structural characterization of the mammalian deoxynucleotide n … · 2013. 10. 9. · 5unité de...

doi:10.1016/j.jmb.2009.10.004 J. Mol. Biol. (2009) 394, 435–447

Available online at www.sciencedirect.com

Structural Characterization of the MammalianDeoxynucleotide N-Hydrolase Rcl and Its StabilizingInteractions with Two Inhibitors

Yinshan Yang1,2,3,4†, André Padilla1,2,3,4†, Chi Zhang5,Gilles Labesse1,2,3,4 and P. Alexandre Kaminski5⁎

© 2009 Elsevier Ltd. All rights reserved.

1CNRS, UMR5048, Centre deBiochimie Structurale, F34090Montpellier, France2INSERM, U554, F34090Montpellier, France3Université Montpellier 1 et 2,IFR3, F34090 Montpellier,France429 rue de Navacelles, 34090Montpellier, France5Unité de Chimie Organique,URA CNRS 2128, InstitutPasteur, Unité de ChimieOrganique, 28 Rue du Dr Roux,75724 Paris Cedex 15, France

Received 6 August 2009;received in revised form25 September 2009;accepted 6 October 2009Available online12 October 2009

*Corresponding author. E-mail addrpierre-alexandre.kaminski@pasteur.† Y.Y. and A.P. contributed equalAbbreviations used: Rcl, gene of d

N-hydrolase Rcl; D69A, the D69A mconstruct; D69N, the D69N mutantconstruct; GMP, guanosine monophnuclear Overhauser effect; NDT, nu2-deoxyribosyltransferase; Ll NDT, Lleichmannii NDT; HSQC, heteronuclcoherence; NOESY, nuclear Overhauspectroscopy; PDB, Protein Data Bacorrelated spectroscopy; PNP, purinphosphorylase; AMN, AMP nucleoshypoxanthine–guanine phosphoribo

0022-2836/$ - see front matter © 2009 E

The gene Rcl encodes a deoxynucleoside 5′-monophosphate N-glycosidasethat catalyzes the hydrolysis of the N-glycosidic bond of the nucleotide togive deoxyribose 5-phosphate and a nucleobase, preferentially a purine.This enzyme is over-expressed in several cancers, and its rate of expressionis correlated to the degree of aggressiveness of tumors, which makes it anew and attractive therapeutic target. We describe here its structuralcharacterization in the presence of two inhibitory substrate mimics. One ofthese ligands corresponds to the monophosphorylated form of acyclovir,which is used in the clinic. This study reveals an important ligand-inducedstabilization of the dimer structure of the enzyme. The original structuralfeatures of Rcl will be helpful for designing new inhibitors.

Keywords: ligand-induced fit; dimeric enzyme; GMP; directed mutagenesis;molecular modeling
Edited by M. F. Summers
ess:fr.ly to this work.eoxynucleotideutant of the short Rclof the short Rclosphate; NOE,cleosideactobacillusear single quantumser enhancement

nk; TOCSY, totale nucleosideidase; HGPRT,syltransferase.

lsevier Ltd. All rights reserve

Introduction

The gene rcl is a c-Myc target identified byrepresentational difference analysis betweencDNAs from fibroblasts that do or do not expressc-myc.1 Since its discovery, the protein Rcl has beenshown to be up-regulated in several cancers such ashuman prostate and breast cancers.2,3 Rcl is amongthe top 50 genes that allow distinguishing betweenbenign and malignant prostate tissues.2 In addition,Rcl is tumorigenic when it is over-expressed inconjunction with VEGF, in Rat1a fibroblasts.4 Rcl isalso a differentially expressed gene in multidrug-resistant gastric carcinoma cells.5 Thus, Rcl is acandidate for the diagnosis and/or prognosis of

d.

http://dx.doi.org/10.1016/j.jmb.2009.10.004

mailto:[email protected]

436 Deoxynucleotide N-Hydrolase Rcl Structure

clinical drug resistance. Rcl is also over-expressed inresponse to chronic administration of the glucocor-ticoid methylprednisolone or estrogen diethylstil-berol, which indicates a role for Rcl in cell growthand/or cell proliferation.6,7 The recent correlation ofthe Rcl expression and the tumor grade reinforcedthis hypothesis.3 This correlation suggests that Rclcould be an oncoprotein and is a potential thera-peutic target. However, its precise role remainspoorly understood.We have recently shown that Rcl is a deoxynucleo-

side 5′-phosphate N-glycosidase, a biochemicalactivity not previously described.8 Rcl hydrolyzesdNMP to form a free nucleobase moiety (N) and 2-deoxyribose 5-phosphate, which is subsequentlyconverted to 2-deoxyribose. The latter has beenshown to possess significant angiogenic activity9

and to be a substrate for glycolysis through itsconversion to glyceraldehyde 3-phosphate. Thisparticular hydrolysis may indirectly increase thedevelopment and malignancy of cancer cell over-expressing Rcl. Rcl may behave like thymidinephosphorylase, which is also known as the angio-genic factor endothelial cell growth factor 1, andconverts thymidine to thymine and 2-deoxyribose.9

