 ....................................................................................... 48 12.1 Data collection and processing ................................................................................................................... 48 12.2 Molecular Replacement .................................................................................................................................. 50 12.3 Structure refinement ........................................................................................................................................ 50 12.4 Structure validation ......................................................................................................................................... 52

PART 5: CONCLUSION AND DISCUSSION ............................................................................................... 55 CHAPTER 13: DISCUSSION ............................................................................................................................................. 56 13.1 Refinement ............................................................................................................................................................ 56 13.2 The mCSF-‐1 structure ...................................................................................................................................... 57 13.3 Free mCSF-‐1 versus CSF-‐1R:mCSF-‐1R ...................................................................................................... 58 13.4 Free mCSF-‐1 versus mCSF-‐1:BARF1 .......................................................................................................... 60

CHAPTER 14: CONCLUSION ........................................................................................................................................... 62 CHAPTER 15: FUTURE PERSPECTIVES ......................................................................................................................... 63 CHAPTER 16: NEDERLANDSTALIGE SAMENVATTING ............................................................................................... 64

PART 6: REFERENCES ................................................................................................................................. 66 PART 7: APPENDICES .................................................................................................................................. 72

i

Abbreviations ADP Anisotropic displacement parameter

AML Acute Myeloid Leukemia

ASU Asymmetric unit cell

Cb Carbenicillin

Cm Chloramphenicol

Coot Crystallographic Object-Oriented Toolkit

CSF Colony-stimulating factor

DC Dendritic cell

EBV Epstein-Barr virus

EDTA Ethylenediaminetetraacetic acid

EM Electron Microscopy

ESRF European Synchrotron Radiation Facility

FEL Free Electron X-ray Laser

Flt3 Fms-related tyrosine kinase 3

GH Growth Hormone

GIST Gastrointestinal Stromal cell Tumor

GnHCl Guanidinium hydrochloride

GSH Reduced glutathione

GSSG Oxidized glutathione

HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

HSC Hematopoietic stem cell

ITD Internal Tandem Duplication

Ig-like Immunoglobulin-like

IL Interleukin

IM Infectious Mononucleosis

IMAC Immobilized metal affinity chromatography

IPTG Isopropyl ß-D-1-thiogalactopyranoside

ITC Isothermal Calorimetry

LLG Log-likelihood gain

ii

MAD Multiple wavelength anomalous diffraction

M-CSF Macrophage Colony Stimulating Factor

MIR Multiple isomorphous replacement

MR Molecular Replacement

NPC Nasopharyngeal carcinoma

PDB Protein Data Bank

PHENIX Python-based Hierarchical Environment For Integrated Xtallography

PMSF Phenylmethanesulfonylfluoride

PTB Phosphotyrosine binding

PTK Protein tyrosine kinase

RA Rheumatoid Arthritis

RFZ Rotation function Z-score

RMS Root Mean Square

RTK Receptor tyrosine kinase

SAD Single wavelength anomalous diffraction

SAXS Small Angle X-ray Scattering

SIR Single isomorphous replacement

SCF Stem cell factor

SEC Size-exclusion chromatography

SH2 Src homology 2

SPR Surface Plasmon Resonance

TFZ Translation function Z-score

TLS Translation-liberation-screw

TNF Tumor necrosis factor

VEGF Vascular Endothelial Growth Factor

XDS X-ray Detector Software

iii

List of figures Figure 1.1: Hematopoiesis model 3

Figure 2.1: Structural features of the four α-helical bundle cytokine family 5

Figure 2.2: Structure of Flt3L 6

Figure 2.3: The structure of human SCF 7

Figure 2.4: Different mRNA transcripts and isoforms of CSF-1 8

Figure 2.5: Structure of CSF-1 8

Figure 2.6: CSF-1 involvement in immunity and inflammation 9

Figure 3.1: Model for receptor dimerization stimulating tyrosine kinase activity 11

Figure 3.2: Flt3 catalytic domain with autoinhibitory loop 12

Figure 3.3: The extracellular domain of c-Kit 13

Figure 3.4: Inhibitor of the catalytic kinase domain of CSF-1R 14

Figure 3.5: The BARF1 structure 16

Figure 4.1: Comparison between Flt3L bound to Flt3 versus the unbound form 18

Figure 4.2: Domain 4 of the Flt3 does not engage in homotypic interactions 18

Figure 4.3: Comparison bound versus unbound SCF 19

Figure 4.4: Structure of CSF-1:CSF-1R complex 20

Figure 4.5: Bound versus unbound CSF-1 20

Figure 5.1: The X-ray crystallography process 22

Figure 5.2: Unit cell parameters 22

Figure 5.3: The Bragg equation and diffraction and resolution limits 23

Figure 5.4: Electron density difference maps 24

Figure 10.1: Induction of the mCSF-1 protein by IPTG 41

Figure 10.2: Purification of mCSF-1 under denaturing conditions 42

Figure 10.3: Elution chromatogram of nickel sepharose purification 43

Figure 10.4: Gel filtration chromatogram of mCSF-1 44

Figure 10.5: Thrombin digestion of mCSF-1 44

Figure 10.6: Source 30Q anion exchange chromatogram mCSF-1 45

Figure 10.7: Checking the Source 30Q purification 45

Figure 11.1: Initial crystallization trials of mCSF-1 46

Figure 11.2: Examples of hits from additive screens mCSF-1 47

Figure 11.3: Hanging drop crystallization 47

Figure 12.1: Diffraction pattern mCSF-1 crystal 48

iv

Figure 12.2: Xtriage output graphs 49

Figure 12.3: The mCSF-1 structure and unit cell after molecular replacement 50

Figure 12.4: R-work and R-free evolution during refinement of mCSF-1 51

Figure 12.5: Backbone torsion angle distribution for NCS related chains in mCSF-1 53

Figure 12.6: Polygon for the mCSF-1 structure 54

Figure 12.7: The refined mCSF-1 structure in the electron density 54

Figure 13.1: Unmodeled blob in mCSF-1 density 56

Figure 13.2: The obtained free mCSF-1 structure 58

Figure 13.3: Free vs. bound mCSF-1 58

Figure 13.4: Side chains mCSF-1 undergo conformational changes upon binding to the CSF-1R

59

Figure 13.5: Comparison free mCSF-1 vs. BARF1:mCSF-1 60

Figure 13.6: BARF1:hCSF-1 versus BARF1:mCSF-1 61

v

Abstract

The Colony stimulating factor 1 (CSF-1) is a pleiotropic cytokine that plays a role in the hematopoiesis by regulating mononuclear phagocytes, which can develop into monocytes, macrophages and dendritic cells. Structurally, CSF-1 is a dimer belonging to the short-chain α-helical bundle cytokines. Its endogenous receptor, CSF-1R, is a member of the receptor tyrosine kinase III family (RTKIII), to which CSF-1 bind by its the distal ends. Binding of the ligand induces dimerization in the receptor, phosphorylation and downstream signaling through effector molecules. The binding paradigm of the CSF-1 is shared by other members of the short-chain α-helical bundle cytokines, Stem cell factor (SCF) and Fms-related tyrosine kinase 3 ligand (Flt3L) and their respective receptors c-Kit and Flt3.

Recently, the CSF-1 protein has been revealed to bind the BARF1 protein, which is expressed during the lytic phase of Epstein-Barr viral infection. In solution, BARF1 forms hexameric rings by specific N-N and C-C interactions. BARF1 was found to be a scavenger of the CSF-1, providing a possible mechanism by which the virus can circumvent the host immune system. Peculiarly the CSF-1 binding epitope is not the same as the one for the CSF-1 receptor and a mechanism other than receptor mimicking must play a role in the modulation of the immune system as the bindings epitope for CSF-1R interaction is still available for signaling. BARF1 binds to both human (h) and mouse (m) variants of CSF-1; however, the signaling through the CSF-1R shows a specific species difference.

During this thesis, the structure of mCSF-1 was determined, as there is to our knowledge no other structure available for the unbound mCSF-1 form. The comparison of the bound versus the unbound form of mCSF-1 to the BARF1 structure could provide insight into the reason of the species difference.

The mCSF-1 protein was recombinantly produced in E. coli and crystallization trials were undertaken after obtaining the native protein using a refolding and extensive purification procedure. One of the crystals diffracted up to 2.6 Å and structure determination was performed by molecular replacement, followed by several rounds of refinement.

vi

Samenvatting De kolonie stimulerende factor 1 (CSF-1) is een pleiotroop cytokine dat een rol speelt in de hematopoëse bij de regulatie van mononucleaire fagocyten, die zich kunnen ontwikkelen naar monocyten, macrofagen en dendritische cellen. Structureel gezien is CSF-1 een dimeer en behoort tot de familie van de korte keten α-helicale bundel cytokines. De endogene receptor, CSF-1R, is een lid van de tyrosine kinase III familie (RTKIII), waaraan CSF-1 bindt met de distale uiteinden. Binding van het ligand induceert receptor dimerizatie, fosforylatie en verdere signalisatie door de werking van effector moleculen. Het bindingsparadigma van CSF-1 wordt gedeeld met andere leden van de korte keten α-helicale bundel cytokines, stam cel factor (SCF) en Fms-gerelateerd tyrosine kinase 3 ligand (Flt3) en hun respectievelijke receptoren c-Kit en Flt3.

Het CSF-1 proteïne is een bindingspartner voor BARF1, een eiwit dat geëxpresseerd wordt gedurende de lytische fase van een Epstein-Barr virus infectie. Het BARF1 vormt in oplossing hexamere ringen door specifieke N-N en C-C interacties. BARF1 vormt een scavenger voor CSF-1, wat een mogelijk mechanisme kan zijn waarmee het virus het immuunsysteem van de gastheer omzeilt. Vreemd genoeg is de CSF-1 bindingsepitoop niet dezelfde als die voor de CSF-1 receptor en een ander mechanisme dan receptor imitatie zal dus een rol spelen in de immuun modulatie aangezien de bindingsepitoop voor CSF-1R interactie nog steeds beschikbaar is. Hoewel BARF1 zowel humaan (h) als muis (m) CSF-1 bindt, is er een specifiek species verschil bij de signalering doorheen de CSF-1R.

Gedurende deze thesis, werd geprobeerd de structuur van mCSF-1 te achterhalen, aangezien er volgens onze kennis geen structuren bekend zijn van de ongebonden mCSF-1 vorm. De vergelijking tussen de ongebonden en gebonden vorm van mCSF-1 aan BARF1 zou dan mogelijks een indicatie opleveren naar de reden van het species verschil.

Het mCSF-1 eiwit werd recombinant geproduceerd in E. coli en kristallisatie pogingen werden ondernomen nadat het natieve eiwit via een heropvouwings- en purificatie procedure werd verkregen. Een van de kristallen diffracteerde tot 2.6 Å en structuur determinatie werd ondernomen via moleculaire vervanging, gevolgd door verschillende rondes van verfijning.

Part 1: Introduction


2

Chapter 1: Introduction to the cytokines

1.1 Hematopoiesis Hematopoiesis is the biological process by which blood cells are produced. A small, self-renewing pool of multipotent hematopoietic stem cells (HSC) that reside in the bone marrow gives rise to diverse cell types of the myeloid and lymphoid kind, such as macrophages, dendritic cells (DC) and B-cells, by a strictly regulated process (Figure 1.1). The hematopoietic growth factors, also known as cytokines, that mediate this proliferation and differentiation play a crucial role in determining the fate of the HSC

The cytokines act on the HSCs and their derived cells by specific interactions with cell-surface receptors. Some cytokines act on different cell lineages or can be cell lineage specific. Cooperation of different cytokines on one cell is also possible to achieve the correct proliferative response.

1.2 The cytokines superfamily The hematopoietic cytokines consist of different proteins such as Colony stimulating factors (CSFs), different interleukins (IL), Tumor Necrosis Factor- α (TNF- α), etc. They influence hematopoiesis by acting during different stages of the process from early differentiation to later on. The Colony-stimulating factor (CSF) for example, has a role in proliferation and maturation of lineage specific precursor cells in the late hematopoiesis (Barreda et al., 2004). Every cytokine elicits a certain signal after specific interaction with complementary surfaces on receptors, which are present on HSCs or derived cell lines.

Next to hematopoietic cytokines, other groups exist as well, such as cytokines that play a role in inflammation (Turner et al., 2010). Based on sequence and structural analysis, the cytokines can be divided into seven distinct groups that include the hematopoietic cytokines and the tumor necrosis factor-like cytokines. Generally, four structural groups are recognized: the helical bundle cytokines, the TNF family, the cysteine-knot growth factor family and the ß-trefoil family (Sprang et al., 1993).

As the helical bundle cytokines represent the biggest and most important family of cytokines, the other families will not be further discussed in this thesis.


3

Figure 1.1: Hematopoiesis model. The hematopoietic stem cells (HCSs) are at the top of the blood cell lineages, having both the ability to differentiate and maintain long-time self-renewal. The multipotent progenitor cells can differentiate further into oligopotent progenitor cells, which will eventually give rise to mature effector cells. The whole process is regulated by cytokines. CLP: Common lymphoid progenitor, CMP: common myeloid progenitor, DC: dendritic cell, GMP: granulocyte/macrophage progenitor, MEP: megakaryocyte/erythroid progenitor, NK: natural killer cell. Figure adapted from (Bryder, Rossi, & Weissman, 2006).


4

Chapter 2: The four α-helical bundle cytokines

Members from the helical bundle family, also known as the four α-helical bundle cytokines, show a similar arrangement of their helices known as an up-up-down-down topology, which has not been found in any other known protein structures. The helical bundle family can be further divides in three groups upon inspection of the present tertiary structures: the short-chain, the long-chain and the interferon-like subfamilies (Figure 2.1). Between the three subfamilies, the helical bundle topology is the same whereas the folds are different (Rozwarski et al., 1994).

The long-chain subfamily has four well-aligned helices that are 20 to 30 residues long, the crossover from helix A to B passes in front of helix D. The crossover connections consist of short helices that are not well aligned with the bundle core. For the short-chain subfamily however, the helices are only 10 to 20 residues long and the crossover between helix A and B occurs behind helix D, thereby forming an integral part of a two-stranded antiparallel ß-sheet contributing to the bundle core. The difference between the crossover parts suggests a different folding pathway of the two subfamilies. The interferons, which make up the third family, have intermediate features: the helix packing angles are closer than in short-chain cytokines but the crossover from helix A to B occurs in front of helix D. The interferon family has one unique feature: the crossover from helix C to helix D forms a fifth α-helix contributing to the bundle core.

There is little amino acid sequence similarity between the members of the α-helical bundle cytokines but they are functionally similar in that they all are extracellular signaling molecules and almost all bind to homologous receptors. They are also structurally similar in that they have a unique topology in their helical bundle and are genetically similar. All members of the family have seemingly diverged from an ancestral protein due to insertions or deletions of exons or introns.

By comparing the cytokines and their mode of binding to their receptors, the information obtained can be extrapolated to other, unknown, structures and thereby gaining insight into a possible paradigm for cytokine-receptor interactions. Also, due to the lack of sequence similarity, the helical bundle family lends itself for the study of protein folding and evolution.


5

Figure 2.1: Structural features of the four α-helical bundle cytokine family. The four helices (A-D) show an up-up-down-down topology where each helix is antiparallel to its neighbors. The amino (N) and carboxyl © termini are indicated. A. Typical fold for the short-chain subfamily with a two-stranded ß-sheet and a crossover connection behind helix D. B. Typical fold for the long-chain subfamily with no ß-strands and a crossover connection in front of helix D. A short helix after αA, involved in receptor binding in GH and G-CSF, is also shown. C. Typical fold for the interferon-like subfamily with key elements a helix in the C-D crossover passing in front of helix D and contributing to the bundle core. Figure adapted from reference (Rozwarski et al., 1994).

2.1 FLT3L Human Fms-related tyrosine kinase 3 ligand or Flt3L is a member of the short-chain α-helical bundle cytokines. In accordance to SCF, it acts directly on stem cells and stimulates early hematopoiesis through activation of a tyrosine kinase receptor class III, Flt3R, present on primitive bone marrow stem cells. By activation of the receptor, Flt3L stimulates stem cell proliferation, mobilization and generation of dendritic cells, which are important for antigen uptake and presentation to T-cells in the lymph nodes.

Flt3L is found to be a noncovalently linked homodimer, and is biologically active both as a soluble form and a transmembrane form, from which the former is derived through proteolytic cleavage. The expression of the protein is very tightly regulated, suggesting a short-range mode of action, even possibly by cell-cell contacts (McClanahan et al., 1996).

The structure of Flt3L has recently been solved (Savvides et al., 2000) to a resolution of 2.2 Å. Special features include a 310-helix, continuous with αC and three proline residues in the middle of the helix connecting it to ß2 (Figure 2.2A). Flt3L contains 3 intramolecular disulfide bridges that play a structural role.

Graddis describes a method for searching for mutations in Flt3L relating to enhanced or decreased activity using a rapid functional screen, whereby thirty-one amino acid substitutions at twenty-four positions were identified (Graddis et al., 1998). In a later study,


6

by taking into consideration for example possible destabilization of the Flt3L, this number was decreased to 9 residues important for bioactivity (Figure 2.2B).

Structurally, Flt3L is most similar to IL-4 and not to the more functionally related MCSF, supporting the hypothesis that the cytokines have diverged enough that structural similarity no longer corresponds to evolutionary distances implied by functional similarity (Savvides et al., 2000).

Figure 2.2: Structure of Flt3L. A. Flt3 (dimensions 75 Å x 35 Å x 25 Å) in its transmembrane form. B. Residues that are solvent accessible and important for bioactivity. Mutations that reduce activity are depicted in red, mutations that increase activity in blue. Figure adapted from (Savvides et al., 2000).

Flt3L can stimulate both proliferation and differentiation of hematopoietic progenitor cells, including proliferation of DCs. Although other cytokines, like GM-CSF, can also have an effect on DC proliferation, Flt3 has the advantage that both myeloid-related and lymphoid-related DCs get induced. Furthermore, Flt3L can stimulate development of immature and mature DCs in humans (Maraskovsky et al., 2000).

DC vaccination is a technique that generates a lot of interest as a potential anti-cancer therapy. The limiting step however is generation of sufficient levels of DCs, as they make up less than 1% of mononuclear cells in the peripheral blood of humans. Anti-tumor responses are mostly regulated by cytotoxic T cells, acting as effector cells through tumor lysis or by stimulating cytokine release. The crucial step in this process is the antigen presentation to immature T cells by antigen-processing cells, like DCs.

By increase the levels of DCs Flt3L can be used as a potential antitumor agent. So far, it has been effectively tested in several tumor models, where it generated a DC response and a clinically visible antitumor effect. Most studies in mouse models have shown an effective dose of 10 mg/day with no cytotoxic effects of administration (Dong et al., 2002). Moreover, the effect of Flt3L is enhanced when combined with specific tumor antigens, thereby eliciting a specific, long-lasting immune response mainly because animals could survive a rechallenge, which was not the case with Flt3L administration alone. Although the upscaling to human cancers could prove to be challenging, early clinical data suggests that Flt3L therapy is well tolerated with minimal side effects and can thus be applied as an anticancer treatment (Maraskovsky et al., 2000).


