Presentation on theme: "NMR Spectroscopy in Structural Biology"— Presentation transcript:
1 NMR Spectroscopy in Structural Biology Atia-tul-Wahab, M. Iqbal Choudhary and Kurt Wüthrich,Dr. Panjwani Center for Molecular Medicine and Drug Research,International Center for Chemical and Biological Sciences,University of Karachi, Karachi-75270, PakistanThe Scripps Research Institute, La Jolla, CA, USA
2 X-Ray VS NMR Structures X-Ray StructuresMolecules 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.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.
3 Protein Structure Determination by NMR The Steps inProtein Structure Determination by NMRSample preparation(a) protein selection(b) gene engineering(c) protein expression(d) protein purification(e) buffer optimization(f ) isotope labeling2. Data collection(a) HSQC(b) amide H/D exchange(c) APSY/ triple-resonance(d) 3D-NOESY3. Data evaluation4. Structure calculation5. Structure refinement6. Structure deposition
4 Automated Projection Spectroscopy (APSY) Sebastian Hiller et al., J. Biomol. NMR (2008)
5 Automated NOE assignment and structure calculation TheAssignCalculateEvaluatecycleAutomated NOE assignmentand structure calculationFig (2003) Progress in NMR Spectroscopy, 43, 105, Guntert.
14 Accurate backbone fold SoftwareGAPROAutomated backbone assignmentsUNIOMATCHASCANATNOS CANDIDInteractive validation of backbone assignmentsChemical shifts adaptation to NOESY spectraAutomated [1H, 1H]-NOESY-based side chain assignments, constraints collection and structure calculationNMR structure solvedAccurate backbone foldInteractive NMR structure refinementNMR structure refinedCYANA
15 Protein NP_ ,A Phage-Related Protein isolated from Bordetella bronchisepticaNo structure was available of the whole familyFunction of the protein was not knownC-terminalC-terminalN-terminalN-terminal4455
16 Amino Acid Sequence GMSQDLIRAAFEKRLSDWAKARTPALPVAWQNTKFTPPAAGVYLRAYV MPAATISRDAAGDHRQYRGVFQVNVVMPIGDGSRSAEQVAAELDALFPVNLVMQSGGLAVRVRTPISNGQPTTGDADHTVPISLGYDVQFYPENumber of amino acid: 141Mol. Wt: 15.2 kDaExperiments:4D-HACANH5D-HACACONH5D-CBCACONH13C-resolved NOESYs (ali & aro)15N-resolved NOESY75.6% backbone assignments68.4% Side chain assignments
21 in Val, Leu, Ile vs Calculated Ring Current Shifs Experimental Chemical Shift Difference from the average of Methyl groupsin Val, Leu, Ile vs Calculated Ring Current ShifsCalculated RCS/ppmObserved CS/ ppm
22 Heteronuclear NOE Results C-terminalN-terminal4II5A518Relative intensity6Q31I7SequenceIVIIIA124G78
29 Conclusion NP_888769.1 is the first representative of unknown family Structure of NP_ was deduced without X-ray coordinatesThe NMR structure shows following featuresTwo α-helix and two β-sheetsA disorder region of 15 amino acid in between the sequenceBinding experiment with Mg++ metal indicated that protein doesnot oligomerized upon addition even 200 mM MgCl2
30 NMR Structure of Protein YP_001336205, From Klebsiella pneumoniae Genome
31 IntroductionYP_ 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_ 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.