We propose that Rcl is involved in tumorigenesis byaltering nucleotide balance, energy metabolism, andadaptation of tumor cells to the hypoxic micro-environment.Rcl is a distant member of the nucleoside 2-

deoxyribosyltransferase (NDT) family (EC 2.4.2.6;pfam 05014). NDT catalyzes the reversible transferof the deoxyribosyl moiety from a deoxynucleosidedonor to an acceptor nucleobase.10 On the contrary,Rcl is a hydrolase (or a transferase using a watermolecule as an acceptor). However, both NDT andRcl are specific for 2′-deoxyribose-containing sub-strates, while the corresponding ribosyl analoguesare inhibitors. Despite an overall low level ofsequence identity, these common functional featuressuggest a similar mode of action.Molecular modeling revealed that a small set of

amino acids that contribute to the active site ofLactobacillus leichmanniiNDT (LlNDT thereafter)11 isconserved in Rcl. These residues include a tyrosineand a glutamate (Y13 and E93 in rat Rcl) thatparticipate in a hydrogen-bond network involvingthe 3′-OH of the substrate sugar moiety. A con-served aspartate (D69 in rat Rcl) points toward thecleavage bond connecting the ribose to the nucleo-base in NDT. Guanosine monophosphate (GMP) hasbeen previously shown as a competitive inhibitor fordGMP (Ki ∼20 μM).8 Similarities of the substrateused by Rcl and NDT were taken into account topredict the orientation of nucleotide in Rcl activesite. Manual docking into the open active site of ourRcl model highlighted no particular clash. Interest-ingly, two polar residues (D72 and N123 in LlNDT),which are involved in the binding of the 5′-OH ofthe sugar in known NDTs, are replaced in Rcl bytwo serines (S87 and S117 in rat Rcl). As theseresidues are smaller and neutral, the active site inRcl could accommodate the large and negatively

charged phosphate group.8 However, Rcl and NDTare significantly divergent (15–20% over 160 aminoacids), precluding accurate molecular modeling ofthe ligand-binding site.We carried out structural studies of RCL bound to

GMP in order to gain new insights into the structureof the active site and the catalytic mechanism andthen to derive new ligands useful for fundamentalresearch, in vivo function detection, and the develop-ment of potential medicinal compounds. Since X-raycrystallography failed to provide structural infor-mation (Steven E. Ealick, personal communication),NMR spectroscopy was chosen first to characterizethe protein in solution. The solution structure wassolved to improve our understanding of therecognition of nucleotides and inhibitory analoguesby Rcl.

Results

Characterization of wild-type and Rcl variants

The wild-type polyhistidine-tagged protein wasover-expressed and purified in large quantities.Above 30 μM, Rcl is mostly dimeric according toprevious analysis of the monomer–dimer equi-librium.8 The first heteronuclear single quantumcoherence (HSQC) spectrum of a 0. 5-mM sample of15N-labeled protein showed a mixture of broad andsharp peaks. This was attributed to the equilibriumbetween the monomeric and the dimeric forms ofthe protein and/or to movement of unstructuredregions. Adding GMP gave a better spreading of theHSQC cross peaks, consistent with a well-foldedprotein. However, even in the presence of GMP,sharp cross peaks and several low-intensity peakswere observed in the HSQC spectrum, indicatingthe presence of flexible regions and/or of residueswith heterogeneous conformations.Beside the polyhistidine tag, some other protein

segments were predicted to contribute to theextended and flexible region. According tosequence comparison and molecular modeling, theN-terminal and C-terminal regions were predictedto be unfolded. Indeed, the amino acid composition(1-MAASGEQAP-9 and 152-PQKTASSSHPSA-163,respectively, in rat Rcl) suggested a high level offlexibility in agreement with the NMR study.Furthermore, orthologues from Danio rerio (zebra-fish) and Xenopus tropicalis (xenopus) are shorterand better match in length the sequences of NDT(Fig. 1). We then produce a truncated form of theprotein by deleting 9 and 12 amino acids at its N-and C-terminal parts, respectively. In addition, thehistidine tag was shifted to the C-terminal part. Thetruncated Rcl (Rclshort) was purified, and its activitywas measured with dGMP as substrate. Thevelocity and affinity parameters of the truncatedform (Vm =0.054 M−1 s−1; Km=90 μM) were veryclose to that measured with the His-tagged and full-length Rcl protein (Vm=0.06 M−1 s−1; Km=48 μM).

Fig. 1. Sequence–structure alignments of Rcl and related enzymes. Secondary structures are shown for Rcl from rat.The mutated aspartate D69 is shown by an up arrow. The catalytic glutamate (E93) and the conserved glycines (G89,G91, and G95) forming a signature of the dimerization interface of NDT and Rcl are highlighted by open and filledcircles, respectively. Residues that are predicted and observed to be in contact with the ligands are marked below byblack and gray stars, respectively. Sequence numbering corresponds to rat Rcl. Sequence names follow nomenclaturefrom UniProt (www.uniprot.org/), and structure codes are from the PDB (http://www.rcsb.org). The symbol ‘b’indicates an N-terminal truncation. The figure was prepared using ESPRIPT (http://espript.ibcp.fr/ESPript/ESPript/).