7

2.2 SCF Stem cell factor (SCF), located on the Steel (Sl) locus, is another member of the dimeric short-chain α-helical bundle cytokines and is produced by various fibroblast-type cells including bone marrow stromal cells. SCF is an early-acting cytokine that signals by binding to its cell-surface bound receptor, c-Kit. It has been shown both in vitro and in vivo that SCF is important for mast cell proliferation and essential for mast cell survival and other cells expressing the receptor (Iemura, M. Tsai, Ando, Wershil, & Galli, 1994).

SCF can be expressed as a membrane-associated form or as a soluble form, where the soluble form exists as a non-covalently associated homodimer (Arakawa et al., 1991). In 2000, the structure of human SCF was solved to a resolution of 2.2 Å using MAD phasing (Jiang et al., 2000), showing the characteristic features of the short chain helical cytokines (Figure 2.3) but again the sequence similarity with other short-chain helical cytokines proved random. In 2007, the mouse SCF structure was solved (Liu, X. Chen, Focia, & He, 2007).

Figure 2.3: The structure of human SCF. The homodimer of stem cell factor is a non-covalently linked structure with head-to-head packing of the two chains (chain A in blue, chain B in orange). Both the N- and C-termini are indicated on chain A. PDB code: 1SCF. Figure generated with PyMol.

Mutations in the SCF gene cause complex phenotypes that include varying degrees of anemia, sterility, lack of coat pigmentation and mast cell deficiency (E. Russell, 1979). In this respect, SCF has been used in clinical trials to treat anemia because it mobilizes peripheral blood progenitor cells. As initial tests proved to be positive, SCF might be used in the future as treatment in for example skin pigmentation disorders (Glaspy, 1993).

2.3 CSF-1 CSF-1, also known as macrophage-CSF (M-CSF) is a member of the Colony stimulating factors (CSF) that form an extended network in the hematopoiesis and inflammation. CSF-1 in particular is a pleiotropic growth factor that regulates mononuclear phagocytes, which can develop into monocytes, macrophages and dendritic cells (DC) (Pixley & Stanley, 2004).

The csf-1 locus is positioned at 1p21-p13 in the human genome. After transcription, different splicing variants of the mRNA can be produced, giving rise to different isoforms of the CSF-1 protein as membrane-bound or secreted forms (Figure 2.4). All the different isoforms have the ability to bind to the endogenous receptor, CSF-1R. An important feature in the CSF-1:CSF-


8

1R complex formation is the apparent species cross-reactivity in signaling. While a human ligand can activate both human and mouse variants of the receptor as well as other primate and non-primate receptors tested thus far; the mouse ligand can only activate the mouse receptor and displays no cross-reactivity (Garceau et al., 2010).

Figure 2.4: Different mRNA transcripts and isoforms of CSF-1. Four different mRNA splice variant are transcribed, which give rise to different CSF-1 isoforms by cleavage and further processing. Figure adapted from reference (Douglass et al., 2008).

As for the unbound CSF-1 structure, only the human form (hCSF-1), which was solved to a resolution of 2.5 Å, was deposited at the PDB. Even though only the Cα-atoms were available, some conclusions could be formed about the general structure (Pandit et al. 1992). In accordance with the other four α-helical bundle cytokines, the CSF-1 consists of 2 monomers dimerized by an interchain disulfide bond (Figure 2.5).

Figure 2.5: Structure of CSF-1. The dimer of CSF-1 contains an interchain disulfide bond at the dimer interface. PDB: 1HMC. Figure rendered with JMol.

Because CSF-1 plays a role in inflammation and immunity, its role in development of different diseases has been extensively studied (Figure 2.6). For example, CSF-1 levels in lupus-associated renal inflammation are elevated causing macrophage recruitment and subsequent macrophage-induced apoptosis. In the development of the autoimmunity disease rheumatoid arthritis (RA) also higher levels of local circulating CSF-1 have been found, correlated with higher levels of infiltrating monocytes and subsequent chronic inflammation.


9

In cases of skin xenografts, the CSF-1 is rapidly produced and is implicated in rejection of the new tissue. Accumulation of CSF-1 dependent macrophages in white adipose tissue of obese mice has been documented, causing chronic low-grade inflammation by producing the inflammatory cytokine Tumor Necrosis Factor α (TNF-α), which stimulates the expansion of the adipose tissue in cooperation with CSF-1. In relation to cancer, high circulating levels of CSF-1 are found in various malignancies and tumor cells can synthesize both the CSF-1 and the CSF-1R to achieve autocrine growth control, enhancing tumor progression and metastasis. In the case of human breast carcinoma progression, CSF-1 may indeed promote metastatic potential (Lin, Nguyen, R. G. Russell, & Pollard, 2001). Expression of the membrane-spanning cell-surface form of CSF-1 can elicit anti-tumoral activity, indicating that the balance between the expression of secreted and cell-surface CSF-1 isoforms could affect tumor progression (Chitu & Stanley, 2006).

Figure 2.6: CSF-1 involvement in immunity and inflammation. CSF-1 regulates the development and activation of mononuclear phagocytes and therefore contributes to viral, fungal, and bacterial immunity. CSF-1 is also involved in the promotion and sustaining inflammation in several diseases. Figure adapted from reference (Chitu & Stanley, 2006).


10

Chapter 3: Receptors for the four α-helical bundle cytokines

3.1 The tyrosine kinase receptors Membrane receptors can be classified into families based upon the ligand that they recognize, the induced biological response and their structure. A large family of cell surface receptors that has intrinsic protein tyrosine kinase activity is called the receptor tyrosine kinases (RTK). These receptors catalyze transfer of the γ-phosphate from ATP to hydroxyl groups of tyrosine residues on target proteins. RTKs play important roles in fundamental cellular processes such as cell metabolism and survival, cell migration and cell proliferation and differentiation (Hubbard, Mohammadi, & Joseph Schlessinger, 1998).

RTKs have several distinct regions: an extracellular domain for ligand binding that is usually glycosylated, a transmembrane helix and a cytoplasmic domain. The cytoplasmic part contains a conserved protein tyrosine kinase (PTK) core and other regulatory sequences. The catalytic activity of RTKs is tightly regulated by protein-tyrosine phosphatases, other protein tyrosine or serine/threonine kinases and autoregulatory mechanisms.

Most RTKs consist of a single polypeptide and are monomeric in the absence of ligand. Members of the insulin receptor subfamily are disulfide-linked dimers consisting of two polypeptides, thereby forming a α2ß2 heterotetramer. Ligand binding to the extracellular part induces dimerization of monomeric receptors or rearrangement in the quaternary structure of the heterotetrameric structures. This process results in autophosphorylation of one to three conserved tyrosine residues in the cytoplasmic part, which in turn stimulates the intrinsic catalytic kinase activity in the receptor or generation of recruitment sites for downstream signaling proteins. These downstream signaling molecules contain special phosphotyrosine recognition domains, such as Src homology 2 domain (SH2) or the phosphotyrosine-binding domain (PTB)(Lemmon & Joseph Schlessinger, 2000).

To prevent autoactivation of the receptor in the absence of ligand, the receptor can be autoinhibited by the so-called A-loop (Figure 3.1). In the case of the insulin receptor, the active site is occupied by a tyrosine prior to autophosphorylation that is only available for trans-autophosphorylation. The inhibition seems to be equilibrium between strong enough to prevent autoactivation and weak enough to permit trans-autophosphorylation between ligand-bound receptors as the insulin receptor can still be activated in the absence of ligand (Hubbard, Mohammadi & Schlessinger, 1998). The presence of the inhibition loop has also been found in another receptor system, where the mutations in the loop were linked to constitutive activation (H. Chen et al., 2007). The presence of this inhibitory mechanism implies that phosphorylated loops in other RTKs may also be autoinhibited by the same method, even if the loops don’t have much sequence similarity.


11

Figure 3.1: Model for receptor dimerization stimulating tyrosine kinase activity. The flexible loop present in the active domain of RTKs can adopt various conformations. A. When in the closed conformation, the loop (red) prohibits receptor dimerization and signaling. B. In the open loop conformation, the substrate ATP can bind and trans-autophosphorylation can occur. C. Phosphorylation in the loop induces an open conformation, enabling receptor dimerization. Figure adapted from reference (Hubbard, Mohammadi & Schlessinger, 1998).

While the four α- helical bundle cytokines do not show any significant sequence similarity, the family of their associated receptors has some similarities: the extracellular segments contain conserved amino acid modules with significant structural and sequence resemblance, which reveal their evolutionary relationship (Bazan, 1990).

The four α-helical bundle cytokines discussed before, SCF, Flt3L and CSF-1, bind to a family of RTKs named the RTKIII family; the cytokines all bind their respective receptors using a common binding mode involving the distal ends of the helical bundles. However, in the past it was thought that they would bind to RTK receptors family I and II, which use a so-called GH–paradigm for binding, named after the growth hormone complex with its receptor. In the GH-paradigm, the interaction sites of the ligand lie on top of the helices, not at the distal ends of the monomers. The paradigm by which the 3 discussed cytokines bind resembles the VEGF (vascular endothelial growth factor) paradigm, used by the cysteine-knot cytokines (S N Savvides, Boone, & Andrew Karplus, 2000).

3.1.1 FLT3 Fms-like tyrosine kinase receptor 3 (Flt3) is a member of the tyrosine kinase receptor III family that is expressed on HSC and early myeloid and lymphoid progenitor cells, mainly B-cell progenitors (Kikushige et al., 2008). Flt3 ligand/receptor signaling thus mainly impacts early hematopoiesis.

The structure of Flt3 was determined to a resolution of 2.1 Å in 2004. The presence of an autoinhibitory loop was confirmed, comprising the complete juxtamembrane (JM) domain where the closed loop folds between two lobes of the inactive kinase fold (Griffith et al., 2004). The structure of Flt3 with the activation/autoinhibition loop can be found in Figure 3.2.


12

Activating mutations in the form of internal tandem duplications (ITD) or point mutations in Flt3 have been detected in 30% of the patients with acute myeloid leukemia (AML). Both mutations lead to constitutively active Flt3 and the resulting signaling enables factor-independent proliferation of growth factor-dependent lymphoid and myeloid cells, supporting the oncogenic role of Flt3 mutations. Drugs have been developed for treatment in AML; however, because different mutations have different sensitivities towards the inhibitors, it is of great importance to determine the precise nature of Flt3 mutations before using receptor tyrosine kinase inhibitors (Grundler et al., 2003).

Figure 3.2: Flt3 catalytic domain with autoinhibitory loop. The autoinhibitory loop consists of the juxtamembrane domain (green) and can fold between the two kinase domains (red and blue) where it will span almost the entire length of the molecule.

3.1.2 C-KIT C-Kit is another member of the tyrosine kinase receptors, subclass III. Even though in many cell types the expression of c-Kit is lost upon differentiation (such as B- and T-cells), mast cells, natural killer (NK) cells and dendritic cells (DCs) retain their expression, suggesting an important role of the c-Kit in these cells.

As is the case for its ligand SCF, c-Kit is produced from different splice variants. The extracellular form of c-Kit consists of 3 Ig-like domains that adopt an elongated and bent conformation, which is more rigid than flexible due to interdomain interactions (Figure 3.3). This conformation keeps the domain 1 bent backwards, which might enable ligand binding to domains 2 and 3 (Liu, X. Chen, Focia, & He, 2007).

As the expression of c-Kit is normally limited to certain types of cells, abnormalities in the expression and function of c-Kit have been associated with a variety of human diseases (Lennartsson, Jelacic, Linnekin, & Shivakrupa, 2005). For example, some tumors are reported to have Kit/SCF autocrine loops, whereas gain-of-function mutations in c-Kit can result in constitutive dimerization and activation, a phenomenon that is for example detected in most of the patients suffering from gastrointestinal stromal cell tumors (GIST). The presence of these mutations is associated with poor prognosis. Several of the mutations are being tested in drug trials using small molecule drugs targeting the SCF:c-Kit interaction (Margulies et al., 2009).


13

Figure 3.3: The extracellular domains of c-Kit. A. The bent conformation between domain 1 and 2 may enable ligand binding. B. The contact region between domains 1 and 2 maintaining the rigid conformation. Figure adapted from reference (Liu, X. Chen, Focia, & He, 2007).

C-Kit has also been linked to the regulating the expression of IL-6, a pleiotropic cytokine with both pro- and anti-inflammatory properties, providing a link between c-Kit and immune responses (P. Ray, Krishnamoorthy, & A. Ray, 2008). The link between DCs and the c-Kit receptor shows a new role in regulating the adaptive immune response. If c-Kit deregulation leads to immune related pathologies such as asthma, c-Kit inhibitors may be used as a treatment for better controlling the disease (P. Ray, Krishnamoorthy, Oriss, & A. Ray, 2010).

3.1.3 CSF-1R The receptor for CSF-1, CSF-1R is also known as Fms and is another member of the RTKIII receptor family. CSF-1R can transmit growth signals as well as a signal for differentiation in the hematopoietic cell lineage. The switch between growth and differentiation is in part caused by presence of certain phosphorylated tyrosine residues in the cytoplasmic tail and subsequent downstream signaling (L. R. Rohrschneider et al., 1997).

In 1993, Wang et al. identified the ligand-binding domains of the receptor for CSF-1 as the extracellular Ig-like domain 3 (Z. E. Wang, Myles, C. S. Brandt, Lioubin, & L. Rohrschneider, 1993). It has also been demonstrated that the C-terminus as well as the extracellular domain around the fourth Ig-like domain are important for dimerization as no crosslinking could occur in the absence of the C-terminus or mutations in the fourth Ig-like domain; rapid internalization and degradation followed quickly (Carlberg & L. Rohrschneider, 1994).

The gene coding for CSF-1R is also known as the c-fms proto-oncogene and resides at the 5q33 gene location. Several mutations in codons of the gene have been associated with the development of cancers. For example, in renal carcinoma whole chromosomal gains of the c-fms gene have been linked to the pathogenesis, where the cancer cells could be hypersensitive to even normal levels of CSF-1R, enabling invasion (Soares et al., 2009). Also, abnormally high CSF1R expression has been associated with aggressive behavior and poor outcome in a variety of malignancies such as epithelial ovarian cancer, where overexpression of CSF-1R indicates poor prognosis (Chambers, Kacinski, Ivins, & Carcangiu, 1997).


14

Several inhibitor drugs have been cocrystallized with the CSF-1R in order to guide the design of small-molecule drugs to treat CSF-1R related diseases. One of the drugs in question binds to the active site of the kinase domain and can inhibit activation and signaling of the CSF-1R, in accordance with the autoinhibitory loop (Schubert et al., 2007) (Figure 3.4).

Figure 3.4: Inhibitor of the catalytic kinase domain of CSF-1R. The inhibitor (a Quinolone, in blue) binds between two kinase lobes. The N- and C-termini and the JM domain (yellow) are indicated. PDB: 2IOV Figure rendered with PyMol.

3.2 The Epstein-Barr virus

3.2.1 Pathology The Epstein-Barr virus (EBV) or human herpes virus 4 (HHV4) is a member of the γ-herpes subfamily of human herpes viruses and infects most of the world’s human population. After infection, the virus persists lifelong in the infected host. Primary infection occurs typically during childhood and remains mostly asymptomatic. However, when primary infection occurs during adolescence or early adulthood, it can results in infectious mononucleosis (IM), a self-limiting lymphoproliferative disease characterized by an expansion of EBV-infected B-lymphocytes. While the majority of acute IM recovers, in immunocompromised and immunosuppressed patients it can lead to lymphoproliferative disease.

EBV is the causative agent of Burkitt’s lymphoma, a childhood cancer common in parts of Africa where malaria is endemic; the EBV sustains the tumors by promoting proliferation and inhibiting cell death (Vereide & Sugden, 2009). The malaria infection may compromise EBV specific immune control, indicating that even in immunocompetent patients, loss of selective components of EBV specific immunity might contribute to malignancies associated with EBV (C. Münz & Moormann, 2008). EBV is also associated with several epithelial cancers including nasopharyngeal carcinoma (NPC), which is endemic in Africa, and other various types of cancerous malignancies (Delecluse, Feederle, O'Sullivan, & Taniere, 2007).

EBV maintains a constant interplay with the host immune system. The immune response upon a primary infection is strong and in most healthy people the virus will be constrained in


15

a strictly latent, immunologically silent status. However, the virus will persist through activation of the lytic replication in some cells, where T and B-cell recognition is evaded by action of EBV proteins ((Rowe et al., 2007). After the lytic phase, the virus may become silent again and establish latency in memory B-cells, where the expression is limited to a small number of proteins, including EBNA1, which is hardly recognized by T-cell. This protein is expressed in all EBV-associated tumors and essential for maintenance of the viral episome. Even though the full-length EBNA1 is not recognized by T-cells, a shorter version is which might be a possible drug target (Voo et al., 2004). Other proteins produced by EBV can dictate the establishment of certain malignancies (Chang, Yu, Mbulaiteye, Hildesheim, & Bhatia, 2009; Zuo et al., 2009).

The treatment of EBV infections can potentially exist of T-cell treatment combined with drugs targeting specific EBV proteins (Merlo et al., 2010). Another study describes how DCs can expand the T-cell signal to high levels; treatment with DCs might be another good immunotherapy of EBV associated malignancies (Subklewe et al., 2005).

3.2.2 BARF1: a new receptor for CSF-1 Recently, a structural genomics study of EBV was undertaken which resulted in four solved structures of proteins, one of which was the BARF1 protein (Tarbouriech, Buisson, et al., 2006). The BARF1 structure was solved to a resolution of 2.3 Å.

Structurally, the BARF1 protein is composed of two domains belonging to the immunoglobulin fold superfamily. In solution, the BARF1 protein forms hexameric rings through contacts involving the C-terminal domains, which interact through an extension of the ß-strand in the Ig-fold to form continuous sheets, and the N-terminal Ig-domains, interacting through side-chain contacts (Tarbouriech, Ruggiero, de Turenne-Tessier, Tadamasa Ooka, & Wim P Burmeister, 2006). The structure of the BARF1 protein can be found in Figure 3.5.

Strockbine et al. proved that BARF1 could bind to the CSF-1 cytokine, where the binding site of the CSF-1 was narrowed down to the helices as all isoforms of the cytokine could interact with the BARF1 protein. When administering BARF1 in mice, the activity of CSF-1 was neutralized, indicating a sequestration role of the BARF1 (Strockbine et al., 1998). By binding the CSF-1, BARF1 may evade the immune system. The binding site has since been clarified as the N-terminal regions between two Ig-like domains of BARF1, a binding site away from the endogenous receptor CSF-1R (Elegheert et al., unpublished). This has raised the question why the BARF1 protein is not simply binding the CSF-1 through blocking the site for receptor binding, but instead binds another site that possibly leaves the option open for residual CSF-1 signaling through CSF-1R. BARF1 is therefore not a classical decoy receptor, which mimics the binding of the endogenous receptor and the possible residual signaling might play a role in the EBV infection cycle.