38 Ribbon representation of the conformer closest to themean coordinates.
39 2D [15N,1H]-HSQC spectrum of a 1 2D [15N,1H]-HSQC spectrum of a 1.4 mM solution of uniformly 15N-labeled YP_ recorded at 600 MHz and 298 K.Cross sections along ω2(1H) through the cross peaks
40 Structure Homologues DALI output No: Chain Z rmsd lali nres %id PDB Description1: 2qzb-B PDB MOLECULE: UNCHARACTERIZED PROTEIN YFEY;2: 2qzb-A PDB MOLECULE: UNCHARACTERIZED PROTEIN YFEY;3: 1su3-A PDB MOLECULE: INTERSTITIAL COLLAGENASE;4: 3kvp-D PDB MOLECULE: UNCHARACTERIZED PROTEIN YMZC;5: 3kvp-C PDB MOLECULE: UNCHARACTERIZED PROTEIN YMZC;6: 1gxd-B PDB MOLECULE: 72 KDA TYPE IV COLLAGENASE;7: 1wmi-A PDB MOLECULE: HYPOTHETICAL PROTEIN PHS013;8: 1wmi-C PDB MOLECULE: HYPOTHETICAL PROTEIN PHS013;9: 3ba0-A PDB MOLECULE: MACROPHAGE METALLOELASTASE;10: 1rtg-A PDB MOLECULE: HUMAN GELATINASE A;11: 1fbl-A PDB MOLECULE: FIBROBLAST (INTERSTITIAL) COLLAGENASE (MMP-1);12: 3bpq-D PDB MOLECULE: RELB;13: 3bpq-B PDB MOLECULE: RELB;14: 1su3-B PDB MOLECULE: INTERSTITIAL COLLAGENASE;15: 2clt-B PDB MOLECULE: INTERSTITIAL COLLAGENASE;16: 2clt-A PDB MOLECULE: INTERSTITIAL COLLAGENASE;17: 2jxy-A PDB MOLECULE: MACROPHAGE METALLOELASTASE;18: 1pex-A PDB MOLECULE: COLLAGENASE-3;19: 1gxd-A PDB MOLECULE: 72 KDA TYPE IV COLLAGENASE;
41 ConclusionWe have determine the structure determination of YP_ from Klebsiella pneumoniae in phosphate buffer at pH 6.0 using automated NMR protocol.YP_ 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.
42 Saturation Transfer Difference (STD) NMR Spectroscopy
43 Selective protein saturation Saturation-Transfer-Difference (STD) NMRGroup Epitope MappingReferencespectrumSelective protein saturationSTD
45 Limitation of STD NMR Spectroscopy SolubilityHigh/ low affinity bindingSpecific and non specific bindings
46 STD (Saturation Transfer Diffusion) Studies on α-Glucosidase Inhibitors46
47 α-Glucosidaseα-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 originThe catalytic role of α-glucosidase makes it a therapeutic target to treat carbohydrate mediated diseases
48 Structural Features of Enzyme 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.MAL12 binding site is composed of Asp214, Glu276, and Asp349 catalytic residues. In addition to the catalytic residues, molecular docking studies confirm Asp68, Tyr71, and Arg439 as important residues in the a-glucosidase inhibitionGuerreiro, L. R., Carreiro, E. P., Fernandes, L., Cardote, T. A., Moreira, R., Caldeira, A. T., Guedes, R. C., Burke, A. J. (2013). Five-membered iminocyclitol α-glucosidase inhibitors: synthetic, biological screening and in silico studies. Bioorganic and Medicinal Chemistry, 21(7):Acarbose – AGI, in active site of modeled Saccharomyces cerevisiae α-glucosidase (Guerreiro et al, 2013).
