1. Applications of Homology Modeling Hanka Venselaar

2. This seminar…. Homology Modeling… Why? What? When? How? And a few real world examples….

3. Hearing loss No structure: MGTPWRKRKGIAGPGLPDLSCALVLQPRAQVGTMSPAI ALAFLPLVVTLLVRYRHYFRLLVRTVLLRSLRDCLSGLRI EERAFSYVLTHALPGDPGHILTTLDHWSSRCEYLSHMG PVKGQILMRLVEEKAPACVLELGTYCGYSTLLIARALPP GGRLLTVERDPRTAAVAEKLIRLAGFDEHMVELIVGSSE DVIPCLRTQYQLSRADLVLLAHRPRCYLRDLQLLEAHAL LPAGATVLADHVLFPGAPRFLQYAKSCGRYRCRLHHTG LPDFPAIKDGIAQLTYAGPG DFNB 63 Sequence:

4. KKIALSDARSMKHALREIKIIRRL DHDNIVKVYEVLGPKGTDLQGELF KFSVAYIVQEYMETDLARLLEQGT LAEEHAKLFMYQLLRGLKYIHSAN VLHRDLPANIFISTEDLVLKIGDF GLARIVDQHYSHKGYLSEGLVTKW YRSPRLLLSPNNYTKAIDMWAAGC ILAEMLTGRMLFAGAHELEQMQLL ETIPVIREEDKDELLRVMPSFVSS ? Why homology modeling? Lab Translation Bioinformatics ATOM 1 N GLN A 117 -42.882 10.838 12.153 1.00 58.09 N ATOM 2 CA GLN A 117 -42.770 10.783 10.668 1.00 58.36 C ATOM 3 C GLN A 117 -41.435 11.371 10.185 1.00 57.07 C ATOM 4 O GLN A 117 -41.264 12.582 10.210 1.00 57.81 O ATOM 5 CB GLN A 117 -43.966 11.532 10.028 1.00 59.40 C ATOM 6 CG GLN A 117 -45.344 10.768 10.084 1.00 62.58 C ATOM 7 CD GLN A 117 -45.254 9.261 9.651 1.00 67.37 C ATOM 8 OE1 GLN A 117 -44.260 8.554 9.948 1.00 68.20 O ATOM 9 NE2 GLN A 117 -46.304 8.778 8.955 1.00 67.47 N ATOM 10 N SER A 118 -40.488 10.545 9.741 1.00 54.71 N ATOM 11 CA SER A 118 -39.144 11.089 9.506 1.00 52.44 C ATOM 12 C SER A 118 -38.389 10.616 8.251 1.00 50.58 C ATOM 13 O SER A 118 -38.692 9.566 7.734 1.00 50.83 O ATOM 14 CB SER A 118 -38.317 10.815 10.736 1.00 52.75 C ATOM 15 OG SER A 118 -38.273 9.437 10.917 1.00 53.04 O ATOM 16 N CYS A 119 -37.428 11.398 7.755 1.00 48.00 N ATOM 17 CA CYS A 119 -36.748 11.070 6.507 1.00 46.41 C ATOM 18 C CYS A 119 -35.339 10.829 6.835 1.00 45.44 C ATOM 19 O CYS A 119 -34.845 11.360 7.805 1.00 45.36 O ATOM 20 CB CYS A 119 -36.721 12.232 5.504 1.00 45.97 C ATOM 21 SG CYS A 119 -38.275 12.940 5.114 1.00 47.29 S ATOM 22 N LEU A 120 -34.657 10.098 5.972 1.00 44.91 N 4

5. Protein structures – 4 levels Primary Secondar y Tertiary Quaternary  Shape of the protein determines its function…..

6. Protein structures…where can we find them? Protein DataBank = www.pdb.orgwww.pdb.org

7. PDB-file: contains the coördinaties for every atom in a protein Visualisation with PDB-viewers -Jmol -PyMol -SwissPDB viewer -YASARA

8. So, 3D Protein-structures provide useful information But…… Not enough protein structures in the PDB database

9. Predictions/Annotations

10. Homology modeling in short… Prediction of structure based upon a highly similar structure 2 basic assumptions: Structure defines function During evolution structures are more conserved than sequence 2 basic assumptions: Structure defines function During evolution structures are more conserved than sequence Use one structure to predict another

11. Homology modeling – When? Example: by 80 residues  30% identity sufficient O

12. Homology modeling in short… Prediction of structure based upon a highly similar structure Add sidechains, Molecular Dynamics simulation on model Unknown structure NSDSECPLSHDG || || | || NSYPGCPSSYDG NSDSECPLSHDG || || | || NSYPGCPSSYDG Model sequence Known structure Back bone copied Copy backbone and conserved residues Model!