16

Figure 3.5: The BARF1 structure. The arrows indicate the interfaces that enable hexamerization with N to N and C to C interactions. Every BARF1 monomer is colored differently. Figure adapted from reference (Tarbouriech, Ruggiero, de Turenne-Tessier, Tadamasa Ooka, & Wim P Burmeister, 2006).

The BARF1 protein is highly expressed during the lytic phase of EBV infection. Several studies have indicated a link between the expression of the BARF1 protein and possible oncogeneity (Sheng, Decaussin, Sumner, & T Ooka, 2001; Wei & Tadamasa Ooka, 1989).

A complexity that hinders the extrapolation to humans is the specific action of the complex. Studies that have been done on complexes of the human (h) and mouse (m) CSF-1 with the BARF1 have tried to characterize the binding constants, and therefore the possibility to sequester the CSF-1, by use of Surface Plasmon Resonance (SPR) and ITC. These have shown that while the combination BARF1:mCSF-1 leaves room for residual signaling to the CSF-1R, the BARF1:hCSF-1 complex has a higher affinity and leaves no room for residual signaling to the CSF-1R. The binding of CSF-1 to the BARF1 is of a very high affinity and as SPR has revealed, CSF-1 is not released after a certain amount of time, as is the case in the interaction between CSF-1 and CSF-1R (Elegheert et al., unpublished). The fact that the mouse variant can still signal through the receptor may be a partial explanation of the attributed oncogenic character of BARF1 in the previous studies, which were all performed in mice.


17

Chapter 4: Cytokine ligand-receptor interaction

4.1 Rationale Cytokines signal through fundamentally similar mechanisms and via overlapping pathways. Only the specific ligand-receptor interaction and the cellular distribution of cytokine receptors dictate the hematopoietic specificity of action. Structure-function studies to gain insight in the binding of the ligand to the receptor and subsequent signaling rely greatly on available three-dimensional models. These studies mainly focus on critical side chains that are responsible for interaction with the receptor. In Chapter 2, it was shown that the presence of some mutations in cytokines or corresponding receptors could cause deregulation of the system and possible associated diseases. Characterization of the interaction sites between ligands and receptors is important for rational manipulation of cytokines for therapeutic benefit. The most popular way of designing drugs targeting protein-protein binding sites is designing peptides that mimic the specific interaction of the natural binding domains, even when their structural scaffold is entirely different from the endogenous interaction partner (DeLano, 2000).

Design of conformationally constrained peptides or small organic molecules that mimic receptor binding can be done by mutational analysis coupled with structure determination. An overview of applied techniques and associated limitations for structure-function studies can be found in (Kaushansky & Karplus, 1993).

4.2 Conformational changes

4.2.1 FLT3 and FLT3L The structure of Flt3L bound to two receptor Flt3 domains 1 to 4 has recently been elucidated up to a resolution of 4.3 Å (Verstraete et al., 2011). Unexpectedly, the ligand-binding epitope is almost exclusively contributed by Flt3 domain 3, in contrast to other RTKIII complexes, where domain 3 of the receptor contributes about half of the epitope for ligand binding.

Flt3L does not undergo any major local structural rearrangements upon binding to the receptor. However, the two subunits of the ligand display a hinge-like rearrangement with an angle of 5-6° in between the two (Figure 4.1).


18

Figure 4.1: Comparison between Flt3L bound to Flt3 versus the unbound form. Despite not undergoing any major structural changes by binding to the receptor, the cytokine ligand Flt3 does show a rigid body movement of the monomers of 5-6°. PDB Flt3L unbound: 1ETE; PDB Flt3L bound: 3QS9. Figure rendered in PyMol.

Another striking feature of the complex is the absence of homotypic receptor interactions between the two receptors, an interaction, following the RTKIII paradigm, which would be contributed by domains 4 of the receptors. In the Flt3:Flt3L case, the domains 4 of the receptors are turned away from each other (Figure 4.2). Sequence inspection revealed that the domain 4 doesn’t posses the conserved structure-sequence fingerprints that are found in all other RTKIII domain homologues.

Figure 4.2: Domain 4 of the Flt3 does not engage in homotypic interactions. Overlay of the backbones of domains 3 and 4 of C-Kit with Flt3 shows that Ig-like domains 4 (D4) of c-Kit connect through a salt bridge as shown by the present arginine residues. This is not the case for Flt3, where domains 4 in both receptor legs are turned away from each other without any interaction. PDB C-Kit:SCF: 2E9W; PDB Flt3D1-4:Flt3L: 3QS7. Figure made in PyMol,


19

4.2.2 C-Kit and SCF The structure of the SCF:c-Kit complex has been recently solved to a resolution of 2.5 Å (Liu, X. Chen, Focia, & He, 2007). Whereas free SCF in both the mouse as the human variant has some disordered and flexible regions (Figure 2.3), including the N-terminus, the bound SCF has good density at the N-terminal region, and another loop gets anchored to the hydrophobic core of SCF (Figure 4.3). These changes are purely induced by binding to the receptor and may be required for c-Kit activation.

C-Kit uses extracellular Ig-like domains 1 to 3 for SCF binding. As domain 4 and 5 were not included in the solved structure, their role in possible receptor dimerization remains to be proven, although this possibility has been questioned because of the large distance between domains 3 in the crystal structure (Figure 4.2).

Figure 4.3: Comparison bound versus unbound SCF. Superimposition of one monomer of unbound mouse SCF (mSCF: PDB: 2O27), human SCF (hSCF, PDB: 1SCF) and bound mSCF to c-Kit (not pictured, PDB: 2O26) are shown as sticks with C, O, N, Cα and Cß atoms present. Green boxes depict biggest conformational changes upon binding to c-Kit. Figure generated with PyMol and adapted from (Liu, X. Chen, Focia, & He, 2007).

4.2.3 CSF-1R and CSF-1 So far, the only structure available for the CSF-1R:CSF-1 complex is a structure composed of the mouse CSF-1 bound to the first 3 extracellular domains of one monomer of the CSF-1 receptor which was solved to a resolution of 2.4 Å (X. Chen, Liu, Focia, Shim, & He, 2008). Curiously, the second monomer of the receptor was not crystallized, which could be an artifact due to the used method where the two proteins were mixed and crystallized rather than first isolating the complex and then attempting crystallization.

The binding of CSF-1 to the receptor is achieved at domain 2 and 3 of the CSF-1R and the interface primarily exists of hydrophilic contacts. Overall, the interaction between CSF-1 and CSF-1R is based on charge complementarity. The structure of the complex can be found in Figure 4.4.


20

Figure 4.4: Structure of CSF-1:CSF-1R complex. The dimeric ligand (blue) is bound to domain 2 and 3 of one monomer of the receptor (orange). Ig-like domain 1 is turned away. PDB: 3EJJ. Figure rendered in PyMol.

No other structures for the CSF-1 complex are available in the PDB. There’s also one structure available for unbound CSF-1 (PDB: 1HMC), but this only consist of Cα atoms (Figure 4.5). Although there’s no structure of unbound mouse CSF-1 available, the article of Chen et al, 2008 describes that upon binding to the receptor, the mouse CSF-1 (mCSF-1) undergoes a conformational change by a rigid body movement of 5°.

Apart from crystallization, studies of the CSF-1:CSF-1R complex by EM (Electon Microscopy), SAXS (Small-Angle X-ray Scattering) and ITC (Isothermal Calorimetry) have revealed assembly and thermodynamics of the complex. The obtained EM and SAXS results confirmed the presence of two receptor monomers bound to one ligand, forming a classical ternary complex (Elegheert et al., unpublished).

Figure 4.5: Bound versus unbound CSF-1. Structures of mouse CSF-1 (mCSF-1) bound to the receptor FMS (PDB: 3EJJ, receptor not pictured) and unbound human CSF-1 (hCSF-1, PDB: 1HMC) are superimposed. Other than the available Cα atoms of hCSF-1, there are no other deposited structures available for unbound CSF-1. Figure generated in PyMol.


21

Chapter 5: X-ray crystallography

5.1 Introduction X-ray crystallography is one of the most used experimental techniques to determine 3-dimensional macromolecular structures. X-rays are very high energy (104 times higher than visible light) electromagnetic waves that are scattered by electrons, termed as Thomson scattering, as they hit periodically arranged atoms in crystals. The electrons in their turn emanate virtual waves that can be interfering constructively or destructively in certain directions. The sum of all the scattering events of independent, single photons then generate the diffraction pattern. In an X-ray diffraction experiment the electron distribution is therefore measured in real space. Form this distribution the average atom positions can be reconstructed.

The process of obtaining a 3-dimensional structure from start to finish is depicted in Figure 5.1. The protein of interest is purified as extensively as possible and is crystallized. Because X-rays are very high intensity beams, they can cause a lot of harm to the crystal by primary and secondary radiation damage. Therefore the crystals are mostly cryo-frozen, and data is collected at 100K. To prevent ice formation during the freezing procedure, a cryo-protectant is used. The crystals are subjected to an X-ray beam, most often coming from a synchrotron source, and the crystal is rotated in the beam to generate diffraction. As the crystal is made up of periodic crystal lattices, the individual scattering events will be amplified and will result in sharp diffraction spots. The intensities of the spots are quantified and the respective structure factors F are determined. Through an inverse Fourier transform the electron density map is calculated into which the unknown structure can be build. After this, the model is adapted to fit the density as best as possible through several rounds of refinement.

5.2 Lattices, unit cells and spacegroups Crystals are made up of an extended three-dimensional arrangement of structural motives according to a lattice. The number of ways to pack molecules in space is limited to 14 Bravais lattices, defined by the relation between their axes and the enclosed angles, and their minimum internal symmetry. The crystal lattice is made up of unit cells, the smallest and simplest volume-element that via translation builds up the whole crystal. Unit cells are defined by three vectors (a, b and c) and three angles (α, ß and γ) (Figure 5.2); and are further made up of asymmetric unit cells (ASU) by symmetry operations.


22

Figure 5.1: The X-ray crystallography process. Figure adapted from slides of Experimental Structural Biology lessons.

The asymmetric unit is the biggest entity of the crystal that has no crystallographic symmetry. One ASU makes up an entire unit cell by symmetry operations, which are limited to rotation and screw axes in proteins crystals. For example, the simplest two-fold screw axis 21 relates one molecule with its symmetry mate by a rotation of 180° around the rotation axis, followed by a translation along ½ of the unit cell axis where the screw axis operates. The translational shift allows a tight and continuous stacking of the molecules in the crystal. The combination of the symmetry operations that result in the original molecule after a certain number of operations, are limited to 32 point groups. Combining the possible point groups with the possible Bravais lattices results in 65 chiral space groups possible for proteins. The most common are the ones containing screw axes that allow for tighter packing as opposed to plain rotation axes; for example the space group of the mCSF-1 protein turned out to be the primitive orthorhombic space group P212121 (see the Results section).

Figure 5.2: Unit cell parameters. The unit cell is formed by three pairs of parallel planes and defined by three vectors (a, b and c) extending from the origin (O) and three angles (α, ß and γ), given rise to the 3 unit cell faces A, B and C (Rupp, 2010).


23

5.3 From diffraction to electron density X-ray diffraction can be treated as reflection on a set of planes in the crystal. Only reflected X-rays that are in phase will get detected, which is summarized by the Bragg equation where λ is the wavelength, θ the reflection angle and d the distance between 2 subsequent planes (Figure 5.3A).

The diffraction data encompass their own space, called the reciprocal space R*, whereas the molecules that give rise to the diffraction exist in real space R. The whole reciprocal space cannot be sampled during one measurement; the extent of the sampled reflections lie on the Ewald sphere; a sphere that can be constructed around the crystal with a radius of 1/λ. Only when a reciprocal lattice point (determined by h, k and l indices) lies on the Ewald sphere, diffraction can occur (Figure 5.3B). The reciprocal space is centrosymmetric and reflects the point group symmetry in the crystal, and in most cases doesn’t need to be sampled completely because of the presence of symmetry related regions.

Figure 5.3: The Bragg equation and diffraction and resolution limits. A. The fundamental diffraction condition is summarized in the Bragg equation (nλ = 2d sinθ). B. When the crystal is rotated by small increments, different reciprocal lattice points will fall on the Ewald sphere (radius 1/λ) and result in diffraction, which can be picked up by the detector (Rupp, 2010).

Inside a unit cell every electron will have a contribution to the resulting scattering. The vector summation of all the diffracted X-rays is the structure factor F. The structure factor relating to every reflection, Fhkl, contains the information that is needed for the construction of the electron density for every x, y and z position in the unit cell. The intensity of the reflection is related to the amplitude of the structure factor squared.

As there is only information about the intensity of the structure factor available, the phases are somehow lost during the measurement. This is known as the phase problem in crystallography. Other methods need to be applied to solve this such as molecular replacement (MR), single/multiple wavelength anomalous diffraction (SAD/MAD), single/multiple isomorphous replacement (SIR/MIR) and direct methods.


24

Data collection is typically a long process because of the small increments in rotation of the crystal. Data can be automatically processed by programs such as XDS (X-ray Detector Software, https://xds.mpimf-heidelberg.mpg.de). XDS uses different jobs to reduce and integrate data by three major steps: 1) spot collection (XYCORR and COLSPOT); 2) indexing (IDXREF) and 3) integration (INTEGRATE and CORRECT). At this point, the gathered data are checked for quality and resolution limit by different kinds of statistical tests and parameters.

When the density is obtained after phasing, the model can be built using a program such as Coot (Crystallographic Object-Oriented Toolkit). Coot generates difference density maps, showing the spatial distribution of the difference between measured and calculated electron density (Fo - Fc). The Fo - Fc map shows positive density (present in the data, but not in the model) and negative density (present in the model but not in the data). Another difference density map is also generated (2 Fo – Fc), which always contains positive density (Figure 5.4).

Figure 5.4: Electron density difference maps. The Fo-Fc difference map is positive (green) or negative (red), whereas the 2Fo-Fc difference density map is always positive (blue). The molecule placed inside the density (orange) is the mCSF-1 protein. Both maps are shown at a 1σ cut-off. Difference density maps generated by Coot, figure generated by PyMol.

Refinement of the model is a process in which the obtained model is adapted to match the data. Several restraints are taken into consideration such as bonding angles and lengths. The structure factors of the obtained model can be calculated and are revised against the observed structure factors through monitoring of the R-values. In the Materials and methods section, the statistics and refinement procedure are looked at in greater detail.

Part 2: Goal

Part 2: Goal

26

Goal

The hematopoietic cytokines and their associated receptors comprise a network of tightly regulated complexes that give rise to different cell types during the development from hematopoietic stem cells to differentiated cells. More and more is revealed about their mechanisms and possible implication with diseases and several recent studies have elucidated the structures of cytokine ligand bound to their specific receptors. In the future, the collaborative work from different fields such as structural biology and cell biology will help to elucidate to entire story surrounding the hematopoietic cytokines.

CSF-1, one of the hematopoietic cytokines has next to its receptor CSF-1R, another binding partner in the form of the hexameric BARF1, a viral protein from the Epstein Barr virus. Binding of CSF-1 to BARF1 is mediated by a site away from the receptor-binding site on CSF-1, leaving room for receptor signaling to still take place. Because residual receptor signaling shows a specific species difference, the BARF1 might play different roles in different species during the Epstein Barr virus infection.

Structure solving by means of X-ray crystallography is a powerful way to visualize the 3D-structure of a protein as it can, for example, given the resolution, provide determination of important residues in protein-protein interactions or give insight into possible functions of the protein.

The goal of this thesis was to determine the structure of the free mouse CSF-1 protein using X-ray crystallography. The protein was to be expressed in a suitable host system, obtained from the producing cells and purified to enable crystallization. Obtained crystals were to be tested by X-ray diffraction and a dataset collected. Using a suitable method to solve the crystallographic phase problem and refinement procedures to ensure correctness of the obtained model, the structure of the free mCSF-1 protein was to be determined.

Also, the obtained structure had to be evaluated with regard to its interaction with BARF1, where binding of mCSF-1 to BARF1 still allows CSF-1 interaction with the CSF-1 receptor. Because no other structures of free mCSF-1 are available to our knowledge, the structure of free mCSF-1 might proof to be a piece of the puzzle that is the hematopoietic cytokine network.

In a more general sense, this thesis also had the purpose of getting acquainted with, and gaining knowledge about the biomolecular crystallography field.

27

Part 3: Materials and methods


28

Chapter 6: Obtaining the construct and overexpression in E. coli

6.1 Introduction During this thesis, the E. coli CodonPlus-RIL strain is used for overexpression of the mCSF-1. The protein can be found in so called inclusion bodies that are nuclear or cytoplasmic aggregates that occur when trying to overexpress heterologous proteins in E. coli. These inclusion bodies need to be extracted from the cells and protein needs to be obtained from within. In most of the cases, the protein is misfolded because no disulfide bridges can be formed in the reducing cytoplasmic environment. Refolding can be done by numerous protocols; in this case strong denaturation by GnHCl followed by rapid dilution and dialysis was chosen. After several purification steps, pure correctly folded and biologically active mCSF-1 is obtained.

The CodonPlus-RIL strain (Novagen, Darmstadt, Germany) is an E. coli BL21(DE) derivative that contains additional tRNAs to achieve higher expression of eukaryotic recombinant proteins. The strain is mostly used for expression of genes coming from AT-rich genomes. The tRNAs that are additionally present are arginine (AGA and AGG), isoleucine (AUA) and leucine (CUA) codons. The BL21 strain carries the T7 polymerase gene and is ideal for expression of genes cloned into vectors containing T7 promoters (such as the used pET15b vector). Selection of the correct colonies was done by adding both carbenicillin, selecting for pET15b presence, and chloramphenicol, selecting for the tRNA carrying plasmid present in the CodonPlus-RIL strain.

The pET15b vector contains a lac promoter and produces a lac inhibitor (LacI), which stops transcription of the mCSF-1 mRNA cloned behind the lac promoter. IPTG, an analogue of lactose, binds to LacI and changes its conformation, hereby preventing the binding of LacI to the promotor. After derepression of the promoter by IPTG, m-CSF1 can be transcribed and translated into protein.

The major advantages of using E. coli as an expression host is that it is a fast and inexpensive way of producing recombinant proteins because the lacks of need for costly media, quick growth and relatively easy purification of the wanted proteins. Therefore, E. coli remains a widespread choice for high-throughput expression and many proteins have been successfully expressed and purified by an appropriate refolding protocol this way. Refolding protocols for all sorts of proteins can be found at the website of Refold, a refolding database (http://refold.med.monash.edu.au/). However, E. coli does not perform mammalian protein glycosylation or post-translational modifications; when such properties are desired, another expression system needs to be found. In such a case, eukaryotic expression systems may be of help such as insect cells, yeast cells or mammalian cell cultures. These systems can perform wanted post-translational modifications and a glycosylation type typical for the used host. As each glycoform has its own pharmacological characteristics, an important hurdle in the development of therapeutic proteins for example, the protein glycosylation pathway has been


29

recently extensively studied and engineered in different expression hosts in an attempt at producing all mammalian complex glycosylation (Jacobs & Callewaert, 2009).