437Deoxynucleotide N-Hydrolase Rcl Structure

The HSQC spectrum of the Rclshort·GMP complexhas good quality, with a reduced number of sharpcross peaks as expected from the removal of the N-and C-terminal flexible parts from the Rclwt. WhileGMP was previously shown to be an inhibitor by

Table 1. NMR experiments acquired for structure calculation

Experiment Nuclei 1H (MHz) x-15N-HSQC 1H, 15N 600 2HNCA 1H, 13C, 15N 600 1CBCACONH 1H, 13C, 15N 600 1HNCO 1H, 13C, 15N 600 1HNCACO 1H, 13C, 15N 600 115N NOESY HSQC 1H, 1H, 15N 600 115N TOCSY HSQC 1H, 1H, 15N 600 113C NOESY HSQC (D2O) 1H, 1H, 13C 700 113C HSQC 1H, 13C 700 1NOESY (D2O) 1H, 1H 800 4Double 13C-half filtered 1H NOESY 1H, 1H 700 1TOCSY (D2O) 1H, 1H 700 215N NOESY HMQCa 1H, 1H, 15N 700 1

Six-hundred-megahertz experiments were recorded using the XWINusing the TOPSPIN Library (v. 1.2).

a Specifically recorded for low-field-shifted NHs.

monitoring the enzymatic activity on a short timecourse,8 several hours of incubation of this Rclshort(and also Rclwt) with GMP revealed slow hydrolysiswith release of free guanine. The additional signaldamping (Supplementary Materials) associated

s and chemical shift assignments

pts y-pts z-pts x-sw y-sw z-sw Mix (ms)

048 256 — 10,802 2372.1 — —500 64 80 8389.3 5282.6 2372.1 —500 92 60 8389.3 11,318.6 2372.1 —500 48 80 8389.3 2113.2 2372.1 —500 60 60 8389.3 2113.2 2372.1 —500 360 66 8389.3 8389.3 2370.9 100500 340 76 8389.3 8389.3 2370.9 40500 200 80 8771.9 8771.9 11,792.5 100500 400 — 8771.9 11,792.5 — —096 480 — 10,000 10,000 — 100500 400 — 9803.2 9803.2 — 100048 512 — 8771.9 8771.9 — 40, 10500 340 72 12,604 12,604 2765.5 100

-NMR library (v. 3.0), and 700-MHz experiments were recorded


with this hydrolytic process was obviously notcompatible with the recording of long-term 3DNMR experiments.

Assignments of Rcl D69A and D69N mutants incomplex with GMP

In order to increase the lifetime of the Rclshort·GMPcomplex, we decided to mutate conserved residuesin the catalytic pocket. Directed mutagenesis wasguided by our molecular model, which highlightedthe putative role of conserved residues. Their role inthe stability of the protein was also taken intoaccount. Among the three known catalytic residues,tyrosine Y13 is themost buriedwhilemutation of theglutamate E93 into alanine did not abolish theactivity.8 The remaining residue was aspartate atposition 69. Its substitution by alanine shouldpreserve the local helical conformation while its

Fig. 2. Assignments of the Rcl D69A·GMP complex. The aThe panel on the left side shows the three low-field-shifted N

mutation to asparagine is predicted to be the mostconservative. Both mutants were able to bind dGMPbut with lower affinities (Km of 0.26 and 0.89 mM,respectively); their Vm values are 6-fold lower(0.01 M−1 s−1). Both Rclshort mutants (D69A andD69N) have similar HSQC spectrum in the presenceof GMP, with no spectral change detectable overseveral days. The spectra are also similar to theRclshort protein, indicating that the mutations did notintroduce major structural rearrangements and thatboth mutants are well suited for recording 3D NMRspectra.The assignments were performed using 3D experi-

ments listed in Table 1 (Materials and Methods).Spectral quality was sufficient to obtain assignmentsof most 15N, NH, HA, 13CA, 13CB, and 13CO reso-nances for both mutants. However, line broadeningwas a handicap for several residues in the stretchesS17–G20, H45–A49, and S117–A118. We completed

ssignments are reported on the 1H–15N HSQC spectrum.Hs.


the sequential assignments of these residues bycomparing the 3D 15N–1H nuclear Overhauserenhancement spectroscopy (NOESY) spectra of thetwo Rcl mutants D69A and D69N. The assignmentsof Rclshort D69A are shown in Fig. 2. Chemical shiftsusing TALOS12 and PREDITOR13 were used to iden-tify secondary structures. The alternating β-strandand α-helix corresponded likely to a Rossmann foldas found in the NDT family.

Preservation of a dimeric interface in NDTsand Rcl

At the time of completion of the assignment, a newstructure of a deoxyribosyltransferase-like proteinwas solved by X-ray crystallography [Protein DataBank (PDB) ID: 3EHD; to be published]. It wasslightly more similar to Rcl than the previousstructures (22% identical instead of 15–18%). This

Fig. 3. Modeling the dimer structures of Rcl. The crystal strmodeling of the Rcl structure in its dimeric form (a). A dim2F672F672F67 (chains A and B; b), 3EHD (chains A and B; c), athe axis of symmetry and drawn using PyMOL molecular sof

prompted us to revise our sequence–structurealignment. A second round of refinement of thealignment of Rcl sequences took also into account thesecondary structure assigned from the NMR chemi-cal shifts. A final sequence–structure alignment wasproduced, including the previously known struc-tures of NDTs. This highlighted that the C-terminalregion is better conserved in structure than expectedpreviously. It also highlighted that it is involved in acommon monomer–monomer interface in all theseenzymes.The distantly relatedTrypanosoma brucei nucleoside

