Clemson CI Symposium - Feb 13, 2013 Establishing a NMR Structural Biology Center at Claflin University. Raj Bhaskaran Department of Chemistry Claflin University, Orangeburg, SC-29115

NMR in CI Symposium NMR in CI Symposium Clemson CI Symposium - Feb 13, 2013 Aim of this talk is two fold: To attract the Cyber Specialists to join hands with Claflin NMR team to mutually collaborate in the areas that require enormous computing and CI efforts; To attract Life Science researchers to interact with the NMR team to explore their problems structurally to provide a functional meaning;... to initiate GEAR CI collaborations.... to initiate GEAR CI collaborations.

Claflin’s NMR Route to Structural Biology and Drug Discovery Research Clemson CI Symposium - Feb 13, 2013 Claflin has the high field 700 MHz NMR Spectrometer- BioNMR/ Metabolomics NSF Major Research Instrumentation grant is applied for a Cryoprobe and Sample Changer Solution Structure Determination and Protein Dynamics- Functional Interpretation based on structural investigations Protein (Enzyme) - Small Molecule (Substrate / Inhibitor) Interactions Protein Structures Derived Functional Interpretation on t. Thermophilus proteome, from Riken, Japan and Filariasis Nematode, Brugia Malayi proteome, (NIH, UIC) Rapid Protein Structure Determination for Structural Genomics Consortium: Structural Bioinformatics (North East Structural Genomics Consortium; Seattle Structural Genomics Center for Infectious Diseases) Computer Aided Drug Discovery: Virtual screening, Data Mining, QSAR Structure Based Drug Design: SAR by NMR, NMR Screening

Structure Based Drug Design Suitable protein target Structure of the target protein Implementation of an easy and reliable HTS assay Identification of a lead compound Computer assisted methods for estimating the affinity of new compounds Access to a synthetic route to produce designed compounds. Knowledge on shape of the pocket to design the shape of the drug. The use of X-ray / NMR structures of Protein-Drug Complexes to design better-fitting, and hence more potent inhibitors Clemson CI Symposium - Feb 13, 2013 New Compound Suggests New Interaction Drug-Protein Complex Biological Activity

Ligands in the Drug Design Process Clemson CI Symposium - Feb 13, 2013 compound libraries natural products directed libraries Identified & Validated Target Protein Production Assays /HTS Hits Chemical tractability Toxicity DrugMetabolism / PharmacoKinetics (genomics, transgenics etc) more structure Structure Ligand-Protein structure Optimised Ligand more chemistry low affinity, high K d high affinity, low K d Free state

Extensive computations involved in Protein NMR Study Clemson CI Symposium - Feb 13, 2013 Isotopically 15 N, 13 C, Labeled NMR sample Acquisition of 3D heteronuclear (H, 15 N, 13 C) NMR experiments Backbone (HN, 15 N, 13 C , H  ) and Side Chain ( 13 C , H  13 C , H , …..) resonance assignment Unambiguous assignment of Nuclear Overhauser Enhancement ( proton-proton interaction within 5 A 0 radius) Structure Determination using Restrained Molecular Dynamics and Simulated Annealing Protocol Structural Refinement using additional experimental restraints Protein Dynamics from 15 N, 13 C  Relaxation experiments

HUMAN ( E219A) MMP-12: 1 H- 15 N HSQC SPECTRUM / NOE CONNECTIVITIES / CSI Clemson CI Symposium - Feb 13, 2013 Backbone NHSQC; CHSQC; TROSY HNCOCA; HNCA HACACONH; HNNCAHA CBCACONH; HNCACB HNCO; HCACOCANH Side chain HBCBCGCD(CE)HD(HE) CCH TOCSY HCCCONH; CCONH HCCH COSY; HCCH TOCSY HCCH TOCSY (aro) NOE N15 EDITED 3D NOESY C13 EDITED 3D NOESY NoesyChsqc (aro) VNMR TOPSPIN NMRPIPE SPARKY AUTO ASSIGN PYMOL / MOLMOL SHIFTX CSI TALOS-PLUS CYANA XPLOR-NIH HADDOCK MODELFREE PROCHECK/PSVS SOFTWARE SOFTWARE NMR EXPTS NMR EXPTS Bhaskaran & VanDoren JBNMR (2006)

First NMR structure of the MMP-12 catalytic domain sans inhibitor Clemson CI Symposium - Feb 13, 2013 Bhaskaran et al. J Mol Biol (2007) 29 unique NOEs ( dotted lines) account for the conformational adjustment active site helix B &  -strands I, III are closer in free state NMR – Green; Inhibitors pull  B away from the far side X-RAY – Red; RCSB PDB code 2POJ 2803 NOEs Used BB RMSD 0.32 A SC RMSD 0.68 A Ramachandran plot statistics: 77.3% in most favored regions 21.5% in allowed regions 0.0% in disallowed regions

Protein Dynamics - rigidity of catalytic domain affect activity upon elastin &  1-AT. High Rigidity Limited Conformational Flexibility Clemson CI Symposium - Feb 13, 2013 MMP-12MMP-3 ps-ns β1 β2 β3 β4 β5 S225 L229 D158 F157 L2/3L3/4 MMP-12 is more rigid than MMP-3. Apparent Trade-off of Activity and Rigidity in MMP12 for Stability and Flexibility in MMP3 Liang, Bhaskaran et al Biophysical Jl. (2010)