Different mammalian cell culture based systems exist, such as Humane Embryonic Kidney 293EBNA cells (HEK) or Henriette Lacks (HeLa) cells. Aricescu et al. describes a time- and cost-efficient high-throughput system for the production of recombinant protein by use of HEK293T cells, which are derived from HEK cells that express the SV40 large T antigen (Aricescu, W. Lu, & Jones, 2006).

6.2 Methods Construct cloning. The mouse CSF-1 construct was cloned into a pET15b vector (vector map can be found in Appendix 1) by Jonathan Elegheert. The expression vector produces a recombinant mCSF-1 with N-terminal His-tag followed by a thrombin cleavage site.

Making electrocompetent cells. BL21(DE3) (CodonPlus-RIL) cells (Stratagene, La Jolla, USA) are grown at 37 °C and harvested through centrifugation (4000 rpm, 4 °C) when the optical density (OD) measured at 600 nm reaches 0.6-1.0 (UVmini-1240 spectrophotometer, Shimadzu). Then a series of washing and resuspension and centrifugation steps follow, after which the cells are resuspended in 10% (v/v) glycerol and kept at -80 °C. A detailed protocol can be found in Appendix 2.

Transformation of the expression construct. Transformation is achieved through electroporation of BL21 (DE3) (CodonPlus-RIL) competent cells (E. coli Pulser, Bio-Rad). A detailed protocol can be found in Appendix 3.

Preculture and overexpression. One colony is grown O/N in 200 mL LB (with Cb/Cm). The following day each time 15 mL of the culture is diluted into 1 L LB with Cb/Cm. When the culture reaches OD600 of 0.6, 1 mM isopropyl ß-D-1-thiogalactopyranoside (IPTG) is added for induction of expression. Overexpression was confirmed by SDS-PAGE. After 3 to 4 h, the culture is pelleted by centrifugation (6000 g, 10 min), resuspended in solubilization buffer (50 mM Tris pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.2 mM PMSF, 1x Complete (Roche)) and kept at -80 °C.


30

Chapter 7: Refolding and purification of mCSF-1

7.1 Introduction A refolding protocol has the aim to obtain correctly folded protein out of a misfolded protein mix. During the inclusion body extraction and breaking through sonication, care was taken to prevent cleavage of the protein by including protease inhibitors in each solubilization step (PMSF, EDTA, Complete). By adding the detergent Triton X-100 in the washing buffer, remaining lipid and cell membranes-associated protein were removed. Intact inclusion bodies were solubilized in a buffer containing the denaturants GnHCl and ß-mercaptoethanol, thereby breaking them open and releasing the contained proteins from within. Afterwards, proteins were completely denatured by adding strong denaturants, such as urea or guanidine hydrochloride. This lowers the transition state barriers, so that while aggregation may be formed faster, it is also unfolded faster, thereby possibly adopting the correct folding state.

During the nickel purification under denaturing conditions, purification of mCSF-1 was achieved by the use of a nickel sepharose column, under denaturing conditions, keeping the protein unfolded during the whole procedure. This form of procedure is also called immobilized metal affinity chromatography or IMAC as the nickel ions are immobilized on a resin and have affinity for His-tags. Elution was achieved by lowering of the pH, as the pI of histidine = 6. At low pH, the His-tag will be positively charged and electrically repulsed by the Ni+ ions in the matrix.

Rapid dilution took place in the presence of 1 M L-arginine, which improves the efficiency of refolding (Verstraete et al., 2009), EDTA and PMSF to prevent breakdown by proteases, and reduced and oxidized glutathione to enable the sampling of the disulfide bonding space of the protein. By working at 4°C, the transition barriers were lowered, where aggregate can be quickly formed but also unformed, ensuring sufficient sampling of the folding space to obtain optimal conformation. After this, dialysis was performed, a necessary step to eliminate arginine, which would otherwise interfere with the following IMAC purification. EDTA was omitted in the final step of buffer change for the same reason.

Further purification steps were all automated with an Äkta system. The elution profiles were followed by absorbance at 280 and 215 nm, the wavelength of aromatic peptides and imidazole absorption, respectively To keep the system from clogging up, all buffers and solutions were first filtered before connection to the system.

The protein solution was first filtered to dispose of particles larger than 0.22 µm, which could contain misfolded aggregates. MgSO4 was added to possibly eliminate residual EDTA.

To remove the imidazole in the protein solution after automated nickel affinity chromatography, a gel filtration was performed. This step also served as an added purification


31

step, in that contaminants are removed on the basis of their weight. This technique is also called size-exclusion chromatography (SEC).

A gel filtration column consists of a matrix of packed beads of a certain diameter. Proteins that are bigger than the diameter pass through unstopped, proteins that are smaller get trapped inside the beads and explore a longer path inside the column, switching between stationary and mobile phase. Proteins are separated based upon their weight and compactness and each protein will elute through a stationary phase at a different rate. Each size exclusion column has a range of molecular weights that can be separated, and in this case a SuperdexTM 75 (GE Healthcare, Diegem, Belgium) was used as the mCSF-1 is a relatively small protein ligand (35 kDa).

The eluted peak from the nickel sepharose purification was first concentrated to a volume of 2 mL to ensure a small injection volume and thereby a small elution volume from the gel filtration because all proteins of the same weight theoretically travel at the same speed through the column. Alternatively, the imidazole can also be removed by use of a desalting column (GE Healthcare, Diegem, Belgium).

The mCSF-1 construct of our pET15b vector carried an N-terminal His-tag, a thrombin cleavage site and the recombinant mCSF-1 protein. To remove the His-tag, thrombin was used, a serine protease for which the optimal cleaving sequence is A-B-Pro-Arg/X-Y with A and B hydrophobic amino acids and X and Y nonacidic amino acids. The cleavage site in the pET15b vector contained the cleavage sequence Leu-Val-Pro-Arg/Gly-Ser (Appendix 1).

To further purify the mCSF-1 protein out of the solution, yet another dimension of purification was used. A Source 30Q column (GE Healthcare, Diegem, Belgium) is an anion exchange chromatography column, which allows elution based on pI differences. In anion exchange, the matrix of the column is positively charged and has affinity for negative charges that pass through. As the pI of mCSF-1 is around 6-6.5, it will be negatively charged at pH 8 and positively charged at pH lower than the pI. To concentrate the sample once again and eliminate residual contaminants, a final gel filtration was performed.

A detailed protocol for refolding and purification of mCSF-1 can be found in Appendix 4.

7.2 Methods Inclusion bodies washing and solubilization. The inclusion bodies are washed by repeating round of sonication in washing buffer (50 mM Tris pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.2 mM PMSF, 1x Complete and 1% Triton X-100) followed by centrifugation at 20800 g. Resolubilization of the inclusion bodies is achieved by adding GnHCl buffer for denaturation (6 M GnHCl, 100 mM NaPO4 pH 8.0, 10 mM Tris, 10 mM ß-mercaptoethanol) and subsequent incubation for 3 h (rotary shaker, room temperature) and centrifugation. The supernatant is collected.

Purification under denaturing conditions. A nickel sepharose column is prepared and equilibrated with 6 M GnHCl, 100 mM NaPO4 pH 8.0, 10 mM Tris. After the supernatant is added, two washing steps follow with equilibration buffer pH 8.0 and pH 6.3, respectively. Elution is achieved by adding equilibration buffer pH 4.5.


32

Refolding by rapid dilution. A cold refolding buffer is set up (100 mM Tris pH 8.0, 1 M arginine, 1 mM EDTA, 3 mM GSH, 1.5 mM GSSG, 0.2 mM PMSF) and the protein solution is added drop wise overnight at 4 °C under constant stirring.

Removal of arginine. Because of the interference of arginine with IMAC, its concentration is lowered by 20 x dialysis with three buffer changes at 4 °C (20 mM Tris pH 8.0, 1 mM EDTA, 1 mM PMSF). In the last step EDTA is not included because it interferes with the subsequent nickel purification.

Prepacked nickel sepharose and gel filtration. A prepacked nickel sepharose column is connected to an ÄKTA purification system (GE Healthcare, Diegem, Belgium) and equilibrated with 50 mM NaPO4 pH 8.0 and 300 mM NaCl. The refolded protein solution is filtered (0.22 µm), supplemented with 300 mM NaCl and 10 mM MgSO4 and brought unto the column. After binding, the protein is eluted with 500 mM imidazole. The protein solution undergoes an extra purification step by gel filtration with buffer 20 mM HEPES pH 7.5 and 150 mM NaCl. (SuperdexTM 75, GE Healthcare, Diegem, Belgium).

Thrombin digest. The His-tag is removed by adding 1U biotinylated thrombin (Novagen, Darmstadt, Germany) and overnight incubation at room temperature. Cleavage can be checked by SDS-PAGE. The protease is removed by adding streptavidin agarose beads and incubation on the rotary shaker for 10 min. The mixture is centrifuged at 500 g for 5 min.

Anion exchange and final gel filtration. After the thrombin digest, the protein solution is desalted on an anion exchange Source 30Q column (GE Healthcare, Diegem, Belgium), with running buffer 20 mM Tris pH 7.5, 150 mM NaCl. Elution takes place by adding elution buffer (20 mM Tris pH 7.5, 1 M NaCl) in increasing concentration in a short time (100% in 10 min). As a final purification step, another gel filtration is undertaken (20 mM HEPES pH 7.5, 150 mM NaCl) (SuperdexTM 75, GE Healthcare, Diegem, Belgium).


33

Chapter 8: Crystallization and handling crystals of mCSF-1

8.1 Introduction When trying to crystallize a protein for the first time, the optimal methods to achieve supersaturation are not known; therefore different test trials can be set up to gain insight in possible indicative conditions. Various commercial kits are available, falling into different categories: 1) sparse-matrix screens that contain conditions which have previously shown to be successful for crystallization; 2) grid screens where specific crystallization conditions are varied in a systematic way and 3) incomplete factorial screens where specific components of crystallization conditions are sampled in a systematic way.

Initial crystallization trials for mCSF-1 were set up with a Mosquito crystallization robot present at the catholic University of Leuven. In a first attempt, several commercial screens were tested: Crystal Screen 1 & II, PEG ion 1 & II, Index (Hampton Research, Aliso Viejo, CA, USA) and PACT (Qiagen, Hilden, Germany). Crystal Screen I and II consist of a sparse-matrix screen sampling salt, organics, pH and polymers. PEG ion 1 is a sparse matrix profile of anions and cations in the presence of PEG 3,350 in the pH range of 4.5 – 9.2, PEG ion II screens a profile of organic acid in presence of PEG 3,350 in pH range 3.7 – 8.8. PACT tests the effect of anions and cations in the presence of PEG, where Index combines all three possible categories of screens.

Additive screens contain small molecules that affect protein solubility and crystallization. They may aid in lattice formation and formation of bigger crystals and keep proteins in solution by manipulating solvent-solvent and solvent-sample interactions. The screens tested were the Additive ScreenTM and Silver BulletsTM (Hampton Research, Aliso Viejo, CA, USA), the latter one having similar contents but more mixtures of amino acids.

In the hanging drop method, a drop is suspended above the reservoir solution using a glass cover sealed of from the environment with grease. In this configuration, the crystals that will be formed will most likely not be affected by the glass plate and fall deeper into the drop.

8.2 Methods Initial crystallization trials and in house reproduction. Crystallization trials of mCSF-1 were performed using a Mosquito® crystallization robot (TTP Labtech, Cambridge, USA), present in the department of pharmaceutical science at the catholic university of Leuven. Several screens were initially set up: Crystal Screen I & II, PEG ion screen I and II, Index (Hampton Research, Aliso Viejo, CA, USA) and PACT (Qiagen, Hilden, Germany). All set up experiments were 96-well plate sitting drop vapor diffusion of 0.2 µL drops (0.1 µL protein + 0.1 µL well solution) with 75 µL reservoir. Pictures were taken of the crystallization drop at


34

the site at regular intervals and could be viewed remotely. After determining the best crystallization conditions achieved by the Mosquito®, in house reproduction of the hits was attempted. This time, the set-up was done by hand in 24-well plate format in sitting drop format of 2 µL drop (1:1) and 300 µL reservoir.

Grid screens. After several attempts of reproducing crystallization conditions, the best three conditions, in correspondence with the plates of the automatic set-up (0.1 M Tris pH 8.0, 0.2 M CaCl2, 20% PEG 6k; 0.1 M NaAc pH 4.6, 8% PEG 4k and 0.2 M NaNO3, 0.1 M Bis-tris propane pH 7.5, 20% PEG 3350) were redone as a grid screen. Variable parameters were pH and precipitant concentration around the hit condition. The set-up was a 24-well plate sitting drop format with 2 µL drop and 300 µL reservoir.

Additive screens. In an attempt to obtain bigger crystals, two screens containing among others volatiles and small peptides that can stabilize protein growth, were set up with the Mosquito®

in Leuven: Additive screenTM and Silver BulletsTM (Hampton Research, Aliso Viejo, CA, USA). The additive in the automated setting was added to the well solution, after which this was mixed with 0.1 µL protein. Later, the best hits were reproduced in threefold in house as a hanging drop format in 24-well plates, 2.2 µL drop (1+1+0.2 µL) and 300 µL reservoir.

Hanging drop screens. The best crystallization condition based on all screens (0.1 M Tris pH 8.0, 0.2 M CaCl2, 20% PEG 6k) was further explored as a grid screen with the same variable parameters as before but now as a hanging drop setup in 24-well plate form, 2 µL drop and 300 µL reservoir. The condition in itself was also repeated in a 24-well plate format (1:1, 300 µL reservoir.

Harvesting of crystals and cryoprotection. Crystals were scored based on appearance: monodispersity, definition of edges and size. The best crystals based on these criteria were selected for cryofreezing. Crystals were scoped out of the drop using a cat-whisker loop and brought into a solution containing the crystallization condition with added cryoprotectant (glycerol, ethylene glycol, PEG 6k or PEG 400). This procedure was repeated into solutions containing increasing amounts of the cryoprotectant. Quality of cryoprotectant was evaluated on prevalent intactness of crystals as they were transferred into higher concentrations of cryoprotectant.


35

Chapter 9: Data collection and solving the structure of mCSF-1

9.1 Introduction The ID-29 beamline at the ESRF facility offers the possibility of a micofocus beam, which enables measurement of microsized crystals or even targeting of different slices in the same crystal, thereby preventing rapid radiation decay to impair data collection. Another powerful addition to the beam line is the Pilatus 6M detector, which allows for shutterless data collection. Furthermore, mounting of the cryopin containing the crystal unto the diffractometer happens completely automated at ID-29, ensuring a fast transition from the liquid nitrogen in the vial to the stream of cold nitrogen present at the beam line.

After mounting a crystal on the diffractometer, a test set of 4 images (oscillation range 1°, oscillation step 90°) was collected and the diffraction pattern was inspected with adxv. The initial diffraction image reveals crucial information about the diffraction quality of the crystal. Primary key parameters are highest resolution, presence of ice rings, spot separation, sharpness of spots and mosaicity.

Initial indexing of the test sets was done with IDXREF, a part of XDS, which assigns a consistent set of unit cell vectors matching the diffraction pattern based on Fourier analysis. XDS tries all of the possible 44 combinations of crystal systems and Bravais centering and ranks them by a penalty function. The most plausible choice is the one with the lowest penalty right before a large jump in penalty for the next possibility. After this initial indexing step, a data collection strategy can be devised based upon the lattice type and Laue group. The higher the lattice symmetry (which determines the minimal symmetry of the reciprocal space) and Laue symmetry, the easier it is to collect a complete data set.

After collection, the dataset was processed by XDS using the complete assembly of jobs. The raw data were corrected and integrated, making sure the correct unit cell parameters and number of frames were used to provide enough completeness. The dataset was then scaled using XSCALE, a step in which the Wilson outliers were removed. The Wilson distribution is an unconditional probability distribution for structure factor amplitudes or intensities; outliers are therefore very improbable and need to be removed to obtain better statistics. Finally, XDSCONV converted the resulting file containing the hkl-indices, structure factor amplitudes and error of the structure factor amplitudes into an mtz format file. The mtz file was subsequently converted to a binary mtz file with f2mtz in CCP4.

Xtriage, a part of PHENIX, was used to check the dataset for various features: detection of possible twinning, anisotropy present in dataset, presence of ice rings etc. After a first run, it was found that the mCSF-1 data were anisotropic, meaning that the scale factor differed by at least a factor 2 in one of more directions. Anisotropy can stall improvement of the R-factor during refinement because of the inclusion of poor reflections and the degradation of electron


36

density map appearance. The server at http://services.mbi.ucla.edu/anisoscale/ performed an ellipsoidal truncation on the mCSF-1 dataset to leave out weak reflections and an anisotropic scaling that resulted in an isotropic dataset where the slope of structure factor amplitude vs. resolution was equal in all directions. Then, the data were sharpened using a negative B-factor to restore the high-resolution reflections that were downscaled during the anisotropic scale.

When obtaining a dataset from a crystal by means of X-rays, the phase information is lost. When a known structure model is available, this can be used to fit into the unit cell of the observed data by rotation and translation until the best solution is found between calculated diffraction data from the replaced model and the observed data from the unknown model. One prerequisite however is at least 30% sequence identity between the unknown and known structure. Because the number of available structures in the PDB keeps increasing, molecular replacement (MR) is a very popular method for solving the phase problem.

Molecular replacement tries to place and score a real space probe given only reciprocal data space by means of a rotation-translational search. This computational approach first searches the 3-dimensional rotation search and once the correct rotation is found, a 3-dimensional translation search is performed. The advantage of this deterministic technique as opposed to an exhaustive 6-dimensional technique (every translation search for every rotation search) is that it is computationally faster. The disadvantage however is that the proposed rotation solution is not always the correct one. The chance of success of MR is increased by implementing maximum likelihood functions (as implemented in Phaser), which allow consideration of model incompleteness and errors in rotation and translation searches.

The success of the MR search is determined by the log-likelihood gain (LLG) of a solution over the average solution, measuring how much better the data can be predicted by the model than by a random distribution of the same atoms. Another score that is given as an output for MR is the Z-score, a score that indicates the number of standard deviations the LLG is above from the mean. Both values are expected to be positive given a good MR solution.

Because the phases for the electron density reconstruction originate entirely from the replaced known structure, the electron density map is highly susceptible to model bias and will primarily reflect model features as opposed to those of the unknown structure. During refinement, several steps can be undertaken to minimize model bias.

The process of refinement has the ultimate goal of obtaining an optimal fit, calculated by a target function, between the model and the observed data by iterative adjustment of variable parameters of the model. As the model improves, the phases and therefore the electron density map will improve as well and refinement can continue into the next round. Cycling between local real space model building (where residues are for example manually fit into the density) and model correction and global reciprocal space refinement (where parameters are refined against the experimental data) will result in the most successful protein structure solving. Reciprocal space refinement takes restraints (prior knowledge) into consideration so that the model features remain within universally expectations of geometry.

A single round of refinement consists of several cycles of parameter adjustments and optimization. The parameters of the model – coordinates, B-factors, and overall parameters such as overall B-factors, bulk solvent corrections, and anisotropy corrections – are refined against all experimental data. The overall fit between the diffraction data and model is quantified by the global linear residual (R-value) between structure facture amplitudes Fobs


37

and Fcalc. The best fit is calculated from a target function, generally a maximum likelihood target.