2-deoxyribosyltransferase (PDB ID: 2F62) is a dimer,as Rcl is. The other known (deoxy)ribosyltransferasesare either tetrameric (PDB ID: 3EHD) or hexameric(PDB ID: 1F8Y), but their complete structure iscomposed of dimer of dimers or of trimer of dimers.In both cases, the dimer resembles that of the simplyhomodimeric structures (see Fig. 3). These dimers

uctures of three distinct NDTs were used for the homologyer template was extracted from the crystal structures ofnd 1F8Y (chains A and B; d). The views were taken alongtware (http://www.pymol.sourceforge.net).

Fig. 4. Local rigidity/flexibility of Rcl bound to GMP.The heteronuclear {1H}–15N NOEs are shown for RclD69A·GMP complex along the sequence. The C-terminusand the long loop between β2 and α2 are clearly flexible(residues 46–64).


share a common interface that is mainly composed ofthree helices (α2, α3, and α4). The central helix α3harbors a motif with two highly conserved glycines

followed by the glutamate belonging to the catalytictriad (G89, G91, and E93 in Rcl). This particularorganization could explain the observed impact ofligand binding on the monomer–dimer equilibriumin Rcl (as described above).To probe the dimer interface, we used mixtures of

labeled and unlabeled proteins, in order to detectintermolecular nuclear Overhauser effects (NOEs)using 13C-filteredNMR experiments. Both D69A andD69N mutant samples were prepared, and 13C-labeled/unlabeled proteins were mixed by ureadenaturation/refolding (seeMaterials andMethods).The control 15N HSQC performed on the D69Amixed sample indicated correct refolding of theprotein. Unexpectedly, the signal-to-noise ratio wasvery low in the 13C-filtered NOESY spectra withthese two mixtures of 13C/13C-labeled mutants, andintermolecular NOEs were unreadable.The 15N-relaxation data were determined for

D69A, and they indicated an overall correlationtime (tc) of roughly 18 ns, which is consistent withRcl being a dimer.14 In parallel, we examined thoseresidues with atypical relaxation parameters (main-ly heteronuclear {1H}–15N NOE; Fig. 4). Flexibleregions correspond either to a long loop (residues46–64) or to residues lying in the vicinity of theactive site and/or of the dimer interface according toour molecular model (e.g., residues 15–20 and 115–

Fig. 5. NOEs involving GMPand Rcl. (Top panel) 13C–1H HSQCshowing the assignments of methylresonances of D69N. (Bottom panel)(F1)13C–(F2)13C-filtered NOESYspectrum shows the NOEs to theGMP ribose protons, H2′, H3′, H4′,and H5′/H5″. As a 10-fold excess ofGMP was added to Rcl, the freeGMP form dominates in the spec-trum plotted at a high level. Theribose 1H assignments are indicatedon the right.

Table 2. Statistics for 10 NMR structures of D69A Rcl incomplex with GMP

NOE restraintsShort range (|i− j|=1) per monomer 928Medium range (1b|i− j|b5)per monomer

662

Long range (|i− j|≥5) per monomer 564Dimer restraints 2×41GMP restraints 2×35Dihedral restraintsa 2×128

Number of NOE violationsN0.5 Å 0.4±0.3 (max, 0.54 Å;

dαN, 129–130)N0.3 Å 1.0±0.3N0.1 Å 4.6±0.7

Dihedral violations (N2°) 0.2±0.2Ramachandran plot statistics (%)b

Φ/Ψ in most favorable regions 80.5Φ/Ψ in additionally allowed regions 16.1Φ/Ψ in generously allowed regions 2.4Φ/Ψ in disallowed regions 1.0

Pairwise RMSD (Å)c

Monomer 0.62±0.04Dimer 0.78±0.07

Structures were calculated using CYANA, refined using CNS,and analyzed using PROCHECK.

a Residues in regular secondary structures were derived fromthe chemical shifts using TALOS and PREDITOR software.

b PROCHECK was used over all the residues.c Main-chain nuclei (N, Cα, C) over the residues 10–42 and

62–153.


120). These data precisely mapped on the proteinsequence the effect of GMP binding, which waspreviously observed in a global manner by NMRand small-angle X-ray scattering data (see above).Altogether, these results highlighted the mobilenature of this dimer interface and the protein regionthat are involved in dimerization.

