nmr spectroscopy in structural biology atia-tul-wahab, m. iqbal choudhary and kurt wüthrich, 1 dr....

NMR Spectroscopy in Structural Biology

Atia-tul-Wahab, M. Iqbal Choudhary and Kurt Wüthrich,

1

Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences,

University of Karachi, Karachi-75270, Pakistan

The Scripps Research Institute, La Jolla, CA, USA

X-Ray VS NMR Structures

Molecules are studied in solution, closer to the native condition found in cell.

Protein folding studies can be done by monitoring NMR spectra.

Chemical or conformational exchange, internal mobility and dynamics at timescales ranging from picoseconds to seconds.

NMR is very efficient in mapping interactions with other molecules, e.g. protein/protein, protein/nucleic acid, protein/ligand or nucleic acid/ligand interactions.

The upper weight limit for NMR structure determination is ~30 kDa.

NMR Structures X-Ray Structures

Crystallization required, potential crystal packing influence the structure, especially on the surface of protein.

Flexible loops may not be visible in crystal structure due to spatial arrangement of electron density.

Above ~ 30 kDa X-Ray is the only technique to solve the structure of proteins.

2

The Steps inProtein Structure Determination by NMR

1. Sample preparation (a) protein selection

(b) gene engineering(c) protein expression(d) protein purification(e) buffer optimization(f ) isotope labeling

2. Data collection(a) HSQC (b) amide H/D exchange(c) APSY/ triple-resonance

(d) 3D-NOESY

3. Data evaluation

4. Structure calculation5. Structure refinement6. Structure deposition

3

Fig. 2 (2003) Progress in NMR Spectroscopy, 43, 105, Guntert.

The

AssignCalculateEvaluate

cycle

Automated NOE assignment

and structure calculation

5

1D 1H-NMR screening1D 1H-NMR screening

Protocol for Automated NMR Structure Determination

1. NMR Sample 2. NMR Structure

Promising protein constructs and

solvent conditions

Promising protein constructs and

solvent conditions

2D [15N, 1H]-HSQC screening2D [15N, 1H]-HSQC screening

Structure quality protein solution NMR profile

Structure quality protein solution NMR profile

Automated backbone assignmentsAutomated backbone assignments

Interactive validation of backbone assignmentsChemical shifts adaptation to NOESY spectra

Interactive validation of backbone assignmentsChemical shifts adaptation to NOESY spectra

Automated [1H, 1H]-NOESY-based side chain assignments, constraints collection and structure calculation

Automated [1H, 1H]-NOESY-based side chain assignments, constraints collection and structure calculation

NMR structure solvedAccurate backbone fold

NMR structure solvedAccurate backbone fold

Interactive NMR structure refinement

Interactive NMR structure refinement

NMR structure refinedNMR structure refined

NMR structure validationNMR structure validationPDBPDB

No

7

PJ03720C

TM0320

A. Folded globular protein

B. Non-globular proteinFolding

PG9814A

C. Aggregated or oligomerized protein

8

[1H, 15N]-HSQC Spectra

TM0320GS13720A

PE00019A

9

Number of expected peaks

Peak number (order with decreasing intensity)

Sig

nal

/No

ise

Signal-to-Noise NMR Profile

10

Sig

nal

/No

ise

Peak number (order with decreasing intensity)

APSY quality

TM0320 NMR Profile

Number of expected peaks

11

Peak Number (order with decreasing intensity)

PE00019A (A6)

0

50

100

150

200

250

300

350

400

450

500

550

600

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160

Sig

nal

/No

ise

88

Number of expected peaks

PE00019A NMR profile

Limited APSY quality

12

NMR Experiments

3 APSY-NMR experiments20 35 20 projections each

3 APSY-NMR experiments20 35 20 projections each

3D-15N-resolved [1H, 1H]-NOESY3D-13C-resolved [1H, 1H]-NOESY (ali)3D-13C-resolved [1H, 1H]-NOESY (aro)

3D-15N-resolved [1H, 1H]-NOESY3D-13C-resolved [1H, 1H]-NOESY (ali)3D-13C-resolved [1H, 1H]-NOESY (aro)