The refinement concept requires that there are more experimental data points n available than model parameters p that need adjustment. If n < p there are too few data points or too much parameters to determine the system. To generate a solution of the structure, there need to be at least n = p observations. However, by adding parameters to the refinement to improve the n/p ratio, by including more restraint for example, the danger of overfitting exists. This can be prevented by cross-validation using the Rfree value. The Rfree value is the R-value of a small test set (about 5% of reflections) that is left out of refinement to validate whether a change in parameterization improves the model or not. The rest of the reflections are included in the refinement and their validation value is termed Rwork. If both Rfree and Rwork improve, the change was beneficial and should be included to improve the model.

Different mathematical optimization strategies exist, and depending on the resolution different methods can be used. For example, a low-resolution dataset can be refined using rigid-body refinement, and the molecule may be correctly placed into the electron density, whereas with a high-resolution dataset refinement at the level of individual anisotropic B-factor may be possible. The choice for incorporating a certain method can also be dependent on the radius of convergence, which describes the ability of an algorithm to escape local minima and approach the global minimum.

For refinement of the mCSF-1 structure, the mtz file with the anisotropically scaled data was always used in each round of refinement against the newly obtained model, preventing model bias.

Because the resolution limit of the mCSF-1 dataset was 2.6 Å, the refinement needed to contain restraints. Unrestrained refinement at such a resolution would be underdetermined because of the low redundancy of the measured data as every atom is described by at least 4 parameters (x, y, z and B).

In cycle 1, the two chains of the mCSF-1 dimer were treated as separate solid entities to be placed in the electron density (rigid body refinement) and all atoms were refinement at the basis of their x, y and z position. The B-factor was refined isotropically; individual anisotropical B-factor refinement was not possible as it consumes 6 parameters per atom. At the end of the refinement, hydrogens were added to the structure by phenix.reduce (Word et al., 1999). These hydrogens were considered as riding hydrogens, as they are covalently attached to the refined N, C and O atoms of the structure. Although the added hydrogens did not contribute to the parameters nor consume data, they did lead to an improvement in model quality.

In cycle 2, stereochemical restraints (secondary structure restraints) were regarded as extra observations so as to increase the data-to-parameter ratio. This kept the bond lengths and angles of α-helices in check. Again, individual atom positions were refinement. This time

R =Fobs!Fcalc

h"

Fobsh"


38

however B-factor refinement was performed by TLS refinement, as isotopic B-factor displacement over the whole structure is inadequate to describe any dynamic molecular movement; long side chains for example move in a more lateral than medial way in respect to their backbone. TLS (translation-liberation-screw) parameterization allows description of the model with fewer parameters than an individual ADP description: it contributes only 20 parameters per group. It provides a more realistic description of molecular motion than individual isotropic B-factors. TLS groups for mCSF-1 were found automatically using phenix.find_tls_groups and incorporated in the subsequent refinement runs.

As of cycle 3, the weights of restraints and X-ray terms were optimized automatically. This way, too high restraints, which will force the model to show ideal geometry, as well as too low restraints, which may not reflect reality, are avoided. Optimal weights are found when Rfree has its relative minimum. Using phenix.refine, the weights in the mCSF1 refinement were adapted automatically to their best values. Also, ordered solvent was incorporated in cycle 3 where water molecules were placed inside previously unaccounted for electron density blobs. In both cycle 4 and 5, the model was mainly inspected for remaining gross errors, improbable rotamers and placement of water atoms.

As a protein is inherently flexible, there will be parts of the model that are well defined and others that aren’t as well defined in electron density. To check the reliability of the model, validation is necessary. The obtained mCSF-1 structure was validated throughout the refinement procedure to ensure the plausibility of the model. The stereochemistry was checked after each cycle: improbable rotamers, Ramachandran outliers, contact clashes etc. and changes were saved as a new model, which was subsequently used in the next round of refinement.

9.2 Methods Data collection. Data collection of the mCSF-1 crystals was performed at the ID-29 beam line at the ESRF, Grenoble, France at a wavelength of 0.972597 Å in combination with the Pilatus 6M detector and microfocus. Crystals were automatically transferred to the diffractometer, the beam (50 x 50 µm, 10% transmission) was centered and an initial set of 4 diffraction images were collected (4 images offset by 90°). A test set was inspected with adxv (http://www.scripps.edu/~arvai/adxv/). The best dataset was collected with an oscillation range and detector distance of respectively 0.1° and 387.84 mm.

Data processing. The dataset was processed using the XDS package (X-ray Detector Software, http://xds.mpimf-heidelberg.mpg.de). The output mtz is converted to a binary mtz using f2mtz, included in the CCP4 package (Collaborative Computational Project, Number 4, 1994).

Anisotropic scaling. Ellipsoidal truncation and anisotropic scaling of the dataset were performed by the Diffraction Anisotropy Server (http://services.mbi.ucla.edu/anisoscale/).

Molecular replacement. Molecular replacement using the deposited mCSF-1 structure in PDB: 3EJJ was performed using Phaser, a module in the PHENIX package (Python-based Hierarchical Environment For Integrated Xtallography) (Adams et al., 2010).


39

Structure refinement. Different rounds of refinement were preformed using phenix.refine, included in the PHENIX suite. After each round, the structure was inspected and altered manually in Coot (Crystallographic Object-Oriented Toolkit) (Emsley & Cowtan, 2004). Changes were saved and used in the next round of refinement.

Visualization of the structure. For visualization and validation of the structure the programs Coot and PyMol (DeLano, W.L. The PyMol Molecular Graphics System (2002) DeLano Scientific, Palo Alto, CA, USA. http://www.pymol.org) were used.

40

Part 4: Results

Part 4: Results

41

Chapter 10: Overexpression of recombinant m-CSF1 in E. coli, refolding and purification

10.1 Cloning of the construct and overexpression Cloning of the full-length mCSF-1 construct in the pET-15b vector between the NdeI and BglII restriction sites (Appendix 1) was performed by Jonathan Elegheert. CodonPlus-RIL strains were transformed by electroporation, and cultures were grown as described in the Methods and Materials section. When the culture reached OD600 = 0.6, indicating the log-phase of growth, high expression of mCSF-1 could be achieved.

Induction of the expression was achieved by adding 1 mM IPTG to the culture. Derepression of transcription and subsequent overexpression of the m-CSF1 protein was checked by SDS-PAGE by loading a sample of the cell culture before and after 3-4 hours of induction by IPTG (Figure 10.1).

Figure 10.1: Induction of the mCSF-1 protein by IPTG. Two samples (20 µL) of cell culture were loaded on SDS-PAGE, one before induction (-IPTG) and one after 3 to 4 hours of induction with IPTG (+IPTG), the gel was stained with Coomassie Blue. IPTG derepresses the inhibition of LacI of mCSF-1 transcription. After induction, the mCSF-1 protein was overexpressed in the cells, as pictured by the presence of the thick protein band visible around 17.5 kDa, representing the monomer form of the protein.

Part 4: Results

42

10.1.1 Nickel affinity purification under denaturing conditions The inclusion bodies were extracted from the cells by alternating sonication steps with centrifugation. A final centrifugation step separated inclusion body debris from soluble proteins.

The His-tag present in the pET15b vector provided a mean for easy purification of the mCSF-1 protein out of the obtained supernatant by nickel affinity purification. To follow the course of the purification, samples were taken during the before loading on the column, after each washing step and after elution and checked on SDS-PAGE (Figure10.2).

As presence of GnHCl in the samples hampers SDS-PAGE by forming a precipitate, the GnHCl was removed using ethanol extraction as described in (Palmer & Wingfield, 2004). The SDS-PAGE suggested the presence of mCSF-1 but also of a lot of contaminants, dictating further purification steps.

Figure10.2: Purification of mCSF-1 under denaturing conditions. Samples (20 µL) were taken before loading on the nickel sepharose column, after washing steps and after elution by pH 4.5 and loaded on SDS-PAGE after eliminating the GnHCl, used for denaturation, by ethanol extraction. The red box indicates the probable weight for monomeric mCSF-1 (17.5 kDa). Despite elimination of many contaminants, still a lot are present at elution. FT: Flowthrough; Supern.: Supernatant

10.1.2 Rapid dilution and dialysis The protein solution obtained after nickel sepharose purification was added drop by drop to a refolding buffer, a technique that is referred to as rapid dilution. After rapid dilution overnight, the protein solution was collected and prepared for dialysis.

10.2 Purification of mCSF-1 As the previous step showed, purity of mCSF-1 was far from ideal after the nickel sepharose purification under denaturating conditions. After refolding, the protein was subjected to

Part 4: Results

43

several purification steps, alternating properties on which elution was based, to eliminate contaminants further.

10.2.1 Affinity chromatography on nickel sepharose The sample was brought onto a prepacked nickel sepharose column and eluted with 500 mM imidazole. The elution peak of several mL was collected and can be found in Figure 10.3.

Figure 10.3: Elution chromatogram of nickel sepharose purification. After refolding of the protein solution, it was loaded unto a nickel sepharose fast-flow column. The column was equilibrated with 50 mM NaPO4 pH 8.0 and 300 mM NaCl. The same buffer, complemented with 500 mM imidazole was used to achieve elution. The peak corresponding to the refolded mCSF-1 was collected.

10.2.2 Gel filtration The gel filtration was performed using a SuperdexTM 75 (GE Healthcare, Diegem, Belgium) with running buffer 20 mM HEPES pH 7.5 and 150 mM NaCl. The resulting chromatogram can be found in Figure 10.4.

Part 4: Results

44

Figure 10.4: Gel filtration chromatogram of mCSF-1. The protein solution containing mCSF-1 was concentrated to 2 mL and injected (0 mL point) unto the column with running buffer 20 mM HEPES pH 7.5 and 150 mM NaCl. Elution was based on weight; the peak corresponding to mCSF-1 (37 kDa) was collected.

10.2.3 Thrombin cleavage of the His-tag and Source 30Q ion-exchange chromatography Using the Thrombin cleavage capture kit (Novagen, Darmstadt, Germany) 1 µL biotinylated thrombin was added to the fractions that were collected after gel filtration and incubated overnight. The cleavage was checked on SDS-PAGE (Figure 10.5). Although contaminants were still present, they were less prevalent.

Figure 10.5: Thrombin digestion of mCSF-1. A sample (20 µL) was loaded on SDS-PAGE before thrombin digest (-T) and after overnight incubation with thrombin (+T). The gel was stained with Coomassie Blue. The red box indicates the mCSF-1 with His-tag (-T) and without His-tag (+T) at approximately 17.5 kDa for the monomeric form.

Part 4: Results

45

In the purification step using 30Q anion exchange chromatography, the process was again followed through the absorbance at 280 nm (Figure 10.6). A broad elution peak was collected and checked on SDS-PAGE for purity (Figure 10.7). After this, another gel filtration was performed (results not shown).

Figure 10.6: Source 30Q anion exchange chromatogram mCSF-1. After equilibration of the column with 20 mM Tris pH 7.5 and 150 mM NaCl, the sample was loaded. Elution was achieved by increasing the salt concentration using 20 mM Tris pH 7.5 and 1 M NaCl; the elution peak corresponding to mCSF-1 had a broad base and was completely collected.

Figure 10.7: Checking the Source 30Q purification. Samples from the broad Source 30Q elution peak (Figure 10.6) were loaded on SDS-PAGE (1 to 5). The mean constituent of the samples was mCSF-1 as shown by the protein bands at 17.5 kDa. Near the end of the elution peak, more contaminants were present (4,5).

Part 4: Results

46

Chapter 11: Crystallization of mCSF-1

11.1 Crystallization trials Crystallization trials were performed as discussed in the Materials and Methods section. After initial screens, several conditions had crystalline appearances of already well-formed crystals. Based on these hits, the consensus for crystallization conditions of mCSF-1 seemed to consist of cations in the presence of PEG. The best matches between automated set-up and manual set-up were again repeated, but this time in a grid screen fashion, where pH and precipitant concentration were varied. The best results are shown in Figure 11.1.

These initial trials showed clear crystal formation, but the morphology was lacking: they were overall very small (maximum around 50 µm x 50 µm x 10 µm), some crystals were growing through each other, most had very poor defined edges and were subsequently very fragile in handling.

Figure 11.1: Initial crystallization trials of mCSF-1. First, crystallization was set up automatically using a Mosquito (A), the best hits were repeated by hand in a grid screen varying pH and precipitate concentration (B). Best matches are shown. Drop volume in A was 0.2 µL, in B 2 µL, reservoir volume 75 and 300 µL, respectively. Different morphologies could be observed: single well defined crystals (A1, A3 and B1), sea urchin shaped (B2) and precipitate (B1 and B3) Crystals were viewed using a microscope equipped with a polarizer, exposing the birefringence of crystalline material. Conditions: A1: 0.1 M Tris pH 8, 20% PEG 6k, 0.2 M CaCl2; A2: 0.1 M NaAc pH 4.6, 8% PEG 4k; A3: 0.2 M NaNO3, 20% PEG 3,350, 0.1 M Bis-tris propane pH 7.5; B1: 0.1 M Tris pH 8.2, 19% PEG 6k, 0.2 M CaCl2; B2: 0.1 NaAc pH 4.7, 9% PEG 4k; B3: 0.2 M NaNO3, 22% PEG 3,350, 0.1 M Bis-tris propane pH 7.5.

Part 4: Results

47

11.2 Additive screens When going back to the previous set-up plates, it was found that based on size and morphology, the best condition seemed to be 0.1 M Tris pH 8, 20% PEG 6k and 0.2 M CaCl2. This condition was eventually chosen for optimization.

When testing the additive screens, bigger crystals seemed to be formed (Figure 11.2), but they were firmly stuck on the bottom of the platform, making it hard to manipulate them. To possible eliminate the influence of the bottom of the platform on crystal formation hanging drops were set up in a further step.

Figure 11.2: Examples of hits from additive screens mCSF-1. The screens were set-up automatically, reservoir 75 µL, drop 0.2 µL (1:1). Crystals showed different morphologies, from cube like (1) to elongated (2). Primary condition in the drop consisted of 0.1 M Tris pH 8, 20% PEG 6k, 0.2 M CaCl2 to which the additive was added. Conditions: 1: 0.01 M magnesium chloride hexahydrate; 2: 1.2% w/v myo-inositol; 3: 0.1 M lithium chloride

11.3 Hanging drop crystallization As discussed by the Materials and methods section, hanging drop crystallization trials were set up. This proved to be the best crystallization set-up, especially the simple Tris, PEG and CaCl2 combination, which contained crystals with 100 µm x 100 µm x 30 µm dimensions (Figure 11.3).

Figure 11.3: Hanging drop crystallization of mCSF-1. The drops were set up manually in a 24-well hanging drop format. Crystals were bigger than in previous set-ups (100 x 100 x 30 µm). Condition: 0.1 M Tris pH 8, 20% PEG 6k, 0.2 M CaCl2 (1 and 2) or contained an included additive (3): 0.33% w/v 1,5-Naphtalenedisulfonic acid disodium salt, 0.33% w/w 2,5-Pyridinedicarboxylic acid, 0.33% w/v 3,5-Dinitrosalicylic acid, 0.02 M HEPES sodium pH 6.8

Part 4: Results

48

Chapter 12: Structure determination of mCSF-1

12.1 Data collection and processing Crystals that were obtained in the previous steps were cryoprotected as described in Materials and methods. An example of a diffraction image obtained at the ID-29 beamline at ESRF from a mCSF-1 crystal can be found in Figure 12.1.

Figure 12.1: Diffraction pattern mCSF-1 crystal. The shown figures were obtained at the ID29 beam line at ESRF. Upper left corner: a mCSF-1 crystal inside a cryoloop centered on the beam (50 x 50 µm). Middle: a diffraction pattern of mCSF-1, the beam center is indicated by the red cross, the beam stop by the white shadow. Diffraction spots were single, resolved and strong (enlargement of the red boxed area in lower right corner).

The test set that proved to be the best in terms of resolution and spot appearance was obtained from a crystal that was set up in a sitting drop fashion. The condition in which this crystal was obtained was 300 µL 0.1 M Tris pH 8, 20% PEG 6k, 0.2 M CaCl2 reservoir solution and a drop of 2.2 µL (1:1:0.1 protein:reservoir:additive). The additive in question was part of the Silver BulletsTM screen and consisted of 0.2% w/v Benzenephosphonic acid, 0.2% w/v Gallic acid, 0.2% w/v Melatonin, 0.2% w/v N-(2-carboxyethyl)-iminodiacetic acid, 0.2% w/v Trimellitic acid and 0.02 M HEPES sodium pH 6.8.

The initial indexing of this crystal provided the following strategy for data collection: starting at spindle angle 170°, rotating over 90° with oscillation range of 0.5°, the collected dataset should have a completeness of data of 99%. The parameters were changed accordingly and a full dataset was collected. The parameters that were obtained from the mCSF-1 dataset can be found in the following Table 1. After running Xtriage again after anisotropic scaling, no anomalies were detected anymore and a couple of statistics are shown in Figure 12.2.

Part 4: Results

49

Table 1: Data collection statistics mCSF-1

X-ray Source ESRF/ID29

X-ray detector Pilatus 6M

Wavelength (Å) 0.972597

Space Group Number (Symbol) 19 (P2(1)2(1)2(1))

Cell parameters (Å)

a, b, c (Å)

α, ß, γ (°)

36.22 68.58 144.39

90.000 90.000 90.000

Matthews coefficient (Å3 Da-1) ** 2.51

Solvent content (%) 51.05

Molecules in asymmetric unit cell 1

Resolution range (Å)* 39.403 – 2.601 (2.73 2.601)

Overall B-factor, Wilson plot (Å2) 53.072

Number of observations* 36857 (5091)

Unique reflections* 11489 (1758)

Completeness (%)* 98.1 (95.8)

Redundancy* 3.21 (2.90)

Mean I/sigma(I)* 10.85 (1.83)

Rmeas (%)* 10.5 (78.2)

*Values in parentheses are for the highest resolution shell

**Determines theoretical number of molecules in ASU (Kantardjieff & Rupp, 2003)

Figure 12.2: Xtriage output graphs. The anisotropically scaled mCSF-1 dataset showed a normal I/sigma (signal to noise) vs. resolution decline (A). The data sanity check (B) showed good fractional completeness over the observed resolution. The Z-score indicates the deviation of values from their expectation value in standard deviations of their sampling distribution.

Part 4: Results

50

12.2 Molecular Replacement For the mCSF-1 dataset, a MR search was performed using the known bound mCSF-1 structure from PDB 3EJJ in Phaser. All possible spacegroups were searched, in order to confirm the presence of screw axes. Spacegroup 19, P212121 containing 3 screw-axes was confirmed. The scores of the MR of mCSF-1 are given in Table 2. A first electron density map and a pdb file containing atom coordinates was written out. The unit cell was visualized and the packing of the mCSF-1 molecules showed a tight arrangement because of the presence of the three screw axes (Figure 12.3).