Family of NMR structures of STT3P C-ter Domain -the Catalytic Subunit of Oligo Sachcharyl Transferase Ave BB rmsd to mean: nm Ave heavy atom rmsd to mean : nm Clemson CI Symposium - Feb 13, 2013 Huang, Bhaskaran, et al Jl.Biol.Chem. (2012) RCSB PDB code “2lgz” It is so far the biggest monomeric helical integral membrane protein structure by NMR / Ost1p binding site Membrane embedding site Catalytic center “WWDYG”motif “DK”motif CW 110 deg

Structural Gallery - Proteins COBROTOXIN (1COD) CARDIOTOXIN III (2CRS) TMH0916 (1NR3) GIP / L-GLUT COMPLEX (2L4T) GIP (2L4S) MMP-12 (2POJ) STT3P (2LGZ) CARDIOTOXIN II (1CRE) Clemson CI Symposium - Feb 13, 2013

PERTURBATION OF MMP12 BY Triple Helical Peptide AND THE MAPPING OF THEM ON THE STRUCTURE OF MMP12. Clemson CI Symposium - Feb 13, 2013 Bhaskaran et al. J Biol Chem.(2008) Binding Isotherm

Clemson CI Symposium - Feb 13, 2013 Protein:Ligand Interface Mapping by NMR The NMR paramagnetic surface protection assay correctly predicts the sites of interaction as confirmed by the loss of function mutation studies Palmier, Bhaskaran et al Jl Biol Chem. (2010)

EVOLUTIONARY TRACE ANALYSIS Evolutionary Trace Method uses a sequence similarity tree of a family of homologous proteins to highlight residues that are statistically under evolutionary pressure and therefore possess certain functional or structural importance for the family Clemson CI Symposium - Feb 13, 2013

BINDSIght Method - maps of binding sites and distinctive sequence to suggest residues tuning specificity Palmier, Bhaskaran et al. J. Biol. Chem. (2010) Clemson CI Symposium - Feb 13, 2013

CI: Scope and Problems for Interaction CI: Scope and Problems for Interaction Clemson CI Symposium - Feb 13, 2013 Pulse program conversion - Varian and Bruker Consolidation of SW packages on NMR structure determination, dynamics and ligand interactions Develop multiuser remote instrumentation to convert into a state level facility A Virtual Web Based File system for managing NMR Data Rapid structure determination package for the structural genomics proteins

BioNMR – External Collaborations BioNMR – External Collaborations Clemson CI Symposium - Feb 13, 2013 Dr. Steven VanDoren, University of Missouri, Columbia, MO : Structural Interactions of Tumor Viral Proteins, E7 and E2F Dr. K.Ramaswamy, UIC, Rockford, IL : Structural Studies of Filarial Proteins Bm-HSP 12.6 Dr. M.Gnanasekar, UIC, Rockford, IL : Structural Studies of Filarial Proteins Bm-TCTP Dr. Peter Myler, Seattle Biomed, Seattle, WA : Structural Genomics of Proteins from Infectious Diseases Dr. Krishna Sharma, University of Missouri, Columbia, MO : Structures of  -Crystallin & Chaperonins of Eye Disease, Cataract Dr. Raghu Kannan, University of Missouri, Columbia, MO : NMR of Gold Nanoparticle Conjugated Bombesin Dr. T. K. S. Kumar, University of Arkansas, Fayetteville, AR : Structural Studies of Fibroblast Growth factor, FGF2 Dr. K. Gunasekaran, University of Madras, Chennai, India : Structural Studies of Filarial Proteins Bm-API Dr. D. Velmurugan, University of Madras, Chennai, India : Structural Genomics of t. thermophilus Proteins Dr. P. Karthe, University of Madras, Chennai, India : NMR structure of Serine glutamate repeat A, a surface adhesin

GEAR- CI: SC-Collaborations GEAR- CI: SC-Collaborations Clemson CI Symposium - Feb 13, 2013 Dr. John Dawson, University of South Carolina : Identification of Dehaloperoxidase-Substrate Binding Sites Dr. Caryn Outten, University of South Carolina : Structural Studies of iron sensing proteins, Fra2 and Grx Dr. Homayoun Valafar, University of South Carolina : NMR Residual Dipolar Coupling analyses Dr. Nick Grossoheme, Winthrop University: Dr. Heather Evans Anderson, Winthrop University: NMR structure of ForkHead Transcription Factor Protein, FOXO1 Dr. Sondra Berger, University of South Carolina : Dr. Angela Peters, Claflin University : Thymidalate Synthase Mutant (R163K)-Ligand Interactions Dr. Michael Sehorn, Clemson University : Dr. Erin Eaton, Francis Marion University : Dr. Karen Buchmuller, Furman University : Dr. Marcello Forconi, College of Charleston : Dr. Esmaeil Jabbari, University of South Carolina : (CRP) NMR Structural Characterization of vasculogenic peptides Dr. Scott Argraves, University of South Carolina : (CRP) NMR Studies of Fibulins, the extracellular matrix proteins

ACKNOWLEDGEMENT ACKNOWLEDGEMENT Clemson CI Symposium - Feb 13, 2013 SC INBRE NASA EPSCoR Space Grant. Mr. Raghav Nagampalli (Fulbright Scholar) Dr. Angela Peters (Claflin) Dr. Verlie Tisdale (Claflin) School of Natural Sciences and Mathematics, Claflin TRUSTWORTHINESS RESPECT RESPONSIBILITY FAIRNESS CITIZENSHIP CARING

THANK YOU ALL Clemson CI Symposium - Feb 13, 2013