Automated backbone assignmentAutomated backbone assignment

Interactive validation of backbone assignmentChemical shifts adaptation to NOESY spectra

Interactive validation of backbone assignmentChemical shifts adaptation to NOESY spectra

Automated [1H, 1H]-NOESY-based sidechain assignment, constraint

collection and structure calculation

Automated [1H, 1H]-NOESY-based sidechain assignment, constraint

collection and structure calculation

NMR structure solvedAccurate backbone

fold

NMR structure solvedAccurate backbone

fold

Interactive NMR structure refinementInteractive NMR structure refinement

NMR structure refinedNMR structure refined

13

Software

GAPROGAPRO

MATCH

ASCAN

ATNOSCANDID

MATCH

ASCAN

ATNOSCANDID

UNIO

Automated backbone assignmentsAutomated backbone assignments

Interactive validation of backbone assignmentsChemical shifts adaptation to NOESY spectra

Interactive validation of backbone assignmentsChemical shifts adaptation to NOESY spectra

Automated [1H, 1H]-NOESY-based side chain assignments, constraints collection and structure calculation

Automated [1H, 1H]-NOESY-based side chain assignments, constraints collection and structure calculation

NMR structure solvedAccurate backbone fold

NMR structure solvedAccurate backbone fold

Interactive NMR structure refinement

Interactive NMR structure refinement

NMR structure refinedNMR structure refined

CYANA14

15

Protein NP_888769.1,

• A Phage-Related Protein isolated from Bordetella bronchiseptica

• No structure was available of the whole family

• Function of the protein was not known

C-terminal

N-terminal

C-terminal

N-terminal

Number of amino acid: 141

Mol. Wt: 15.2 kDaExperiments:

4D-HACANH5D-HACACONH5D-CBCACONH

13C-resolved NOESYs (ali & aro)15N-resolved NOESY

75.6% backbone assignments

68.4% Side chain assignments

GMSQDLIRAAFEKRLSDWAKARTPALPVAWQNTKFTPPAAGVYLRAYVMPAATISRDAAGDHRQYRGVFQVNVVMPIGDGSRSAEQVAAELDALFPVNLVMQSGGLAVRVRTPISNGQPTTGDADHTVPISLGYDVQFYPE 

16

17

18

C-terminal

N-terminal

67

76

84

94

134

125

Sequence

CA

-CB

(p

pm

)

Secondary Structure Elements

19

C-terminal

N-terminal

67

76

84

94

134

125

20

Heteronuclear NOE Results

Sequence

A51

A124G78

Q31

N-terminalC-terminal

II

I

III IV

Rela

tive

in

ten

sity

22

C-terminal

N-terminal

C-terminal

N-terminal

C-terminal

N-terminal

C-terminal

N-terminal

23

C-terminalN-terminal

C-terminal

N-terminal

24

20 NMR conformersof PE00019A

C-terminal

N-terminal

C-terminal

N-terminal

C-terminal

N-terminal

C-terminal

N-terminal

25

26

Stereo View of side chain

Structure Homologues

λ bacteriophage

S. Typhimrium

27

28

Binding with Mg++ metal

29

Conclusion

• NP_888769.1 is the first representative of unknown family

•Structure of NP_888769.1 was deduced without X-ray coordinates

•The NMR structure shows following features

Two α-helix and two β-sheets

A disorder region of 15 amino acid in between the sequence

Binding experiment with Mg++ metal indicated that protein does not oligomerized upon addition even 200 mM MgCl2

30

NMR Structure of Protein YP_001336205, From Klebsiella pneumoniae Genome

• YP_001336205.1 is the first structural representative of the domain of unknown function DUF3315 (PF11776),

• Consists of 283 sequences from 112 different species.

• The 9.4 kDa polypeptide YP_001336205.1 was selected with emphasis on members of Pfam families with no structure representative.

• Isolated from Klebsiella pneumoniae, a Gram-negative bacterium, a pathogen causing nosocomial pneumonia in immunocompromised patients as well as urinary tract infections (UTI), septicemia, and liver abscesses.