Figure 12.3: The mCSF-1 structure and unit cell after molecular replacement. A. One mCSF-1 molecule and the corresponding unit cell. B. Packing of the mCSF-1 molecules as viewed along the a-axis in space group P212121. C. Packing of the mCSF-1 molecules as viewed along the c-axis. Symmetry mates within a radius of 20 Å are shown. Unit cell dimensions: a = 36.22 Å, b = 68.58 Å, c = 144.39 Å; α, ß, γ = 90°. Figure generated in PyMol.

12.3 Structure refinement After obtaining the first electron density map, a first inspection showed that some regions of density correspond better with the model than others. Different cycles of refinement were performed using phenix.refine in the PHENIX suite. A summary of the used procedures in each cycle can be found in Table 3. The complete list of non-default parameters that served as input for the different refinement cycles can be found in Appendices 5 to 9.

Table 2: Molecular replacement scores mCSF-1

Log-likelihood gain (LLG) 730

RFZ (rotation function Z-score) 10.8

TFZ (translation function Z-score) 26.7

Packing clashes in spacegroup 19 0

Part 4: Results

51

Table 3: Refinement cycles for mCSF-1 and used procedures

Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 5

Individual sites

True True True True True

Rigid body True False False False False

Individual ADP

True False False False False

TLS False True True True True

Ordered solvent

False False True True True

Secondary structure restraints

False True True True True

Optimized target weights

False False True True True

Figure 12.4: R-work and R-free evolution during refinement of mCSF-1. Both R-work and R-free improved progressively as the model became more complete and more parameters were included. Refinement at cycle 5 seemed to reach a minimum for lowest R-free value.

Part 4: Results

52

The refinement statistics for mCSF-1 are given in Table 4.

The evolution of Rwork and Rfree given the different refinement cycles is shown in Figure 12.4. At cycle 5, the best model is reached. The gap between Rwork and Rfree evolved from 0.2 to 0.04 over the 5 cycles and indicated a good model. Too wide a gap indicates that there still is room for improvement; while too small a gap might indicate overparameterization. However, the gap itself is not a sharp discriminator for refinement quality, as it displays a wide variance over resolution when all PDB entries are taken into account.

Table 4: Refinement statistics mCSF-1

Refinement program Phenix.refine (PHENIX suite)

Residues refined 293

Non-hydrogen protein atoms refined 4678

Number of water molecules 22

Resolution (Å) 39.40 – 2.60

Reflections in cross-validation set (random, %) 5

R-work* 0.2553 (0.3662)

R-free* 0.2921 (0.4059)

Average B-factor (Å2) 41.30

RMS angles (°) 0.776

RMS bonds (Å) 0.0035

Ramachandran plot appearance

Favored regions (%) 92.73

Allowed regions (%) 7.72

Disallowed regions (%) 0

*Values in parentheses are for the highest resolution shell

12.4 Structure validation Because the mCSF-1 protein consisted of 2 identical monomers, their non-crystallographic symmetry (NCS) could also be used as a validation for the structure. The two monomers are related to each other and should have the same general geometry. Significant pair-wise differences could indicate regions of questionable φ, ψ-angles. The backbone φ, ψ- torsion angles were not restrained during refinement and thus were an independent means of validation. No significant differences could be found between chain A and B of the mCSF-1 structure (Figure 12.5).

Part 4: Results

53

Figure 12.5: Backbone torsion angle distribution for NCS related chains in mCSF-1. The phi-psi (φ, ψ) points of corresponding residues between chain A (white squares) and chain B (blue squares) are connected with lines. No significant differences of torsion angles between the two chains were found, except for the termini and the flexible loop at residues 95-99. Mainly populated areas of the plot are the ß-region (upper left) and the right-handed α-helical region (middle left).

Another way of validating the structure was to compare it to other PDB structures that were solved at the same resolution limit (2.6 Å). The statistics of the dataset were set out against the ones from the PDB and interconnected. The result is a so-called polygon, where the statistics are indicated on a ruler, colored according to frequency. If a point is positioned on a red part of the ruler, it lies outside of the typical values. A well-refined structure will have a small and roughly equilateral polygon (Urzhumtseva, Afonine, Adams, & Urzhumtsev, 2009). For the mCSF-1 structure, the overall polygon for 2.6 Å resolution was reasonably good; however some statistics were clearly positioned on the larger side of the spectrum. The fact that the refinement didn’t converge to a lower Rwork/Rfree and average B-factor was probably due to the anisotropy in the dataset. Because the dataset was limited to 3.2 Å in one position, the polygon was recalculated with a resolution cut-off of 3.2 Å; the resulting polygon showed better comparison to the deposited structures, although now some were considered outliers for the 3.2 Å resolution. (Figure 12.6) The structure of the refined mCSF-1 with the corresponding electron density can be found in Figure 12.7.

Part 4: Results

54

Figure 12.6: Polygon for the mCSF-1 structure. A. Standard polygon displaying statistics of the dataset (in black) versus the upper and lower limit of the structures solved at the same resolution (in red). Resolution up to 2.6 Å was included. B. Same polygon as before, but now with a resolution cut-off at 3.2 Å to correct for the anisotropy present in the dataset.

Figure 12.7: The refined mCSF-1 structure in the electron density. The mCSF-1 dimer (chain A = green, chain B = pink) was placed inside the electron density. Water molecules are shown as yellow spheres. Density map cut-off 1 σ. Figure rendered in PyMol.

55

Part 5: Conclusion

and discussion

Part 5: Conclusion and discussion

56

Chapter 13: Discussion

13.1 Refinement Although the mCSF-1 structure reached reasonable Rwork/Rfree values, it could have been possible that this was just a local energy minimum and the global minimum had yet to be reached. Stopping the refinement before reaching a convergence is bad practice and should be avoided; however the refinement has to be stopped at one point. That point is based on three criteria: 1) no more significant and interpretable differences in the electron density maps remain; 2) no more unexplained significant deviations from stereochemical target values remain and 3) the model makes chemical and biological sense.

For the mCSF-1 structure points 2 and 3 were fulfilled as discussed in the Results: Structure validation section. However, there were still some differences in the electron map present that were unaccounted for. These were mostly unmodeled blobs that were too big to be caused by water molecules (Figure 13.1). When evaluating the crystallization condition that was used for the diffracting crystal, the additive could have been a good candidate for explaining the presence of the blobs. The additive in question (condition 84 from Silver BulletsTM) contained several small aromatic molecules, which could possibly fit into the unmodeled electron densities.

Figure 13.1: Unmodeled blob in mCSF-1 density. The mCSF-1 molecule (green) around chain A residue 9 (His) and its symmetry mate (orange) encompassed an area of density that could not be accounted for by water molecules. This density could have been due to the presence of additives used in the crystallization cocktail. Density map cut-off 1 σ. Figure rendered in PyMol.

To fulfill condition 1 to define the stopping moment for refinement, the suspecting molecules could be modeled inside the electron density to confirm their presence. However, because the resolution limit is 2.6 Å, no electron density at the level of individual atoms is visible, which


57

would make it hard to differentiate between similar small aromatics such as benzenephosphonic acid and gallic acid.

The flexible loop in the mCSF-1 structure around residue 95-99 proved to be difficult to place inside the electron density as it is inherently unstructured and has subsequent higher B-factors. The detail of the data at 2.6 Å was not sufficient to see the possible conformations of the flexible loop. Furthermore, the 22 water molecules already placed into the density of mCSF-1 probably don’t reflect reality, as about one water molecule per residue can be expected for the average structure (Kleywegt et al., 1997). Due to the average resolution limit of 2.6 Å, the number of discrete solvent molecules that could be placed correctly was lower than expected.

Due to the anisotropy in the dataset, Rwork and Rfree were not optimal for the 2.6 Å resolution limit and stalled at higher than expected values. To achieve better R-values, other refinement procedures could be tried to escape from a possible local energy minimum. While further refinement procedures were attempted during this thesis, none delivered better R-values. Another possibility would naturally be to try and obtain better crystals, with lower anisotropic diffraction. If the crystals packed better and grew larger in every direction, the resulting diffraction would be at a higher resolution, which would benefit the electron density map in terms of detail and enabling discriminatory placement of similar structures.

13.2 The mCSF-1 structure Although the used PDB 3EJJ model for molecular replacement essentially contained the same sequence as the free mCSF-1, it is always useful to check whether the obtained structure actually fits expectations. Differences in the two structures could reveal parts in the protein that play an important role in receptor binding.

The overall structure of the obtained free mCSF-1 was evaluated based on the knowledge of short-chain four α-helical bundle cytokines (Figure 13.2). As expected, the free mCSF-1 structure consisted of a dimer, linked together by one interchain disulfide bond. The helices were arranged in a typical up-up-down-down topology and are about 20 amino acids long. A small beta sheet was part of the bundle core. The crossover from helix A to B, which consisted of the first ß-strand, passed behind helix D. The obtained mCSF-1 protein thus was structurally very plausible. The chemical plausibility was shown in the Results section.


58

Figure 13.2: The obtained free mCSF-1 structure. The helices are all colored in a different way: helix A in blue, helix B in orange, helix C and D in green and yellow respectively. The small ß- sheet is colored in red. The interchain disulfide bond is depicted in purple and shown as a stick representation. Figure generated in PyMol.

13.3 Free mCSF-1 versus CSF-1R:mCSF-1R The obtained structure of free mCSF-1 is useful for comparison to its bound counterpart. Differences between the two structures might reveal insights into mechanistic actions of binding and important interaction parts of the molecules.

Comparison between the free mCSF-1 and mCSF-1 bound to one monomer of the CSF-1R (PDB 3EJJ) revealed that the bound versus the unbound mCSF-1 structure differed by an angle of about 8° (Figure 13.3). This is in correspondence to the reported tilt of the unbound versus bound mCSF-1 structure as reported by Chen et al., 2008. Upon binding the receptor, mCSF-1 undergoes a conformational change at the hinge domain between the two monomers of the ligand.

Figure 13.3: Free vs. bound mCSF-1. The refined mCSF-1 structure (red and blue) was superimposed with the structure of bound mCSF-1 to domains 1 to 3 of the CSF-1R (PDB 3EJJ) (grey). Chain A served as the anchor of the alignment; the resulting two B chains made an angle of about 8° in respect to each other. Figure generated in PyMol.


59

Surprisingly, the bound mCSF-1 structure depicted in Figure 9.3, is tilted downwards towards the membrane upon binding to the receptor, rather than upwards as described in the Chen et al., 2008 paper. Of course, as the 3EJJ structure only contains one receptor monomer, one chain of the ligand will have more conformational freedom than the other. The tilting that is observed may be partly due to this conformational freedom in one of the ligand monomers, and this may explain why both upwards and downwards tilting have been perceived.

The 3EJJ structure offers the possibility to compare unbound ligand monomers but also ligand monomers bound to the receptor. When the two structures of bound versus unbound mCSF-1 were aligned using the receptor bound monomer as anchor, it was clear that some side chains also change conformation upon binding to the receptor, although not as extensively as depicted in Chen et al, 2008.

Figure 13.4: Side chains mCSF-1 undergo conformational changes upon binding to the CSF-1R. In red the 3EJJ structure, in green the obtained free mCSF-1 structure. Figure rendered in PyMol.


60

13.4 Free mCSF-1 versus mCSF-1:BARF1 Upon binding to BARF1, the mCSF-1 undergoes a slightly bigger conformational change. The angle of the tilt is now about 13° (Figure 13.5). This tilt could lock the CSF-1 in a certain restrained conformation enabling high affinity binding and hindering subsequent release of the mCSF-1 ligand as indicated by the scavenging of mCSF-1 upon BARF1 exposure. As the tilt doesn’t change the distal ends of the cytokine, it is unlikely that the binding of mCSF-1 by BARF1 would completely abolish the binding to the CSF-1R, therefore leaving room for residual signaling through the receptor, as described by Elegheert et al., unpublished.

Figure 13.5: Comparison free mCSF-1 vs. BARF1:mCSF-1. Overlaying the refined structure of free mCSF-1 (yellow) to the bound mCSF-1 structure (blue) at the level of chain A of the mCSF-1 dimer clearly showed a change in conformation in mCSF-1 upon binding to BARF1. The angle between the two structures was about 13°. Figure rendered in PyMol.

In the human variant however, no signaling through the hCSF-1 receptor is detected after BARF1:hCSF-1 complex formation. The comparison of both BARF1:mCSF-1 and BARF1:hCSF-1 may reveal a difference that explains the possibility of residual signaling in one complex but not the other. The overlay of BARF1:hCSF-1 and BARF1:mCSF-1 is shown in Figure 13.6. A small rigid body shift of about 5° in the cytokine ligand is visible; however, in both of the cases no rearrangements around the distal ends of the ligand are detected. Thus, residual signaling through the CSF-1R is theoretically possible in both cases. Both ligand structures are very similar and, based on their structures bound to BARF1, the unbound hCSF-1 ligand will probably show the same rigid body shift of 13° after binding by BARF1.


61

As the ligands are very similar and the BARF1 protein used in experiments naturally had the same sequence, the question remains why exactly the binding of BARF1 to the human ligand leaves no room for CSF-1R signaling while in the mouse variant, signaling through the receptor can still occur.

Some caution is advised however as the EBV only infects primates. The situation of mCSF-1 bound to BARF1 could therefore be an artificial, induced one, as the BARF1 protein doesn’t normally occur in the context of a murine system. Still, the difference in residual signaling is present and a reasonable explanation remains to be found.

Figure 13.6: BARF1:hCSF-1 versus BARF1:mCSF-1. The human CSF-1 bound to BARF1 is shown in red, the mouse variant in blue. A small difference in structure of about 5° at the right monomer of the ligand is visible. Figure rendered with PyMol.


62

Chapter 14: Conclusion

During this thesis, protein crystallization and structure determination of free mCSF-1 was undertaken through all stages of protein crystallography. Overproduction of recombinant mCSF-1 in E. coli and subsequent refolding and purification resulted in pure natively folded mCSF-1. The yield was sufficient enough to set up crystallization trials, several of which contained crystals of tens to a hundred micrometer dimensions. The structure of the mCSF-1 was solved using molecular replacement to a resolution of 2.6 Å

The CSF-1 protein structure in its free form has not yet been extensively studied, probably because of the lack of clinical relevance in relation to pathologies. However, the structure determination of the BARF1 protein and discovery of its binding to the CSF-1 protein sheds a new light on the relevance of the structure of free CSF-1. The BARF1 binds to the CSF-1 dimer using a different interaction interface than the one used for CSF-1 receptor binding. Peculiarly, when human CSF-1 binds to BARF1, there is no residual signaling detected through the CSF-1 receptor; in contrast to the case of murine CSF-1, where signaling is still observed.

By determining the free mCSF-1 structure, the comparison could be made with the BARF1:mCSF-1 complex. However, the question of the reason for the species difference still remains, as there is no high-resolution structure yet available for the human CSF-1.

The comparison of the obtained mCSF-1 to the receptor bound form, revealed some conflicts in regards to an earlier study. Further studies of the mCSF-1 in or out of complex with its binding partners may help in determining the nature of the observed difference.


63

Chapter 15: Future perspectives

A lot of proteins that play a role in the hematopoiesis have already been solved using X-ray crystallography and remain the subject of many current studies because of their important role in the development of blood cells lineages and their implication in different pathologies.

As more structures, in particular of complexes become available, more information can be gathered regarding their binding mode and possible function. However so far, the protein complexes important in hematopoiesis have proven quite difficult to crystallize because of their size and subsequent fragile crystals containing large solvent channels. For the characterization of these complexes, other methods can be used such as SAXS and EM for the structural level and ITC and SPR for the thermodynamics level.

In the future, the collaborative effort of these different fields in science combined with, for example, cellular studies and bioinformatics, will elucidate the whole cytokine network at the level of the genome, proteome, interactome etc. Recent studies have tested new X-ray techniques for the high-resolution 3D structure determination of single molecules in solution, such as use of a Free electron X-ray Laser (FEL). However, still a lot of technical hurdles need to be overcome for these methods to become readily applicable (Henderson, 2002).

The obtained mCSF-1 structure in its free form is, as far as our knowledge, the only known unbound CSF-1 structure. In the future it can be further characterized in terms of binding to CSF-1R and BARF1. Obtaining a higher resolution structure of the mCSF-1 can also be pursued.

To fully understand the BARF1 interplay with the immune system, the high-resolution structure of unbound human CSF-1 could also be crystallized in the future. This can be further extended to other species, as demonstrated by recent studies investigating the BARF1 protein in the macaque (Ohashi et al., 2011). Furthermore, the characterization of the binding interface between hCSF-1 and the BARF1 protein may lead to rational drug design. Treatment based on targeting of the interface may restore the immune response lost due to CSF-1 sequestration.


64

Chapter 16: Nederlandstalige samenvatting

Hematopoëse is het sterk gereguleerde proces waarbij bloedcellen worden aangemaakt en gedifferentieerd naar meer gespecialiseerde cellen. Dit proces wordt voornamelijk gestuurd door cytokines, die cel signalisatie mediëren door binding met specifieke receptoren op het celoppervlak.

De kolonie stimulerende factor 1 (CSF-1) is een pleiotroop cytokine dat mononucleaire fagocyten stuurt naar verdere ontwikkeling tot bv. dendritische cellen en macrofagen. Verhoogde levels van circulerend CSF-1 is gelinkt aan verschillende immunologische en inflammatoire aandoeningen zoals reumatoïde artritis.

Op structureel vlak behoort CSF-1 tot de familie van de korte keten α-helicale bundel cytokines en is bijgevolg dimerisch. De endogene receptor voor CSF-1, CSF-1R, is een lid van de tyrosine kinase III familie (RTKIII), waaraan CSF-1 bindt met de distale uiteinden. Binding van het ligand induceert receptor dimerizatie op het celoppervlak, fosforylatie in het cytoplasmatisch deel en verdere signalisatie door de werking van effector moleculen.

Het bindingsparadigma van CSF-1 aan CSF-1R wordt gedeeld met andere leden van de korte keten α-helicale bundel cytokines, stam cell factor (SCF) en Fms-gerelateerd tyrosine kinase 3 ligand (Flt3) en hun respectievelijke receptoren c-Kit en Flt3. Het bindingsparadigma is echter anders dan oorspronkelijk gedacht en is gerelateerd aan het bindingsmechanisme dat gebruikt wordt door cysteïne knoop factoren. Mutaties in de RTKs kunnen constitutieve activatie veroorzaken, die met verschillende ziektes worden gerelateerd.

Het CSF-1 proteïne is naast zijn endogene receptor ook een bindingspartner voor BARF1, een eiwit dat geëxpresseerd wordt gedurende de lytische fase van een Epstein-Barr virus infectie.

Het Epstein-Barr virus (EBV) infecteert het grootste deel van de menselijke bevolking. Hoewel meestal asymptotisch, kan infectie in immunogecomprimeerde patiënten tot andere aandoeningen leiden. Zo is het de oorzaak van Burkitt’s lymfoom, wat vooral voorkomt in Afrika waar malaria endemisch is.

