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Structure Prediction of human Histamine 4 Receptor (hH4R)

Structure Prediction of human Histamine 4 Receptor (hH4R). Charlie Seto, Bioinformatics Summer Institute, CSULA Ravi Abrol & Soo-Kyung Kim, MSC, CalTech William Goddard, MSC, CalTech. Outline. Why Study GPCRs? Objective H4R Structure Prediction & Building Ligand docking Future Work.

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Structure Prediction of human Histamine 4 Receptor (hH4R)

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  1. Structure Prediction of human Histamine 4 Receptor (hH4R) Charlie Seto, Bioinformatics Summer Institute, CSULA Ravi Abrol & Soo-Kyung Kim, MSC, CalTech William Goddard, MSC, CalTech

  2. Outline • Why Study GPCRs? • Objective • H4R • Structure Prediction & Building • Ligand docking • Future Work

  3. “Core of Modern Medicine” • GPCRs have cell signaling functions • 40% of new drugs target GPCRs • Novartis: Zelnorm (5-HT4) • Eli Lilly: Zyprexa (5-HT2) • Schering-Plough: Clarinex (H1) • GlaxoSmithKline: Zantac (H2) (Source: Modern Drug Discovery, “It’s a GPCR World”, Nov 2004)

  4. H4R (Histamine H4 Receptor) • All H4 functions still not known • Together with H1, asthma? • H4 is unique • Dissimilar to H1, H2, H3 • Known Histamine receptor ligands weak against H4R. • New ligands needed to optimize drugs! • Or, to increase specificity Histamine

  5. Objective of your Project • Predict 3D structure of Histamine H4 Receptor (H4R) from AA sequence • Validate structure with known ligands, Histamine etc. • Good structure for drug design • Other questions • Difference of H4R vs. other Histamine receptors?

  6. TM Prediction • Perform BLAST with H4R sequence • “Broad search”, E-value = 0.1 • Run multiple-sequence alignment • Develop hydrophobicity profile by averaging BLASTed sub-profiles • Prediction of TM regions based on hydrophobicity • Get hydrophobic center, controls TM “depth”

  7. Hydrophobicity Profile Large hydrophilic domain implies cytosolic loop TM2 poorly defined

  8. Other PredictM output • PredicTM’s BLAST output • Nearest relative was H4R in Mouse & Rat • ~79-80% identity (#1 & #2) • Histamine 3-R, paralog • ~55% identity, #6 • Human β-2 Adrenergic (template) • TM ~33% identity, #179 • Bovine Rhodopsin (template) • ~22% identity, #1249

  9. “Final” TM Sequence • TM 1: 15-RVTLAFFMSLVAFAIMLGNALVILA-39 • TM 2: 51-SYFFLNLAISDFFVGVISIPLYIPHTLFEWDFGK-85 • TM 3: 88-VFWLTTDYLLCTASVYNIVLISY-111 • TM 4: 131-VLKIVTLMVAVWVLAFLVNGPMILV-155 • TM 5: 169-EWYILAITSFLEFVIPVILVAYFNMNIYWS-203 • TM 6: 303-KSLAILLGVFAVCWAPYSLF-327 • TM 7: 336-KSVWYRIAFWLQWFNSFVN-359

  10. Helix Kinking • Have: TM sequences • Need: 3D structure • First: Predict helix kinking • Helix kinking caused by Pro & Gly residues, stabilized by Ser & Thr Pro71 Right: TM2, minrmsd method. Pro75

  11. Helix Building 0° 15° • Compare helices to template (human β2 ADR) • Template determines angle of insertion into membrane, orientation of TMs 30°

  12. Rotation • Bihelix Pairing: Adjacent helices paired off, rotated (12 combinations) • Bihelix data aggregated to build 7-TM structures

  13. Visual Analysis • Visually check H-bonds • Check for GPCR motifs • NPxxY • 1-2-7 networks, etc • Structure Activity Data • Example: Mutate Asp94…no activity, therefore…Asp94 important! (Assess angle and coordinate)

  14. Asp94 (3.32) Asn147 (4.57) Theorized imidazole binding pocket Glu182 (5.46)

  15. Ligands • “Build” in Maestro • Special pre-reqs for selecting ligands • Need experimental data for binding • Big Picture: Ligands are to validate structure! Imetit, agonist

  16. Current Progress • Analysis continues • Missing expected interhelical interactions • Causes being investigated • A highly polar motif on TM2 may have shifted the structure • Not found on other Histamines, or templates • Will continue with pre-docking • Assess ligand affinity data after docking • Determine if structure “good/bad” by comparison to experimental data

  17. Special Thanks • Mentor: Ravinder Abrol & Soo-Kyung Kim, MSC, Caltech • PI: William Goddard, MSC, Caltech • Other members of Biogroup, MSC • The SoCalBSI faculty team • Dr. Sandra Sharp • Dr. Wendie Johnston • Dr. Jamil Momand • Dr. Nancy Warter-Perez • …and others • Funding provided by:

  18. References • D. FILMORE. “It’s a GPCR World” Modern Drug Discovery Nov 2004 • N. SHIN, E. COATES, N. J. MURGOLO, K. L. MORSE, M. BAYNE, C. D. STRADER, and F. J. MONSMA, JR. “Molecular Modeling and Site-Specific Mutagenesis of the Histamine-Binding Site of the Histamine H4 Receptor” Mol Pharmacol 62:38–47, 2002

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