Introduction

YP_001336205

Number of amino acid: 83

GAAGIDQYAL KEFTADFTQF HIGDTVPAMY LTPEYNIKQW QQRNLPAPDA

GSHWTYMGGN YVLITDTEGK ILKVYDGEIF YHR 

10 20 30 40 50

60 70 80

ω1(15N)ppm

ω2(1H)ppm

33

Experiments:

4D-HACANH5D-HACACONH5D-CBCACONH

13C-resolved NOESYs (ali & aro)15N-resolved NOESY

82.2% backbone assignments

89.2% Side chain assignments

YP_001336205

Statistics GS13720A

Validation Table

Secondary Structure ElementsC

A-C

B (

pp

m)

Sequence

C-terminal

N-terminal

Ribbon representation of theconformer closest to themean coordinates.

39

2D [15N,1H]-HSQC spectrum of a 1.4 mM solution of uniformly 15N-labeled YP_001336205.1 recorded at 600 MHz and 298 K.

Cross sections along ω2(1H) through the cross peaks

No: Chain Z rmsd lali nres %id PDB Description 1: 2qzb-B 3.0 3.0 62 147 8 PDB MOLECULE: UNCHARACTERIZED PROTEIN YFEY; 2: 2qzb-A 2.9 3.0 58 145 9 PDB MOLECULE: UNCHARACTERIZED PROTEIN YFEY; 3: 1su3-A 2.3 5.1 49 415 8 PDB MOLECULE: INTERSTITIAL COLLAGENASE; 4: 3kvp-D 2.3 2.4 40 49 13 PDB MOLECULE: UNCHARACTERIZED PROTEIN YMZC; 5: 3kvp-C 2.3 2.5 39 45 13 PDB MOLECULE: UNCHARACTERIZED PROTEIN YMZC; 6: 1gxd-B 2.3 3.0 42 623 12 PDB MOLECULE: 72 KDA TYPE IV COLLAGENASE; 7: 1wmi-A 2.3 3.2 54 88 15 PDB MOLECULE: HYPOTHETICAL PROTEIN PHS013; 8: 1wmi-C 2.3 3.1 54 88 15 PDB MOLECULE: HYPOTHETICAL PROTEIN PHS013; 9: 3ba0-A 2.2 5.0 51 365 8 PDB MOLECULE: MACROPHAGE METALLOELASTASE; 10: 1rtg-A 2.2 3.6 46 203 13 PDB MOLECULE: HUMAN GELATINASE A; 11: 1fbl-A 2.1 5.1 49 367 8 PDB MOLECULE: FIBROBLAST (INTERSTITIAL) COLLAGENASE (MMP-1); 12: 3bpq-D 2.1 4.0 53 86 11 PDB MOLECULE: RELB; 13: 3bpq-B 2.1 4.0 56 85 16 PDB MOLECULE: RELB; 14: 1su3-B 2.1 5.1 48 416 8 PDB MOLECULE: INTERSTITIAL COLLAGENASE; 15: 2clt-B 2.1 5.1 49 367 8 PDB MOLECULE: INTERSTITIAL COLLAGENASE; 16: 2clt-A 2.1 4.9 49 367 8 PDB MOLECULE: INTERSTITIAL COLLAGENASE; 17: 2jxy-A 2.1 4.9 50 194 8 PDB MOLECULE: MACROPHAGE METALLOELASTASE; 18: 1pex-A 2.1 4.8 48 192 10 PDB MOLECULE: COLLAGENASE-3; 19: 1gxd-A 2.1 3.5 45 624 13 PDB MOLECULE: 72 KDA TYPE IV COLLAGENASE;  

Structure HomologuesDALI output

• We have determine the structure determination of YP_001336205.1 from Klebsiella pneumoniae in phosphate buffer at pH 6.0 using automated NMR protocol.

• YP_001336205.1 exhibited a new structure fold and is the first representative of a new Pfam family of unknown function DUF3315 (PF11776).

• The protein showed a well-define globular structure comprises an anti-parallel β-sheet, an anti-parallel β-hairpin which is located perpendicularly to the β-sheet and five 310-helices which surround the core of the protein.