Het BARF1 vormt in oplossing hexamere ringen gevormd door de interactie van Immunoglobine(Ig)-achtige dimeren door specifieke N-N en C-C interacties. Aangezien circulerend CSF-1 na toediening van BARF1 helemaal wordt gebonden, vormt het BARF1 een scavenger voor CSF-1. Dit kan een mogelijk mechanisme kan zijn waarmee het virus het immuunsysteem van de gastheer omzeilt. Het BARF1 eiwit wordt gelinkt aan verschillende eiwitten en zou een oncogeen effect hebben.

Vreemd genoeg is de CSF-1 bindingsepitoop niet dezelfde als die voor de CSF-1 receptor en een ander mechanisme dan receptor imitatie zal dus een rol spelen in de immuunmodulatie aangezien de bindingsepitoop voor CSF-1R interactie nog steeds beschikbaar is.


65

Hoewel BARF1 zowel humaan (h) als muis (m) CSF-1 bindt, is er een specifiek species verschil bij de signalering doorheen de CSF-1R. Als BARF1 het humaan CSF-1 bindt, wordt geen residuele signalisatie waargenomen doorheen de CSF-1R. Dit in tegenstelling tot het muis systeem, waar de residuele signalisatie wel wordt waargenomen. De reden voor dit verschil is nog niet opgehelderd. Karakterisatie van alle proteïnen die een rol spelen in dit verhaal kan mogelijks een antwoord opleveren. Hoge-resolutie methoden zoals X-straal kristallografie, die een 3D beeld geven van de structuur van een eiwit, zijn hier een essentieel hulpmiddel bij.

Gedurende deze thesis werd het mCSF-1 recombinant geproduceerd in E. coli en vervolgens geëxtraheerd uit aanwezige inclusion bodies, heropgevouwen en opgezuiverd. Daarna werd tot kristalliseren overgegaan, zowel door middel van een kristallizatierobot als manuele opzettingen. Bekomen kristallen werden blootgesteld aan X-stralen en getest op diffractie. De beste dataset werd gecollecteerd tot 2.6 Å resolutie.

Voor structuurbepaling werd een beroep gedaan op moleculaire vervanging, waarin de afwezige fase-informatie in de dataset wordt ingevuld door die van een gekend homoloog model. In het geval van mCSF-1, werd de moleculaire vervanging uitgevoerd met een structuur die dezelfde mCSF-1 molecule bevatte, maar in complex met een van de CSF-1 receptor monomeren. De resulterende elektron densiteits map en een eerste model werden hieruit verkregen.

Verfijningprocedures hebben tot doel het model zodanig aan te passen dat het zo goed mogelijk overeenkomt met de data. Verschillende rondes van verfijning werden ondernomen voor de structuurbepaling van mCSF-1. Het verloop van verfijning werd in de gaten gehouden via de R-factoren, die bepalen of een gemaakte verfijning het model beter doen correleren met de data of er enkel sprake is van overfitting.

De bekomen structuur van het vrije mCSF-1 eiwit werd vergeleken met zowel de gebonden versie aan CSF-1R en aan BARF1. Mogelijke verschillen kunnen belangrijke regio’s van interactie blootleggen of indicatief zijn voor de eventuele gevolgen van interactie. In geval van vergelijking tussen gebonden mCSF-1 aan CSF-1R en vrij mCSF-1, werden er enkele verschillen gevonden in tegenspraak tot de literatuur. In geval van BARF1:mCSF-1 complex en vrij mCSF-1 werd een duidelijke tilt van het ligand waargenomen bij binding aan de N-terminale BARF1 domeinen. Helaas konden er geen sluitende conclusies getrokken worden over de species verschillen van humaan en muis CSF-1 in interactie met BARF1, aangezien er geen hoge resolutie structuren beschikbaar zijn voor humaan CSF-1 en aangezien de tilt waarschijnlijk dezelfde is bij zowel mens als muit. De reden van het species verschil in BARF1 signalisatie blijft een onopgeloste vraag.

66

Part 6: References

Part 6: References

67

Adams, P. D., Afonine, P. V., Bunkóczi, G., Chen, V. B., Davis, I. W., Echols, N., et al. (2010). PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta crystallographica. Section D, Biological crystallography, 66(Pt 2), 213-21. doi: 10.1107/S0907444909052925.

Arakawa, T., Yphantis, D. a, Lary, J. W., Narhi, L. O., Lu, H. S., Prestrelski, S. J., et al. (1991). Glycosylated and unglycosylated recombinant-derived human stem cell factors are dimeric and have extensive regular secondary structure. The Journal of biological chemistry, 266(28), 18942-8. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/1717457.

Aricescu, a R., Lu, W., & Jones, E. Y. (2006). A time- and cost-efficient system for high-level protein production in mammalian cells. Acta crystallographica. Section D, Biological crystallography, 62(Pt 10), 1243-50. doi: 10.1107/S0907444906029799.

Bazan, J. F. (1990). Structural design and molecular evolution of a cytokine receptor superfamily. Proceedings of the National Academy of Sciences of the United States of America, 87(18), 6934-8. Retrieved from http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=54656&tool=pmcentrez&rendertype=abstract.

Bryder, D., Rossi, D. J., & Weissman, I. L. (2006). Hematopoietic Stem Cells: The Paradigmatic Tissue-Specific Stem Cell. American Journal Of Pathology, 169(2), 338-346. doi: 10.2353/ajpath.2006.060312.

Carlberg, K., & Rohrschneider, L. (1994). The effect of activating mutations on dimerization, tyrosine phosphorylation and internalization of the macrophage colony stimulating factor receptor. Molecular biology of the cell, 5(1), 81-95. Retrieved from http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=301011&tool=pmcentrez&rendertype=abstract.

Chambers, S. K., Kacinski, B. M., Ivins, C. M., & Carcangiu, M. L. (1997). Overexpression of epithelial macrophage colony-stimulating factor (CSF-1) and CSF-1 receptor: a poor prognostic factor in epithelial ovarian cancer, contrasted with a protective effect of stromal CSF-1. Clinical cancer research : an official journal of the American Association for Cancer Research, 3(6), 999-1007. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/9815777.

Chang, C. M., Yu, K. J., Mbulaiteye, S. M., Hildesheim, A., & Bhatia, K. (2009). The Extent of Genetic Diversity of Epstein-Barr Virus and its Geographic and Disease Patterns: A Need for Reappraisal. Virus research, 143(2), 209–221. Elsevier. doi: 10.1016/j.virusres.2009.07.005.The.

Chen, H., Ma, J., Li, W., Eliseenkova, A. V., Xu, C., Neubert, T. A., et al. (2007). A “Molecular Brake” in the Kinase Hinge Region Regulates the Activity of Receptor Tyrosine Kinases. Molecular cell, 27(5), 717–730. Elsevier. Retrieved May 1, 2011, from http://linkinghub.elsevier.com/retrieve/pii/S1097276507004406.

Chen, X., Liu, H., Focia, P. J., Shim, A. H.-R., & He, X. (2008). Structure of macrophage colony stimulating factor bound to FMS: diverse signaling assemblies of class III receptor tyrosine kinases. Proceedings of the National Academy of Sciences of the United States of America, 105(47), 18267-72. doi: 10.1073/pnas.0807762105.

Chitu, V., & Stanley, E. R. (2006). Colony-stimulating factor-1 in immunity and inflammation. Current opinion in immunology, 18(1), 39-48. doi: 10.1016/j.coi.2005.11.006.

Collaborative Computational Project, Number 4 (1994) The CCP4 suite: programs for protein crystallography. Acta Crystallogr D Biol Crystallogr 50: 760-763.

DeLano, W. L. (2000). Convergent Solutions to Binding at a Protein-Protein Interface. Science, 287(5456), 1279-1283. doi: 10.1126/science.287.5456.1279.

Part 6: References

68

Delecluse, H.-J., Feederle, R., OʼSullivan, B., & Taniere, P. (2007). Epstein Barr virus-associated tumours: an update for the attention of the working pathologist. Journal of clinical pathology, 60(12), 1358-64. doi: 10.1136/jcp.2006.044586.

Dong, J., McPherson, C., & Stambrook, P. (2002). Flt-3 Ligand: A Potent Dendritic Cell Stimulator and Novel Antitumor Agent. Cancer biology & therapy, 1(5), 486. Retrieved May 1, 2011, from http://www.ncbi.nlm.nih.gov/pubmed/12496473.

Douglass, T. G., Driggers, L., Zhang, J. G., Hoa, N., Delgado, C., Williams, C. C., et al. (2008). Macrophage colony stimulating factor: not just for macrophages anymore! A gateway into complex biologies. International immunopharmacology, 8(10), 1354-76. doi: 10.1016/j.intimp.2008.04.016.

D. R. Barreda, P. C. Hanington, and M. Belosevic. Regulation of myeloid development and function by colony stimulating factors. Dev. Comp. Immunol., 28(5):509–554, 2004.

Emsley, P., & Cowtan, K. (2004). Coot: model-building tools for molecular graphics. Acta Crystallographica Section D: Biological Crystallography, 60(12), 2126–2132. International Union of Crystallography. Retrieved June 9, 2011, from http://scripts.iucr.org/cgi-bin/paper?s0907444904019158.

Garceau, V., Smith, J., Paton, I.R., Davey, M., Fares, M.A., Sester, D.P., Burt, D.W., and Hume, D.A. (2010). Pivotal Advance: Avian colony-stimulating factor 1 (CSF-1), interleukin- 34 (IL-34), and CSF-1 receptor genes and gene products. J Leukoc Biol 87, 753-764.

Glaspy, J. (1993). Clinical applications of stem cell factor. Current opinion in hematology, 3(3), 223-9.

Graddis, T. J., Brasel, K., Friend, D., Srinivasan, S., Wee, S., Lyman, S. D., et al. (1998). Structure-function analysis of FLT3 ligand-FLT3 receptor interactions using a rapid functional screen. The Journal of biological chemistry, 273(28), 17626-33. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/9651358.

Griffith, J., Black, J., Faerman, C., Swenson, L., Wynn, M., Lu, F., et al. (2004). The structural basis for autoinhibition of FLT3 by the juxtamembrane domain. Molecular cell, 13(2), 169-78. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/14759363.

Grundler, R., Thiede, C., Miething, C., Steudel, C., Peschel, C., & Duyster, J. (2003). Sensitivity toward tyrosine kinase inhibitors varies between different activating mutations of the FLT3 receptor. Blood, 102(2), 646. American Society of Hematology. doi: 10.1182/blood-2002-11-3441.Supported.

Henderson, R. (2002) Excitement over X-ray lasers is excessive. Nature 415, 833

Hubbard, S. R., Mohammadi, M., & Schlessinger, Joseph. (1998). Autoregulatory Mechanisms in Protein-tyrosine Kinases. The Journal of biological chemistry, 273(20), 11987-11990.

Iemura, a, Tsai, M., Ando, a, Wershil, B. K., & Galli, S. J. (1994). The c-kit ligand, stem cell factor, promotes mast cell survival by suppressing apoptosis. The American journal of pathology, 144(2), 321-8. Retrieved from http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1887147&tool=pmcentrez&rendertype=abstract.

Jiang, X., Gurel, O., Mendiaz, E. a, Stearns, G. W., Clogston, C. L., Lu, H. S., et al. (2000). Structure of the active core of human stem cell factor and analysis of binding to its receptor kit. The EMBO journal, 19(13), 3192-203. doi: 10.1093/emboj/19.13.3192.

Kantardjieff, K. A., & Rupp, B. (2003). Matthews coefficient probabilities: improved estimates for unit cell contents of proteins, DNA, and protein–nucleic acid complex crystals. Protein science, 12(9), 1865–1871. Wiley Online Library. doi: 10.1110/ps.0350503.tially.

Part 6: References

69

Kaushansky, K., & Karplus, P. a. (1993). Hematopoietic growth factors: understanding functional diversity in structural terms. Blood, 82(11), 3229-40. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/8241495.

Kikushige, Y., Yoshimoto, G., Miyamoto, T., Iino, T., Mori, Y., Iwasaki, H., et al. (2008). Human Flt3 is expressed at the hematopoietic stem cell and the granulocyte/macrophage progenitor stages to maintain cell survival. Journal of immunology (Baltimore, Md. : 1950), 180(11), 7358-67. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/18490735.

Kleywegt GJ, & Jones TA (1997) Model building and refinement practice. Methods Enzymol. 277, 208-230.

Lemmon, M. a, & Schlessinger, Joseph. (2000). Cell signaling by receptor tyrosine kinases. Cell, 141(7), 1117-34. doi: 10.1016/j.cell.2010.06.011.

Lennartsson, J., Jelacic, T., Linnekin, D., & Shivakrupa, R. (2005). Normal and oncogenic forms of the receptor tyrosine kinase kit. Stem cells (Dayton, Ohio), 23(1), 16-43. doi: 10.1634/stemcells.2004-0117.

Lin, E. Y., Nguyen, a V., Russell, R. G., & Pollard, J. W. (2001). Colony-stimulating factor 1 promotes progression of mammary tumors to malignancy. The Journal of experimental medicine, 193(6), 727-40. Retrieved from http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2193412&tool=pmcentrez&rendertype=abstract.

Liu, H., Chen, X., Focia, P. J., & He, X. (2007). Structural basis for stem cell factor-KIT signaling and activation of class III receptor tyrosine kinases. The EMBO journal, 26(3), 891-901. doi: 10.1038/sj.emboj.7601545.

Maraskovsky, E., Daro, E., Roux, E., Teepe, M., Maliszewski, C. R., Hoek, J., et al. (2000). In vivo generation of human dendritic cell subsets by Flt3 ligand. Blood, 96(3), 878-84. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/10910900.

Margulies, D., Opatowsky, Y., Fletcher, S., Saraogi, I., Tsou, L. K., Saha, S., et al. (2009). Surface Binding Inhibitors of the SCF-KIT Protein-Protein Interaction. ChemBioChem, 10(12), 1955–1958. Wiley Online Library. doi: 10.1002/cbic.200900079.Surface.

McClanahan, T., Culpepper, J., Campbell, D., Wagner, J., Franz-Bacon, K., Mattson, J., et al. (1996). Biochemical and genetic characterization of multiple splice variants of the Flt3 ligand. Blood, 88(9), 3371-82. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/8896402.

A.J. McCoy, R.W. Grosse-Kunstleve, P.D. Adams, M.D. Winn, L.C. Storoni & R.J. Read "Phaser Crystallographic Software" J. Appl. Cryst. (2007). 40, 658-674

Merlo, A., Turrini, R., Dolcetti, R., Martorelli, D., Muraro, E., Comoli, P., et al. (2010). The interplay between Epstein-Barr virus and the immune system: a rationale for adoptive cell therapy of EBV-related disorders. Haematologica, 95(10), 1769-77. doi: 10.3324/haematol.2010.023689.

Münz, C., & Moormann, A. (2008). Immune escape by Epstein-Barr virus associated malignancies. Seminars in cancer biology (Vol. 18, p. 381–387). Elsevier. doi: 10.1016/j.semcancer.2008.10.002.Immune.

Ohashi et al. Cloning of the Epstein-Barr virus-related rhesus lymphocryptovirus as a bacterial artificial chromosome: a loss-of-function mutation of the rhBARF1 immune evasion gene J. Virol..2011; 85: 1330-1339

Palmer, I., & Wingfield, P. T. (2004). Preparation and extraction of insoluble (inclusion-body) proteins from Escherichia coli. Current protocols in protein science / editorial board, John E. Coligan ... [et al.], Chapter 6, Unit 6.3. doi: 10.1002/0471140864.ps0603s38.

Part 6: References

70

Pandit J., Bohm A., Jancarik J., Halenbeck R., Koths K., Kim S.H. (1992) Three-dimensional structure of dimeric human recombinant macrophage colony-stimulating factor. Science 258: 1358-1362

Pixley, F. J., & Stanley, E. R. (2004). CSF-1 regulation of the wandering macrophage: complexity in action. Trends in cell biology, 14(11), 628-38. doi: 10.1016/j.tcb.2004.09.016.

Ray, P., Krishnamoorthy, N., Oriss, T. B., & Ray, A. (2010). Signaling of c-kit in dendritic cells influences adaptive immunity. Annals of the New York Academy of Sciences, 1183, 104-22. doi: 10.1111/j.1749-6632.2009.05122.x.

Ray, P., Krishnamoorthy, N., & Ray, A. (2008). Emerging functions of c-kit and its ligand stem cell factor in dendritic cells: regulators of T cell differentiation. Cell cycle (Georgetown, Tex.), 7(18), 2826-32. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/18787413.

Rohrschneider, L. R., Bourette, R. P., Lioubin, M. N., Algate, P. a, Myles, G. M., & Carlberg, K. (1997). Growth and differentiation signals regulated by the M-CSF receptor. Molecular reproduction and development, 46(1), 96-103. doi: 10.1002/(SICI)1098-2795(199701)46:1<96::AID-MRD15>3.0.CO;2-1.

Rowe, M., Glaunsinger, B., Leeuwen, D. van, Zuo, J., Sweetman, D., Ganem, D., et al. (2007). Host shutoff during productive Epstein-Barr virus infection is mediated by BGLF5 and may contribute to immune evasion. Proceedings of the National Academy of Sciences of the United States of America, 104(9), 3366-71. doi: 10.1073/pnas.0611128104.

Rozwarski, D. a, Gronenborn, a M., Clore, G. M., Bazan, J. F., Bohm, a, Wlodawer, a, et al. (1994). Structural comparisons among the short-chain helical cytokines. Structure (London, England : 1993), 2(3), 159-73. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/8069631.

Rupp, B (2010) Biomolecular crystallography: principles, practice and application to structural biology. Garland Science

Russell, E. (1979). Hereditary anemias of the mouse: a review for geneticists. Advanced genetics, 20, 357-459.

Savvides, S N, Boone, T., & Andrew Karplus, P. (2000). Flt3 ligand structure and unexpected commonalities of helical bundles and cystine knots. Nature structural biology, 7(6), 486-91. doi: 10.1038/75896.

Schubert, C., Schalk-Hihi, C., Struble, G. T., Ma, H.-C., Petrounia, I. P., Brandt, B., et al. (2007). Crystal structure of the tyrosine kinase domain of colony-stimulating factor-1 receptor (cFMS) in complex with two inhibitors. The Journal of biological chemistry, 282(6), 4094-101. doi: 10.1074/jbc.M608183200.

Sheng, W., Decaussin, G., Sumner, S., & Ooka, T. (2001). N-terminal domain of BARF1 gene encoded by Epstein-Barr virus is essential for malignant transformation of rodent fibroblasts and activation of BCL-2. Oncogene, 20(10), 1176-85. doi: 10.1038/sj.onc.1204217.

Soares, M. J., Pinto, M., Henrique, R., Vieira, J., Cerveira, N., Peixoto, A., et al. (2009). CSF1R copy number changes, point mutations, and RNA and protein overexpression in renal cell carcinomas. Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc, 22(6), 744-52. doi: 10.1038/modpathol.2009.43.

Sprang, S. R., & Bazan, J. F. (1993). Cytokine structural taxonomy and mechanisms of receptor engagement. Current opinion in structural biology, 3(6), 815-827.