NMR structure of Rcl D69A·GMP

At this step, we used a combined strategy con-sisting of generating models for the monomer withor without bound GMP and for the dimer withoutGMP. Distance restraints were collected using 3D1H–15N NOESY HSQC, 3D 1H–13C NOESY HSQCat 600 MHz, and 2D 1H–1H NOESY spectra at800 MHz. We used the capability of introducingambiguous NOE constraints and also of modeling asymmetrical homodimer in the program CYANA.15

NOE constraints were first checked for theirconsistency using monomer models. As a matter offact, several constraints, between H66 and A123 andthose between I65 and A118, were systematicallyviolated, by more than 1.5 Å, when they consideredas intramonomer. These NOEs were subsequentlyassigned to intermonomer constraints, and theresulting dimer structures were checked for NOEviolations especially for residues at the interface asdeduced from the quaternary-structure conserva-tion (see above). After several runs of structurecalculation, many intermolecular NOEs could beassigned using this combined strategy. The fewremaining NOEs, which involved the central helix(third helix) in the dimer, were treated as ambiguousconstraints.We also recorded 13C-filtered 2D NOESY experi-

ments in the presence of a 10-fold excess of GMP inorder to identify protein–ligand interactions by so-called transferred NOEs. In these spectra, a set oftwo resonances for each proton of the GMP wasobserved. The broader one (and smaller in intensity)was assigned to the bound form of GMP, while theintense and sharp one corresponds to the free form.Exchange cross peaks between bound and free GMPsignals were readily observed in total correlatedspectroscopy (TOCSY) spectra with 40 ms mixingtime but not seen when the mixing time wasreduced to 10 ms, indicating a slow exchange rateof the ligand (N25 and b100 s−1). Consequently,transferred NOEs could be recorded using a NOESYexperiment using a mixing time of 100 ms. In the13C-filtered 2D NOESY spectrum shown in Fig. 5,we reported the intermolecular NOEs between theGMP and methyl resonances of the protein.Finally, CYANA models were refined using

CNS,16 introducing non-crystallographic symmetryoperators, and using all of the nonambiguous NOEconstraints issued from the CYANA results (∼17distance constraints per residue), including intra-/intermonomer constraints and constraints with theGMPmolecule. Dihedral angle constraints were alsoadded to this final stage of the minimization(Materials andMethods). We selected 10 conformers

with the lowest NOE target function value, and thestatistics are shown in Table 2.The overall shape (Fig. 6) of the dimer is oblate,

with a maximum diameter of about 60 Å. In theorthogonal direction, the maximum extension isabout 34 Å. This global shape agrees with the tensoranalysis deduced from relaxation data analysis.17

They are also compatible with the Rg value of 33 Åand the maximal distance of ∼65 Å as determinedfrom small-angle X-ray scattering experiments (datanot shown).Rcl adopts a Rossmann fold as the other member

of the NDT family. The monomer is composed offive buried β-strands alternating with five α-helices.This overall structure is predicted to be conserved inother Rcls beside a long and flexible loop connectingstrand β2 and helix α2 (residues 46–64). Threehelices (α2, α3, and α4) are buried at the monomer–monomer interface. The central helices α3–α3′makeextensive contacts between each other in a compactmanner (Cα–Cα distance for Y92–Y92′, ∼4.2 Å).Similarly, the two glycines G91 and G95 are found inclose contacts, which explain their conservationamong Rcl sequences.

Ligand-binding site

The global architecture of the active site in Rclresembles that in known NDTs. It is mainly com-posed of the N-terminal region from one monomerand a handful of residues from the secondmonomer.The protein residues participating in the recognitionof GMP are well defined by various NOE contacts(listed in Table 3 and shown in Fig. 7). The overall

Fig. 6. NMR structure. (a) NMR structure (10 conformers) of Rcl D69A·GMP dimer in cyan and blue. The GMPs are inred. (b) Secondary-structure elements are indicated. One monomer is colored from yellow to red and the other is coloredfrom green to blue; the GMPs are in sticks.


orientation of the nucleotide in Rcl appeared similarto that of bound nucleoside in NDTs. It is composedof three main parts recognizing each moiety of theligand: the nucleobase, the ribose, and the phosphategroup.The guanine showed little interaction, but for its

proton H8, which makes extensive NOE contactswith the residues I18, I65, and A118′. A fewadditional contacts place the guanine base onto theinner face of the second α-helix. The nucleobase isstacked between H45 and I65, which constitute thestems of the long loop joining strand β2 and helix α3.The ribose-binding pocket is central and includes

the catalytic residues (Y13, D69, and E93). The riboseprotons have NOEs with the residues I18, H45, V46,I65, A69, and V90 from one monomer and with theresidueM119′ from the othermonomer. Importantly,the ribose moiety makes extensive hydrophobiccontacts with the side chain of I18, according to thenumerous NOE contacts observed (Fig. 7 andTable 3). Hydrogen bonding may connect the O2′atom of the ribose and the side chains of residues Y13and H45.

Table 3. NOE contacts between D69A and the GMP

Residues

Guanine

H8 N2H2 H1′ H2′

I65 Hβ1, CH3γ1, CH3δ CH3γ

I18 CH3γ, CH3δ Hβ, CH3γ, CHH45 Hδ2, Hɛ1V46 CH3γ

1,2

I65 Hβ, CH3γ, CH3δ CH3γA69 CH3βW72 Hɛ3V90A118′ CH3βM119′ CH3ɛ

The phosphate group interacts with several sidechains (S17 and S117′) and the N-terminus of helicesα3 and α4′ at the dimer interface. The NHs of I18(12.55 ppm), R19 (10.78 ppm), andA118′ (11.27 ppm)are unusually low field shifted. In the structure, theyare found near the nucleotide phosphate group. Thestrong chemical shift displacements of these amideprotons indicate that they are hydrogen bonded tothe negatively charged phosphate. Additionally, thetwo serines S17 and S117′ are also found close to thephosphate group, and there, hydroxyl groups mayform hydrogen bonds.The cross peaks for theRcl·Acyclovir-P complex are

at identical positions as those of the Rcl D69N·GMPcomplex in their respectiveHSQC spectra (Fig. 8). Thelow-field-shifted peaks originating from the residuesin contact with the phosphate group in the GMPcomplex are also observed for the Rcl·Acyclovir-Pcomplex. This suggested thatAcyclovir-P bindsRcl ina similar manner as GMP does.The catalytic residue E93 lies in the middle of the

GMP-binding site and is close to the O3′ atom of theribose (2.7–3.0 Å distances with the carboxylate).