Conclusion

Saturation Transfer Difference (STD) NMR Spectroscopy

42

Saturation-Transfer-Difference (STD) NMRSaturation-Transfer-Difference (STD) NMRGroup Epitope MappingGroup Epitope Mapping

Saturation-Transfer-Difference (STD) NMRSaturation-Transfer-Difference (STD) NMRGroup Epitope MappingGroup Epitope Mapping

Reference

spectrum

STDSTD

Selective

protein

saturation

STD For Epitope Mapping and STD For Epitope Mapping and Binding StudiesBinding Studies

Solubility

High/ low affinity binding

Specific and non specific bindings

Limitation of STD NMR Limitation of STD NMR SpectroscopySpectroscopy

STD (Saturation Transfer Diffusion) STD (Saturation Transfer Diffusion) Studies on Studies on

α-Glucosidase Inhibitors α-Glucosidase Inhibitors

α-Glucosidase

α-Glucosidase is present in the brush border membrane of the small intestine. It catalyzes the final step of carbohydrate digestion so that its inhibition suppresses the release of glucose from dietary origin

The catalytic role of α-glucosidase makes it a therapeutic target to treat carbohydrate mediated diseases

Saccharomyces cerevisiae α-glucosidase (modeled) with Maltose as substrate in active site (Protein Model Portal).

Saccharomyces cerevisiae iso-maltase (PDB-3AJ7) used for modeling with Maltose as substrate in active site.

Acarbose – AGI, in active site of modeled Saccharomyces cerevisiae α-glucosidase (Guerreiro et

al, 2013).

Structural Structural Features of Features of

EnzymeEnzyme

48

αα-Glucosidase Inhibition in Diabetes-Glucosidase Inhibition in DiabetesAcarbose as an example of AGI

49(Arungarinathan et al., 2011)

Inhibitors of α-glucosidase delay the rate of conversion of disaccharide into monosaccharide. As a result, the postprandial blood glucose level is maintained at a lower level, leading to a decreased insulin demand.

This approach is useful to manage glycemic index, independent to insulin in diabetic patients.

Can be used as anti-obesity drugs

Also have anti-viral drugs

α-Glucosidase Inhibitors

How α-Glucosidase Inhibitors Work?

-GLUCOSIDASE INHIBITORY ACTIVITY

α-Glucosidase (EC 3.2.1.20) an exo type glycosylase that release α-glucoside from the non-reducing end side of the substrate.

The aim of anti-diabetic therapy, both in insulin dependent diabetes mellitus and non-insulin dependent diabetes mellitus, is to achieve normoglycaemia (normal serum glucose level).

Mechanism of Action of α- Glucosidase Inhibitors

Inhibition of the intestinal enzymes that break down the carbohydrates thus delay the absorption and digestion of carbohydrates in the gut

Specifically target meal-related (postprandial) hyperglycemia, an independent risk factor for cardiovascular complications

Control the glucose levels independently of insulin

The effect on glycated hemoglobin (GHb) are comparable to metformin or thiazolidines

Mechanism of Action of α- Glucosidase Inhibitors

α-Glucosidase inhibitors (AGI) as initial treatment for patients with Type 2 Diabetes

Cause no hypoglycemic events

Cause no weight gain

Potential to be used as anti-obesity agents

(Z)

HO

OH

HO

HN

OH

O

O

O O

O

OH

HO

OH

HO

CH3

OH

HO

OH

OH

OH

Acarbose

H2O

DMSO

H2ODMSO

CH3

Sugar protons

Sugar protons

A

B

Protein

irradiation

poin

t

56

(Ren et al., 2011)

IC50±SEM = 906±6.3 µM + Non-cytotoxic against 3T3

cell line

N

OH

OHHO

H OH

.HCl

1-deoxynojirimycin

H2O

DMSO

H2O

DMSO

A

B

Protein

irradiation

poin

t

57

Competitive Inhibition

Inhibitor (DNJ) concentrations

A: Line-weaver-Burk Plot of DNJ1/[S]