49 α-Glucosidase Inhibition in Diabetes Acarbose as an example of AGIType 1 : with damage to β pancreatic cells which in turn leads to insulin deficiency, thus increases the level of sugar in blood.Type 2 : it is associated with resistance to insulin and insulin secretary defects causing relative deficiency. Obese, aged and people with less physical activity are prone to this type of diabetes. Women, hypertensive and dyslipidemic patients are at higher risk of NIDDM. Genetic factors also contribute to this type of diabetes but clear picture is not yet available.Persistent hyperglycemia tends to glycate the proteins. These glycated proteins, in turn, start to accumulate the advance glycation endproducts (AGEs).By far, the fda approved clinically used α-glucosidase inhibitors for niddm (voglibose, acarbose and miglitol) are competitive and reversible inhibitors of α-glucosidase. (Α-glucosidase inhibition in NIDDM)Why we need better agi??Tcurrent management with agi is associated with gastric side effects which includes flatulence, gastric cramps, diarrhea, etc. Another problem is frequent dosing i.E. AGI has to be taken with every meal. This also increases the cost of the therapy and patient compliance.(Arungarinathan et al., 2011)
50 α-Glucosidase Inhibitors 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 drugsAlso have anti-viral drugs
52 -GLUCOSIDASE INHIBITORY ACTIVITY α-Glucosidase (EC ) 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).52
53 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 gutSpecifically target meal-related (postprandial) hyperglycemia, an independent risk factor for cardiovascular complicationsControl the glucose levels independently of insulinThe effect on glycated hemoglobin (GHb) are comparable to metformin or thiazolidines53
54 Mechanism of Action of α- Glucosidase Inhibitors α-Glucosidase inhibitors (AGI) as initial treatment for patients with Type 2 DiabetesCause no hypoglycemic eventsCause no weight gainPotential to be used as anti-obesity agents54
55 Types of Inhibition Mixed-type Competitive Non-competitive plus mixed-type inhibitor1/VmaxappMixed-typeCompetitiveComp inhi. Type of inhibition in which Km of the enzyme is not affected and inhibitor binds to active site.Mixed type Type of inhibition in which inhibitor acts on both, Km and Vmax and inhibitor can bind to active site or any other site of enzyme.Non comp Type of inhibition in which Vmax of the enzyme and inhibitor remains same and inhibitor binds to any other site, blocking the active site to perform action.Un-comp Type of inhibition which does not allow product or substrate to leave active site as enzyme-substrate complex is inhibited. Kmapp may be lowered or increased relative to Km and Vmaxapp of enzyme is lowered.Non-competitiveUncompetitive
56 + Non-cytotoxic against 3T3 cell line A AcarboseIC50±SEM = 906±6.3 µM+ Non-cytotoxic against 3T3 cell lineASugar protonsDMSOCH3H2OProtein irradiation pointBThe diagrammatic representations of the hydrogen bonds (dashed lines) and hydrophobic interactions (dashed-lined semicircles) formed by MGAM-C with inhibitor acarbose. NE2 of His1584 and OD2 of Asp1279 form hydrogen bonds with chemical groups OH Y=tyrosin w=trp F=phe R=Arg P=prolineSugar protons(Ren et al., 2011)DMSOH2O