GMP

Ribose

H3′ H4′ H5′/H5″

3d Hβ, CH3γ, CH3δ HN, Hβ, Hγ, CH3γ, CH3δ

CH3γ1,2

CH3γ2 CH3γ

2

Fig. 7. The GMP-binding site. Sticks and labels show the side chains of the residues close to the GMP. Broken linesindicate possible H-bonds.


The ribose 3′ hydroxyl can form a hydrogen bond tothe glutamate E93 and is in the vicinity of thehydroxyl group of the tyrosine Y13. This resemblesthe situation previously observed in otherNDTs.11,18 In the wild-type enzyme, the 2′-hydroxylof GMP will be in close contact with the side chainsof residues D69 and W72. The presence of the extra

Fig. 8. HSQC of Rcl (magenta), Rcl·GMP (black), and Rcl·phosphate group, and some of the other shifted residues are

hydroxyl group may decrease the activation of theC1′ by the aspartate D69 carboxylate. It may alsolead to a misorientation of the ribose, preventingefficient enzymatic activity. Determining the struc-ture of Rcl in complex with other ligands shouldprovide additional restraint to guide the design ofbetter inhibitors.

Acyclovir-P (blue). Circles show residues in contact to thealso indicated.


Discussion

The structure of Rcl in complex with a micromolarligand, GMP, was determined and corresponds to adimer as observed for T. brucei NDT.19 It preciselydefined the structure of the active site and thespecific protein–nucleotide interactions. A similarcatalytic triad composed of a tyrosine, an aspartate,and a glutamate is present in Ll NDT (Y7, D72, andE98) and in Rcl (Y13, D69, and E93) (Fig. 9).Mutagenesis of these residues to alanine affectssimilarly the affinity of these two enzymes for theyrespectively susbtrate Rcl (to be described else-where). However, the role of Glu E93 as a nucleo-phile in Rcl was questioned as the E93A mutantretained the same catalytic efficiency as the wildtype.8 Nevertheless, the conservative mutation intoglutamine (E93Q) totally abolished the enzymeactivity (to be described elsewhere). NDT hydro-lyzes deoxyribonucleosides reversibly while nodeoxyribonucleotides could be synthesized by Rclwith deoxyribose 5-phosphate and a purine base assubstrates. The irreversibility of this reaction mightbe explained by the rather weak interactions of Rclwith the nucleobase in agreement with its weakspecificity. Despite this variation, the overall orien-tation of the substrate into the enzyme active site ishighly similar in Rcl and NDT. Our NMR and enzy-matic data suggest that Rcl binds in the samemanner as two substrate-mimicking inhibitors, acy-clovir monophosphate and phospho-Immucillin-H,which are the monophosphorylated forms of twodrugs currently used in the clinic or in clinical trials.Altogether, these results confirmed the divergentevolution of Rcl from previously known NDTs.

Fig. 9. Schematic representation of the NDT (a) and Rcl (b)broken lines.

The conservation of the overall topology and of theactive sites of Rcl with N-deoxyribosyltransferasehas suggested a similar catalytic mechanism. Thetransfer of 2′-deoxyribosyl group between bases byNDT proceeds via a ping-pong bi–bi mechanism20

involving a covalent deoxyribosyl intermediate, andthe carboxylate of the glutamyl amino acid atposition 98 is the nucleophile.21,22 In the absence ofan acceptor base, NDT catalyzes the slow hydrolysisof 2′-deoxynucleosides.23 The existence of a deoxy-ribose 5-phosphate–enzyme intermediate was alsoaddressed by usingmethanol as nucleophile solvent.No methyl-deoxyribose 5-phosphate was detected(data not shown), indicating that either the covalentdeoxyribose 5-phosphate–enzyme intermediatedoes not exist or it differs from those described forLl NDT.21

Rcl is the first member of the NDT family active ondeoxyribonucleoside 5′-phosphates that is charac-terized at the structural level. Another member ofthis novel nucleotide monophosphate hydrolasesubfamily is BlsM, which was previously shown tohydrolyze dCMP as well as CMP.24 Sequencecomparisons highlighted Rcl and BlsM as atypicalmembers of the N-(deoxy)ribosyltransferase-likesuperfamily.8 Such a divergent evolution is remi-niscent to that of the nucleoside phosphorylasefamily composed of purine nucleoside phosphory-lase (PNP), 5′-deoxy-5′-methylthioadenosine phos-phorylase, and AMP nucleosidase (AMN).25 AMNplays a critical role in prokaryotes by regulating theintracellular level of AMP.26 It catalyzes the irrever-sible hydrolysis of AMP to yield adenine and ribose5-phosphate.27 PNP, AMN, and 5′-deoxy-5′-methylthioadenosine phosphorylase display low

active sites. Potential hydrogen bonds are indicated with


sequence similarities but share the overall topology ofthe catalytic domain and some of the key active-siteamino acids.25 While functionally related, theseenzymes are structurally unrelated to NDT and Rcl.However, PNP and the related hypoxanthine–guanine phosphoribosyltransferase (HGPRT) exem-plified the use of the mechanism-based approach asan attractive way to design exquisite inhibitors.22,28,29