-12 -10 -8 -6 -4 -2 0 2 4 6 8 10 12

1/V

-0.2

0

0.2

0.300 mM

0.200 mM

0.100 mM

0.025 mM

0.00 mM

IC50±SEM = 279.715±4.73 µM + Non-cytotoxic against 3T3

cell line

A

B

Reported Activities:

HypnoticSkin whiteningAnticancerAntiangiogenesisAntioxidant

NH

NH

O

SO

R1

R2

R3

R4

R5

NH

NH

O

SO

R

DMSO

Protein

irradiation

p

oint

DMSO

H2O

H2O

224

1' 2

7

NH6

5

NH 43

O

SO

O1

5'4'

3'2'

H-1

H-1

H-4',5'

H-4',5'

I-------Impurities------I

I-------Impurities------I

A

B

H-3'

H-3'

59

1/[S]-12 -10 -8 -6 -4 -2 0 2 4 6 8 10 12

1/V

-0.2

0

0.2

Competitive Inhibition

DMSO

Protein

irradiation

poin

t

DMSOH2O

H2O

201

1'

1

2

7

NH6

5

NH 43

O

SO

2'

3'

4'

5'

6'HO

H-3',5'

H-3',5'

H-1

H-2',6'

H-2',6' H-1

A

B

60

-10 0 10

1/V

-0.8

-0.6

-0.4

-0.2

0

0.2

0.4

0.6

0.8

Mixed-type Inhibition

Reported Activities:

AntioxidantAlzheimer's diseaseAntibacterialAntimalarialAntidiabeticAntiparasitic

NH

N (E)

O

HO

R4

R1

R2

R3

R5R6

175

5'

4'

3'

2'

1'

6'1

NH2

N3

4

(E)

O

1''

6''

5''

4''

3''

2''

HO

Br

HO

Br

H2O

H2O

DMSO

DMSO

H-6''H-2',6'

H-3',5'

H-4''

H-4

H-2',6' H-3',5'

H-4

A

B

Protein

irradiation

poin

t

62

1/[S]-12 -10 -8 -6 -4 -2 0 2 4 6 8 10 12

1/V

-0.2

0

0.2

Mixed-type Inhibition

176

5'

4'

3'

2'

1'

6'1

NH2

N3

4

(E)

O

1''

6''

5''

4''

3''

2''

HO

OH

HO

CH2CH3

H2O DMSOH-6''

H-2',6' H-3'',4''

H-3',5'

CH2

CH3

H2ODMSOH-6'' H-3',5'

A

B

Protein

irradiation

p

oint

63

1/[S]-12 -10 -8 -6 -4 -2 0 2 4 6 8 10 12

1/V

-0.4

-0.2

0

0.2

0.4

Non-Competitive Inhibition

5'

4'

3'

2'

1'

6'1

NH2

N3

4

(E)

O

1''

2''3''

4''

5''

6''

HO

OH

OEt

184 – Non-inhibitor

H2O

H2O

DMSO

DMSO

A

B

Protein

irradiation

p

oint

No STD Signals were No STD Signals were observedobserved

64

Solubility

High/Low

affinity

No interaction or no

inhibition

A

B

Reported Activities:

AntimicrobialInsecticidalAntioxidantAntibacterialAndrogen LigandAntidiabetic

NH

N

O

N R5

R4

R3

R2

R1

NH

N

O

NR5

R4

R3

R2

R1

259

DMSO

H2O

B

4'1

O

NH2

N3

CH4

1''

6''

5''

4''3''

2''

OH

OH

5'

6'

N1'

2'

3'

(Z)

Protein

irradiation

p

oint

A

B

H-2',6'

H-5''H-3', 5'

H-6''

H-2''

H-4

H-2',6' H-5''

H-3', 5' H-2''

H2O

DMSO

66

1/[S]

-10 0 10

1/V

-0.8

-0.6

-0.4

-0.2

0

0.2

0.4

0.6

0.8

1

Mixed-type Inhibition

67

On Going Projects

•YP_040532.1 Thioredoxin protein•YP_041423.1 Hypothetical phage protein•YP_039780.1 Putative glycine cleavage H protein•YP_041699.1 Putative membrane protein•YP_041210.1 Putative membrane protein

68