59 Competitive Inhibition 224ADMSOH-4',5'I Impurities------IH-1H2OH-3'Protein irradiation pointBCOMPOUND 224 HAD A FURAN RING INSTEAD OF BENZENE RING. THIS STRUCTURE HAD SHOWN RESULTS COMPARABLE WITH 205 I.E. THE FURAN IS LESS INTERACTIVE SINCE THE H-1', 3' AND H-4', 5' HAS REDUCED PEAK INTENSITY.DMSOH-1Competitive InhibitionH-4',5'I Impurities------IH2OH-3'
60 Mixed-type Inhibition 201Mixed-type InhibitionH-2',6'H-3',5'H-1H2ODMSOAProtein irradiation pointBMIXED TYPE OF INHIBITION IS REFERRED AS INTERACTION OF LIGAND WITH CATALYTIC POCKET AS WELL AS THE PROTEIN SURFACE. COMPOUND 201 AND 205 SHOWED THE SIMILAR PATTERN. H-1 PROTON, WHICH WAS SEEN INTERACTIVE IN ALL THE OTHER COMPOUNDS OF THIS SERIES, DEMONSTRATED LESSER (201) AND NO (205) INTERACTION WITH THE PROTEIN. IT CAN ALSO BE SEEN THAT BOTH THESE COMPOUNDS HAVE HYDROXYL GROUP ON BENZENE RING LIKE 199, WHICH MAY BE INTERACTING WITH THE WATER RESIDUES IN THE ACTIVE SITE TO STABILIZE THE LIGAND. HYDROGEN BONDING IS ALSO POSSIBLE WITH AVAILABLE HYDROPHOBIC OR HYDROPHILIC PARTNER IN THE ACTIVE POCKET. IT IS ALSO POSSIBLE THE CH OF TYROSINE PRESENT IN ACTIVE POCKET AS HYDROPHOBIC AMINO ACID, CH-Π INTERACTION IS TAKING PLACE BETWEEN THE BENZENE AND TYROSINE.H-3',5'DMSOH-2',6'H-1H2O
62 Mixed-type Inhibition 175H-4Mixed-type InhibitionAH-6''DMSOH-2',6'H-4''H2OH-3',5'Protein irradiation pointBIT WAS OBSERVED THAT H-2', 6' AND H-3', 5' ARE INTERACTIVE PROTON PEAKS. FROM THIS OBSERVATION, WE CAN SAY THAT BENZENE RING NEAR TO CARBONYL END IS MORE INTERACTIVE AND IS MORE IMPORTANT FOR RENDERING THE ACTIVITY. IT IS ALSO OBSERVED THAT H-4'' AND H-6'' IS ALSO SHOWING WEAK INTERACTIONS. THUS WE CAN ASSUME THAT THE PART OF COMPOUND WHICH IS RESPONSIBLE FOR ACTIVITY IS PROBABLY THE BENZENE PARTDMSOH-4H-2',6'H-3',5'H2O
63 Non-Competitive Inhibition 176AH-3',5'H-6''CH3H2ODMSOH-2',6'CH2H-3'',4''Protein irradiation pointBTHE INTERACTIVE PARTS OF THE LIGAND 176 178 WERE APPARENTLY FOUND TO BE BOTH THE BENZENE RINGS OF 176 178. FROM THIS REPEATED PATTERN IN 176 178, WE CAN ASSUME THAT PROBABLY THE PROTONS OF BENZENE RINGS ARE ACTING LIKE INTERACTIVE ANCHORS TO STABILIZE THE COMPOUND ON PROTEIN SURFACE TO GIVE THE ACTIVITY.Non-Competitive InhibitionH-6''H-3',5'DMSOH2O
64 No interaction or no inhibition 184 – Non-inhibitorADMSOH2OBNo STD Signals were observedProtein irradiation pointNo interaction or no inhibitionDMSOSolubilityHigh/Low affinityH2O
66 Mixed-type Inhibition 259DMSOAH-2',6'H-2''H-4Mixed-type InhibitionH-6''H-5''H-3', 5'H2OBB. COMPOUND WAS FOUND TO MIXED TYPE OF INHIBITOR, INTERFERING TO THE FACT THAT IT HAS AN INTERACTION CAPABILITY WITH BOTH, CATALYTIC SITE AS WELL AS ENZYME SURFACE. TO IDENTIFY THE MOLECULE(S) RESPONSIBLE FOR INTERACTION, STD NMR SPECTROSCOPY WAS CARRIED OUT. IT WAS OBSERVED THAT PROTONS OF HETEROCYCLIC RING HAVING N AS ITS COMPONENT, WERE INTERACTING SINCE ALL THE PEAKS APPEARED IN THE DIFFERENCE SPECTRA. MOREOVER, PROTONS AROUND THE HYDROXYL FUNCTIONALITY WERE ALSO INTERACTING AS SEEN THROUGHOUT THE STUDY. THIS MAY SUGGEST THAT THE OH WHICH IS CAPABLE OF HYDROPHOBIC AS WELL AS HYDROPHILIC INTERACTION IS PLAYING ROLE IN CONTACT. THE CATALYTIC RESIDUES ASP AND GLU CAN BE STABILIZED BY SUCH OH GROUPS, MAKING THEM NON-INTERACTIVE WITH THE SUBSTRATE. THIS CAN BE THE POSSIBLE MECHANISM BY WHICH THIS PARTICULAR INHIBITOR IS ACTING.Protein irradiation pointDMSOH-3', 5'H-2''H-2',6'H-5''H2O
67 On Going Projects YP_040532.1 Thioredoxin protein YP_ Hypothetical phage proteinYP_ Putative glycine cleavage H proteinYP_ Putative membrane proteinYP_ Putative membrane protein