Indeed, PNP is now targeted by Immucillin-H, whichrecently entered clinical trials.30–32 While 5′-phospho-Immucillin-H is a nanomolar inhibitor of HGPRT,29

its much lower activity (Ki of 370 μM) against Rclsuggests that the reaction intermediate is distinct fromthe ribooxocarbenium ion of HGPRT.33 In thiscontext, additional experiments defining the cataly-tic mechanism of Rcl are required. However, thesuccess of the transition-state theory on the formerexamples paves the way for the design of efficientinhibitors of Rcl.

Materials and Methods

Protein production and characterization

Samples of uniformly 15N-, 13C-, and 15N–13C-labeledRcls-Hiswere produced inM9minimalmedium containing15NH4Cl and 13C glucose as sole nitrogen and carbonsources, respectively. Proteins were purified as previouslydescribed.8 The concentrations of protein samples were6–10 mg/mL in 50 mM sodium phosphate buffer, pH 6.0.

Construction of a truncated N-terminal and C-terminalHis-tagged Rcl

Oligonucleotides RCLupdel (5′-GGAATTCCATATG-CGCCGCTCCGTGTACTTCTG) and RCLdodel (5′-CGGGATCCGTCGACAAGATATGCCTCAAAGTACG)were used in a PCR reaction with plasmid pET28aRcl asthe DNA template. The parameters used were 1 cycle of5 min at 95 °C; 25 cycles of 30 s at 95 °C, 30 s at 53 °C, and1 min at 72 °C; and 1 cycle of 10 min at 72 °C. The PCRproduct was purified by using the QIAquick PCRPurification Kit (Qiagen) and then digested with NdeIand SalI (underlined) enzymes over 2 h at 37 °C andrepurified. Each PCR product was ligated with plasmidpET24a digested with the same restriction enzymes. Theligation mixtures were used to transform strain DH5α.The plasmid with the correct sequence, pET24aRcls, wasused to transform strain Bli5.

Construction of the D69A and D69N mutants

Each mutant was constructed by two successive PCRs.The first PCR was realized by using oligonucleotidesD69N (5′-GTTCATCCATGAGCAGAACCTGAACTGG-CTGCA) or D69A (5′-CATCCATGAGCAGGCCCTGAA-CTGGCTGCAG) and T7prom (5′-CGCGAAATTAATA-CGACTCACTATAGGGG); oligonucleotides InvD69N(5′-TGCAGCCAGTTCAGGTTCTGCTCATGGATGAAC)or InvD69A (5′-CTGCAGCCAGTTCAGGGCCTGCT-CATGGATG) and T7term (5′-GGGGTTATGCTAGTTA-TTGCTCAGCGG) were used in a PCR reaction withplasmid pET24aRcls as DNA template. The parameters

used were the same as above except that the annealingtemperature was dependent on the couple of oligonucleo-tides used.Oligonucleotides T7prom and T7term were used in a

second PCR using aliquots of the first one, that is, D69N-T7prom and InvD69N-T7term or D69A-T7prom andInvD69A-T7term as DNA template. The amplified DNAfragments were purified by using the QIAquick PCRPurification Kit (Qiagen) and then digested with NdeI andSalI enzymes over 2 h at 37 °C and repurified. Each PCRproduct was ligated with plasmid pET24a digested withthe same restriction enzymes. The ligation mixtures wereused to transform strain DH5α. The plasmid with thecorrect sequence, pET24aRcls D69A or pET24aRcls D69N,was used to transform strain Bli5.

Kinetic measurements

The enzyme activity was determined spectrophoto-metrically by incubating the enzyme with dGMP and byfollowing the production of deoxyribose 5-phosphate (oneof the reaction products) as described previously.8 Theinitial velocity of the reaction was measured either at avariable concentration of dGMP, both in the absence andpresence of inhibitors, or at a fixed concentration of dGMPand variable concentrations of inhibitors, allowing thetesting of the nature of the inhibition.

Molecular modeling

Rcl sequence was used to search for compatibletemplates in the PDB database. Rcl and its close ortho-logues were manually aligned to NDT of known structureusing ViTO34 in either the monomeric or the dimericconfiguration. The most significant variations corre-sponded to the C-terminal region. Partial models wereobtained using SCWRL3.0,35 and refined models werededuced using MODELLER 7v7.36 Further refinementwas performed using recently solved crystal structure(e.g., PDB ID: 3EHD (chains A and B).