Strockbine, L. D., Cohen, J. I., Farrah, T., Lyman, S. D., Wagener, F., DuBose, R. F., et al. (1998). The Epstein-Barr virus BARF1 gene encodes a novel, soluble colony-stimulating factor-1 receptor. Journal of virology, 72(5), 4015-21. Retrieved from http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=109629&tool=pmcentrez&rendertype=abstract.

Part 6: References

71

Subklewe, M., Sebelin, K., Block, A., Meier, A., Roukens, A., Paludan, C., et al. (2005). Dendritic cells expand Epstein Barr virus specific CD8+ T cell responses more efficiently than EBV transformed B cells. Human immunology, 66(9), 938-49. doi: 10.1016/j.humimm.2005.07.003.

Tarbouriech, N., Buisson, M., Géoui, T., Daenke, S., Cusack, S., & Burmeister, Wim Pascal. (2006). Structural genomics of the Epstein-Barr virus. Acta crystallographica. Section D, Biological crystallography, 62(Pt 10), 1276-85. doi: 10.1107/S0907444906030034.

Tarbouriech, N., Ruggiero, F., Turenne-Tessier, M. de, Ooka, Tadamasa, & Burmeister, Wim P. (2006). Structure of the Epstein-Barr virus oncogene BARF1. Journal of molecular biology, 359(3), 667-78. doi: 10.1016/j.jmb.2006.03.056.

Turner, S. L., Mangnall, D., Bird, N. C., Xqqlqj, M. E. B.-zajdel, Wkh, L. Q., Khsdwrfhooxodu, K., et al. (2010). Effects of Pro-Inflammatory Cytokines on the Production of Soluble Fractalkine and ADAM17 by HepG2 Cells. J Gastrointestin Liver Dis, 19(3), 265-271.

Urzhumtseva, L., Afonine, P. V., Adams, P. D., & Urzhumtsev, A. (2009). Crystallographic model quality at a glance. Acta crystallographica. Section D, Biological crystallography, 65(Pt 3), 297-300. doi: 10.1107/S0907444908044296.

Vereide, D., & Sugden, B. (2009). Proof for EBVʼs Sustaining Role in Burkitt's Lymphomas. Seminars in cancer biology (Vol. 19, p. 389–393). Elsevier. doi: 10.1016/j.semcancer.2009.07.006.Proof.

Verstraete, K., Koch, S., Ertugrul, S., Vandenberghe, I., Aerts, M., Vandriessche, G., et al. (2009). Efficient production of bioactive recombinant human Flt3 ligand in E. coli. The protein journal, 28(2), 57-65. doi: 10.1007/s10930-009-9164-5.

Verstraete, K., Vandriessche, G., Januar, M., Elegheert, J., Shkumatov, A. V., Desfosses, A., et al. (2011). Structural insights into the extracellular assembly of the hematopoietic Flt3 signaling complex. Blood. doi: 10.1182/blood-2011-01-329532.

Voo, K. S., Fu, T., Wang, H. Y., Tellam, J., Heslop, H. E., Brenner, M. K., et al. (2004). Evidence for the presentation of major histocompatibility complex class I-restricted Epstein-Barr virus nuclear antigen 1 peptides to CD8+ T lymphocytes. The Journal of experimental medicine, 199(4), 459-70. doi: 10.1084/jem.20031219.

Wang, Z. E., Myles, G. M., Brandt, C. S., Lioubin, M. N., & Rohrschneider, L. (1993). Identification of the ligand-binding regions in the macrophage colony-stimulating factor receptor extracellular domain. Molecular and cellular biology, 13(9), 5348-59. Retrieved from http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=360234&tool=pmcentrez&rendertype=abstract.

Wei, M. X., & Ooka, Tadamasa. (1989). A transforming function of the BARF1 gene encoded by Epstein-Barr virus. The EMBO Journal, 8(10), 2897. Nature Publishing Group. Retrieved May 1, 2011, from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC401352/.

Word, et. al. (1999) J. Mol. Biol. 285, 1735-1747.

Zuo, J., Currin, A., Griffin, B. D., Shannon-Lowe, C., Thomas, W. a, Ressing, M. E., et al. (2009). The Epstein-Barr virus G-protein-coupled receptor contributes to immune evasion by targeting MHC class I molecules for degradation. PLoS pathogens, 5(1), e1000255. doi: 10.1371/journal.ppat.1000255.

72

Part 7: Appendices

Part 7: Appendices

73

Appendix 1: Vectormap pET15b Novagen, Darmstadt, Germany

Part 7: Appendices

74

Appendix 2: Preparing electrocompetent cells 1. Inoculate a colony in 2 mL LB (10 g bactotrypton/L; 5 g NaCl/L; 5 g yeast extract/L)

2. Prepare a preculture: bring the 2 mL culture into 50 mL LB, incubate O/N shaking at 200 rpm at 37 °C

3. Dilute 1/100: bring 2 mL culture in 200 mL LB. Let cells grow at 37 °C under continuous shaking until OD600 of 0.600 to 1.000

4. Cool the cells down on ice for minimally 20 min

5. Spin down at 4000 rpm, 4 °C, 10 min. Divide 200 mL over 6 sterile tubes

6. Take off supernatant (work in flow for sterility)

7. Resuspend pellet in (6x) 30 mL cold water

8. Spin down 4000 rpm, 4 °C, 10 min

9. Discharge supernatant quickly an resuspend pellet in (6x) 15 mL cold water


11. Discard supernatant immediately, pellet is very loose!

12. Resuspend in 3 mL 10% (v/v) glycerol. Divide over 2 mL Eppendorf tubes


14. Take off supernatant with pipet en resuspend pellet in 600 µL 10% (v/v) glycerol

15. Aliquot 600 µL in fractions of 40 µL

16. Keep at -80 °C

17. Check for contamination by plating out on LB agar with antibiotic (0.1 mg/mL Cb or other) and incubate O/N

Part 7: Appendices

75

Appendix 3: Transformation of expression construct 1. Add 1 to 5 µL DNA (1mg/mL) solution to 40 µL cell culture

2. Transfer suspension to an ice-cooled electroporation cuvette

3. Electroporate cells at 2.5 kV, 25 µF and 200 Ohm

4. After pulse, resuspend cells quickly with 1 mL SOC medium (or alternatively LB)

5. Transfer to a falcon and incubate for 1 h at 37 °C constantly shaking at 200 rpm

6. Plate out 20 to 100 µL of cell culture on LB-plates that contain the appropriate antibiotic (0.1 mg/mL Cb, 0.025 mg/mL Cm), incubate O/N at 37 °C.

Part 7: Appendices

76

Appendix 4: Refolding protocol for mCSF-1 from 1 L bacterial expression culture (developed by Elegheert J. and Verstraete K.) 1. Expression

a. Expression plasmid: pET15b-mCSF-1

i. Cloned between NdeI and BglII sites

b. Expression strain: CodonPlus RIL: Cb/Cm selection markers

c. Expression conditions:

i. LB with carbenicillin and chloramphenicol, 37 °C

ii. Induction with 1 mM IPTG at OD600 = 0.6

iii. Harvesting after 4 h

2. Washing of inclusion bodies

a. Centrifuge culture and resolubilize in 10 mL solubilization buffer

i. 50 mM Tris pH 8.0

ii. 100 mM NaCl

iii. 1 mM EDTA

iv. 0.2 mM PMSF

v. 1x Complete (Roche)

b. Lyse by sonication (4x 1 min, 30%) on ice

c. Separate inclusion bodies of soluble protein: 10 min, 20800 g, 4 °C

d. Solubilize pellet, containing the inclusion bodies, in 30 mL washing buffer


ii. 100 mM NaCl

iii. 1 mM EDTA

iv. 0.2 mM PMSF

v. 1x Complete (Roche)

vi. 1% Triton X-100

e. Sonicate 15 seconds on ice to dissolve pellet

f. Centrifuge again and repeat the washing step two times

3. Solubilization of the inclusion bodies

a. Dissolve pellet in 30 mL GnHCl buffer (There is no EDTA added because of its interference with IMAC)

i. 6 M GnHCl

ii. 100 mM NaPO4 pH 8.0

Part 7: Appendices

77

iii. 10 mM Tris

iv. 10 mM ß-mercaptoethanol

b. Sonicate 5 seconds until dissolved

c. Incubate 3 h on a rotary shaker at room temperature

d. Centrifuge 30 min, 20800 g, 4 °C

e. Collect supernatant in 50 mL falcon

4. Histidine-affinity purification under denaturing conditions

a. Prepare a nickel sepharose column (5 mL matrix is sufficient)

b. Equilibrate with equilibration buffer (approximately 3 column volumes)

i. 6 M GnHCl

ii. 100 mM NaPO4 pH 8.0

iii. 10 mM Tris

c. Add supernatant to the column

d. Wash with 15 mL equilibration buffer pH 8.0 and 15 mL equilibration buffer pH 6.3

e. Elute with equilibration buffer pH 4.5

5. Refolding by rapid dilution

a. Prepare a cold refolding buffer (4°C) (to obtain a protein concentration of 0.2-0.3 mg/mL)


ii. 1 M arginine

iii. 1 mM EDTA

iv. 3 mM GSH

v. 1.5 mM GSSG

vi. 0.2 mM PMSF

b. Add the protein solution drop wise to the buffer at 4 °C under constant stirring, incubate overnight

6. Removal of arginine by dialysis

a. Dialyze 20x with 3 buffer changes (20 mM Tris pH 8.0, 1 mM EDTA, 1 mM PMSF) at 4 °C. In the final step, EDTA is omitted to prevent chelation of nickel ions in IMAC. Arginine needs to be removed because it also interferes with IMAC; the final concentration is preferably under 1 mM.

7. Nickel sepharose to capture the refolded protein

a. Filter the solution through a 0.22 µm filter

Part 7: Appendices

78

b. Add 300 mM NaCl and 10 mM MgSO4 (for conductivity and removing residual EDTA)

c. Prepare a prepacked nickel sepharose column and connect to Äkta system (GE Healthcare, Diegem, Belgium)

i. Optional: before and after use, the column can be regenerated:

1. Wash with water

2. Add stripping buffer (20 mM Tris pH 7.5, 500 mM NaCl, 50 mM EDTA)

3. Wash with water

4. Add 0.1 M NiSO4

d. Equilibrate column with bindingsbuffer

i. 50 mM NaPO4

ii. 300 mM NaCl

e. At sample to column, flow speed 3 mL/min

f. Elute with 500 mM imidazole

g. Desalting is achieved by gel filtration (SuperdexTM 75, GE Healthcare, Diegem, Belgium) (20 mM HEPES pH 7.5, 150 mM NaCl)

8. Thrombin digest

a. Once all the imidazole is removed, the thrombin digest can be started. Add 1 µL biotinylated thrombin (1 U/µL) to the mixture (Novagen, Darmstadt, Germany)

b. Incubate O/N at room temperature

c. Check the cleavage on SDS-PAGE

d. Add 400 µL streptavidin agarose beads

e. Incubate 10 min on rotary shaker

f. Remove beads by centrifugation, 5 min at 500 g. (Optional: by gravity flow)

9. Anion exchange

a. To lower the salt concentration in the protein sample after the thrombin digest, an anion exchange is performed. A Source 30Q column is used (GE Healthcare, Diegem, Belgium) with running buffer 20 mM HEPES pH 7.5, 150 mM NaCl. The matrix of the column consists of Q (Quaternary ammonium) groups bound to the polystyrene/divinylbenzene resin. After the sample is loaded, the protein is eluted by increasing the elution buffer to 100% over a period of 15 min (20 mM HEPES pH 7.5, 1 M NaCl).

10. Final gel filtration

a. To ensure purity and concentration of the protein, a final gel filtration is performed (SuperdexTM 75, GE Healthcare, Diegem, Belgium) (20 mM HEPES pH 7.5, 150 mM NaCl).

Part 7: Appendices

79

Appendix 5: Refinement cycle 1 mCSF-1 #phil __ON__ refinement {

crystal_symmetry { unit_cell = 36.21900177 68.59999847 144.4089966 90 90 90 space_group = "P 21 21 21"

} input {

pdb { file_name = "/Users/anaisbekaert/Documents/UGent/mCSF1project/PHENIX/phaseraniso/mCSF1_ID29_phaseraniso.1.pdb" }

xray_data { file_name = "anisotropyscaled.mtz"

labels = "FP_ISOB,SIGFP_ISOB" r_free_flags {

file_name = "anisotropyscaled.mtz" label = "FreeRflag"

test_flag_value = 1 }

} }

output { prefix = "mCSF1_ID29_phaseraniso.1_refine" serial = 1

} electron_density_maps {

map_coefficients { map_type = "2mFo-DFc"

mtz_label_amplitudes = "2FOFCWT" mtz_label_phases = "PH2FOFCWT"

fill_missing_f_obs = True

} map_coefficients {

map_type = "2mFo-DFc" mtz_label_amplitudes = "2FOFCWT_no_fill" mtz_label_phases = "PH2FOFCWT_no_fill" }

map_coefficients { map_type = "mFo-DFc"

mtz_label_amplitudes = "FOFCWT" mtz_label_phases = "PHFOFCWT"


map_type = "anomalous" mtz_label_amplitudes = "ANOM"

mtz_label_phases = "PANOM" }

map { map_type = "2mFo-DFc"

fill_missing_f_obs = True }


} map {

map_type = "mFo-DFc" }

} refine {

strategy = *individual_sites *individual_sites_real_space *rigid_body \

*individual_adp group_adp tls *occupancies group_anomalous

sites {

Part 7: Appendices

80

rigid_body = "chain A"

rigid_body = "chain B" }

} main {

number_of_macro_cycles = 4 }

real_space_refinement { lockit_parameters {

finishing_geometry_minimization { cycles_max = 25

} real_space_target_weights {

number_of_samples = 5

} lbfgs_max_iterations = 25

} }

secondary_structure { h_bond_restraints {

substitute_n_for_h = True }

} }

#phil __OFF__

Part 7: Appendices

81



} input {

pdb { file_name = "/Users/anaisbekaert/Documents/UGent/mCSF1project/PHENIX/phaseraniso/mCSF1_ID29_phaseraniso.1_refine_001_H.pdb" }





} }
















} map {


} refine {

strategy = *individual_sites individual_sites_real_space rigid_body \

individual_adp group_adp *tls *occupancies group_anomalous

sites {

Part 7: Appendices

82



adp { tls = "chain 'A' and (resseq 1:12)"

tls = "chain 'A' and (resseq 13:32)" tls = "chain 'A' and (resseq 33:45)"



tls = "chain 'B' and (resseq 4:32)" tls = "chain 'B' and (resseq 33:63)"



} }

main { secondary_structure_restraints = True

random_seed = 2772306 }




number_of_samples = 5 }

lbfgs_max_iterations = 25 }

} secondary_structure {

h_bond_restraints {


helix { selection = "chain 'A' and resseq 5:7"

helix_type = alpha pi *3_10 unknown }

helix { selection = "chain 'A' and resseq 13:24" }





helix { selection = "chain 'B' and resseq 13:24"

} helix {

selection = "chain 'B' and resseq 46:63" }


} helix {

Part 7: Appendices

83


helix { selection = "chain 'B' and resseq 140:144" }

sheet { first_strand = "chain 'A' and resseq 33:38" strand {

selection = "chain 'A' and resseq 103:108"

sense = parallel *antiparallel unknown

bond_start_current = "chain 'A' and resseq 108"

bond_start_previous = "chain 'A' and resseq 33"

}

} sheet {

first_strand = "chain 'B' and resseq 33:38"

strand { selection = "chain 'B' and resseq 103:108" sense = parallel *antiparallel unknown bond_start_current = "chain 'B' and resseq 108" bond_start_previous = "chain 'B' and resseq 33" }

} }

} #phil __OFF__

Part 7: Appendices

84



} input {

pdb { file_name = "/Users/anaisbekaert/Documents/UGent/mCSF1project/PHENIX/phaseraniso/mCSF1_ID29_phaseraniso.1_refine_002.pdb" }





} }
















} map {


} refine {



sites {

Part 7: Appendices

85






tls = "chain 'A' and (resseq 103:148)" tls = "chain 'B' and (resseq 4:12)"




} }

main { ordered_solvent = True

secondary_structure_restraints = True random_seed = 2864671

} tls {

max_number_of_iterations = 10 }

adp_restraints { iso {

sphere_radius = 1.55 }

} mask {

solvent_radius = 0.8 }

target_weights {

optimize_wxc = True optimize_wxu = True

bonds_rmsd_max = 0.015 angles_rmsd_max = 2

} real_space_refinement {

lockit_parameters { finishing_geometry_minimization {

cycles_max = 25 }

real_space_target_weights { number_of_samples = 5

} lbfgs_max_iterations = 25

} }

secondary_structure { h_bond_restraints {



helix_type = alpha pi *3_10 unknown }




Part 7: Appendices

86

}




} helix {



} helix {

selection = "chain 'B' and resseq 110:130"

} helix {


} sheet {

first_strand = "chain 'A' and resseq 33:38"

strand {





} }

sheet { first_strand = "chain 'B' and resseq 33:38" strand {



bond_start_current = "chain 'B' and resseq 108"

bond_start_previous = "chain 'B' and resseq 33"

} }

} }

#phil __OFF__

Part 7: Appendices

87



} input {






} }
















} map {


} refine {



sites {

Part 7: Appendices

88





tls = "chain 'A' and (resseq 103:147)" tls = "chain 'B' and (resseq 1:45)"



} }

main { nqh_flips = False

ordered_solvent = True secondary_structure_restraints = True


tls { max_number_of_iterations = 10

} adp_restraints {

iso { sphere_radius = 1.55

} }

peak_search { max_number_of_peaks = 4659

} alpha_beta {

interpolation = False }

mask {


target_weights { optimize_wxc = True

optimize_wxu = True wxc_scale = 0.05

wxu_scale = 0.1 bonds_rmsd_max = 0.015

angles_rmsd_max = 2 }







h_bond_restraints { substitute_n_for_h = True

} helix {

selection = "chain 'A' and resseq 5:7" helix_type = alpha pi *3_10 unknown

} helix {


} helix {

Part 7: Appendices

89

selection = "chain 'A' and resseq 46:63" }





} helix {



} helix {


} helix {


}






} }






} }

} }

#phil __OFF__

Part 7: Appendices

90



} input {






} }
















} map {


} refine {



sites {

Part 7: Appendices

91








} }

main { nqh_flips = False

ordered_solvent = True secondary_structure_restraints = True


tls { max_number_of_iterations = 10

} adp_restraints {

iso { sphere_radius = 1.55

} }

peak_search { max_number_of_peaks = 4659

} alpha_beta {

interpolation = False }

mask {


target_weights { optimize_wxc = True

optimize_wxu = True wxc_scale = 0.05

wxu_scale = 0.1 bonds_rmsd_max = 0.015

angles_rmsd_max = 2 }







h_bond_restraints { substitute_n_for_h = True

} helix {

selection = "chain 'A' and resseq 5:7" helix_type = alpha pi *3_10 unknown

} helix {


} helix {

Part 7: Appendices

92

selection = "chain 'A' and resseq 46:63" }





} helix {



} helix {


} helix {


}






} }






} }

} }

#phil __OFF__