Small-angle X-ray scattering

Data were collected at beamline X33, at DeutschesElektronen-Synchrotron, European Molecular BiologyLaboratory, Hamburg, at 10 °C, using a wavelength of1.5 Å. Data analysis and ab initio shape calculations wereperformed using PRIMUS, GNOM, and GASBOR.37

NMR

The NMR samples were concentrated at 0.5–1.0 mMprotein, in 50 mM phosphate buffer, pH 7.2, in 90% H2O/10% D2O (or 100% D2O), with 5 or 10 mM GMP. Internalreference 2,2-dimethyl-2-silapentane-5-sulfonate wasadded to the sample.

Labeled–unlabeled mixed samples

An Rcl-D69A sample was prepared by mixing 5.5 mg ofunlabeled protein with 4.6 mg of 13C/15N-labeled proteinin 0.5 mL added to 4. 5 mL urea at 8.9 M. The Rcl-D69Nsample was prepared bymixing 6/6mg of unlabeled/13C-labeled proteins in 0.5 mL added to 4.5 mL urea at 8.9 M.The samples were left for 1 h at room temperature. Afterovernight dialysis against 2 L of 50 mM phosphate buffer,pH 7.5, and 2 mM DTT, a second overnight dialysis was

‡http://www.rcsb.org


performedwith the same bufferwithout DTT. The sampleswere concentrated and H2O was replaced by D2O usingCentricon® 5000 with 50 mM phosphate, pH 7.5, and10 mM GMP in D2O buffer. A control 15N–1H HSQCexperiment was recorded with the D69A sample prior toreplacing H2O by D2O.

15N backbone amide NMR relaxation data

15N-T1,15N-T2, and heteronuclear {1H}–15N NOE relax-

ation data were acquired at 300 K on a Bruker Avance600-MHz spectrometer, processed, and analyzed aspreviously described38 for 250-μL samples containing0.7 mM D69A and 5 mM GMP.

Assignment of D69A and D69N and structurecalculation for GMP-D69A

Spectra were acquired on 600- and 700-MHz AvanceBruker and 800-MHz INOVA Varian spectrometersequipped with triple-resonance (1H, 15N, 13C) z-gradientcryo-probes (Table 1). NMR data were processed usingGIFA (v 4.0)39 and XWIN-NMR (v. 3.0) andwere analyzedusing strip plots with Cindy (v 1.2), an in-house software.A manual reordering of the sequential stretches wasperformed. The remaining residues were assigned usingstrip plots of the 3D 15N NOESY HSQC experiments. Side-chain assignment was carried out using 2D NOESY and2D TOCSY experiments with D2O samples at 800 and700 MHz, respectively. The side-chain 1H resonances werealmost completely assigned. Only the NH of the first 2residues and those of 45, 47, and 48 remained unassigned.NOEs were classified as strong, medium, and weak (2.4,

3.6, and 4.4 Å upper-bound constraints, respectively).Strong dαN NOEs in the β-strands were assigned to 2.2 Å.Structure calculations were performed with CYANA(v. 2.1)15 using the distance restraints from 3D 15N NOESYHSQC and 3D 13C NOESY HSQC experiments.At the initial stage, the structure of the monomer was

calculated using dihedral angle restraints, NOEs inferredfrom the regular secondary structures, and nonambiguousNOEs belonging to the monomer, according to themolecular model based on structure–sequence homology.The long-distance constraints were checked for theirconsistency by listing the NOE, dihedral constraintsviolations, and van der Waals clashes. In parallel, thedimer structure calculations were initiated and theconflicting NOEs, deduced from the monomer structurecalculations, were introduced as intermonomer con-straints. The NOE constraints involving the GMP werechecked on the monomer structure. With the final set ofNMR constraints, CYANA was used to calculate 100structures, of which the 20 conformers with the lowesttarget function were refined by CNS (v. 1.2).16 We used thenon-crystallographic symmetry energy term in all therefinement. The first CYANA structure was refined inCNS with two GMP molecules, and all the NMRconstraints, including those involving GMP, were intro-duced. For this docking, the GMP was placed at variouspositions from the protein (50- to 150-Å distances), and thestructure was submitted to high-temperature (1000 K)torsion angle dynamics, slow cooling, and 200 steps ofPowell minimization. The resulting GMP coordinateswere then introduced in each of the 20 CYANA con-formers, and structure refinement was done in CNS using200 steps of torsion angle dynamics at 250 K and slowcooling, followed by 200 steps of Powell minimization.The final 10 conformers were selected with the lowest

NOE and dihedral angle violations and are the structuresdiscussed herein and deposited (with PDB IDs). Structureswere validated using PROCHECK.40

Data deposition

The refined models and structure factors have beendeposited in the Research Collaboratory for StructuralBiology‡ under the PDB accession number 2KLH.

Acknowledgements

Financial support from the TGE RMNTHC Fr3050for conducting the research is gratefully acknowl-edged. We are grateful to Dr. Adrien Favier whokindly recorded NMR experiments on a Varian 800spectrometer at I.B.S. Jean-Pierre Ebel (Grenoble,France). We acknowledge help from Prof. ChristianRoumestand for recording and analyzing the relaxa-tion data.

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tted for publication, the structure of apo-Rcl was8. PMID: 19720067). Both structures are similar withabsence of GMP.

structural characterization of the mammalian deoxynucleotide n … · 2013. 10. 9. · 5unité de...

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