13. The 8 steps of Homology modeling

14. 1: Template recognition and initial alignment

15. BLAST your sequence against PDB Initial alignment Best hit is usually your template

16. 1: Template recognition and initial alignment 2: Alignment correction

17. Functional residues  conserved Use multiple sequence alignments Deletions  shift gaps CPISRTGASIFRCW CPISRTA---FRCW CPISRT---AFRCW CPISRTGASIFRCW CPISRTA---FRCW CPISRT---AFRCW CPISRTAAS-FRCW CPISRTG-SMFRCW CPISRTA--TFRCW CPISRTAASHFRCW CPISRTGASIFRCW CPISRTA---FRCW CPISRTAAS-FRCW CPISRTG-SMFRCW CPISRTA--TFRCW CPISRTAASHFRCW CPISRTGASIFRCW CPISRTA---FRCW Both are possible Multipe sequence alignment Correct alignment  Sequence with known structure  Your sequence

18. 2: Alignment correction Core residues  conserved Use multiple sequence alignments Deletions in your sequence  shift gaps Known structure FDICRLPGSAEAV Model FNVCRMP---EAI Model FNVCR---MPEAI S G P L A E R C IV C R M P E V C R M P E  Correct alignment F-D- -A-V

19. 1: Template recognition and initial alignment 2: Alignment correction 3: Backbone generation

20. Making the model…. Copy backbone of template to model Make deletions as discussed (Keep conserved residues)

21. 1: Template recognition and initial alignment 2: Alignment correction 3: Backbone generation 4: Loop modeling

22. Known structure GVCMYIEA---LDKYACNC Your sequence GECFMVKDLSNPSRYLCKC Loop library, try different options

23. 1: Template recognition and initial alignment 2: Alignment correction 3: Backbone generation 4: Loop modeling 5: Sidechain modeling

24. 5: Side-chain modeling Several options Libraries of preferred rotamers based upon backbone conformation

25. 1: Template recognition and initial alignment 2: Alignment correction 3: Backbone generation 4: Loop modeling 5: Sidechain modeling 6: Model optimization

26. Molecular dynamics simulation Remove big errors Structure moves to lowest energy conformation

27. 1: Template recognition and initial alignment 2: Alignment correction 3: Backbone generation 4: Loop modeling 5: Sidechain modeling 6: Model optimization 7: Model validation

28. 7: Model Validation Second opinion by PDBreport /WHATIF Errors in active site?  new alignment/ template No errors?  Model!

29. 1: Template recognition and initial alignment 2: Alignment correction 3: Backbone generation 4: Loop modeling 5: Sidechain modeling 6: Model optimization 7: Model validation 8: Iteration

30. Model! 1: Template recognition and initial alignment 2: Alignment correction 3: Backbone generation 4: Loop modeling 5: Sidechain modeling 6: Model optimization 7: Model validation 8: Iteration

31. 8 steps of homology modeling 1: Template recognition and initial alignment 2: Alignment correction 3: Backbone generation 4: Loop modeling 5: Side-chain modeling 6: Model optimization 7: Model validation 8: Iteration Alignment Modeling Correction

32. Hearing loss Structure! MGTPWRKRKGIAGPGLPDLSCALVLQPRAQVGTMSPAI ALAFLPLVVTLLVRYRHYFRLLVRTVLLRSLRDCLSGLRI EERAFSYVLTHALPGDPGHILTTLDHWSSRCEYLSHMG PVKGQILMRLVEEKAPACVLELGTYCGYSTLLIARALPP GGRLLTVERDPRTAAVAEKLIRLAGFDEHMVELIVGSSE DVIPCLRTQYQLSRADLVLLAHRPRCYLRDLQLLEAHAL LPAGATVLADHVLFPGAPRFLQYAKSCGRYRCRLHHTG LPDFPAIKDGIAQLTYAGPG DFNB 63 Sequence:

33. Mutation: Tryptophan 105 -> Arginine Hydrophobic contacts from the Tryoptohan are lost, introduction of an hydrophilic and charged residue

34. The three mutated residues are all important for the correct positioning of Tyrosine 111 Tyrosine 111 is important for substrate binding Published in Nature Genetics: 2008 Oct 26.

35. Voorbeeld: C-terminale deletie van 10 aa in Dectine Afdeling: Interne geneeskunde of Internal Medicine >Dectin_1_Isoform_a MEYHPDLENLDEDGYTQLHFDSQSNTRIAVVS EKGSCAASPPWRLIAVILGILCLVILVIAVVL GTMAIWRSNSGSNTLENGYFLSRNKENHSQPT QSSLEDSVTPTKAVKTTGVLSSPCPPNWIIYE KSCYLFSMSLNSWDGSKRQCWQLGSNLLKIDS SNELGFIVKQVSSQPDNSFWIGLSRPQTEVPW LWEDGSTFSSNLFQIRTTATQENPSPNCVWIH VSVIYDQLCSVPSYSICEKKFSM

36. MSQSTQTNEFLSPEVFQHIWDFLEQPICSVQPIDLNFVDEPSEDGATNKI EISMDCIRMQDSDLSDMWPQYTNLGLLNSMDQQIQNGSSSTSPYNTDHAQ NSVTAPSPYAQPSSTFDALSPSPAIPSNTDYPGPHSFDVSFQQSSTAKSA TWTYSTELKKLYCQIAKTCPIQIKVMTPPPQGAVIRAMPVYKKAEHVTEV VKRCPNHELSREFNEGQIAPPSHLIRVEGNSHAQYVEDPITGRQSVLVPY EPPQVGTEFTTVLYNFMCNSSCVGGMNRRPILIIVTLETRDGQVLGRRCF EARICACPGRDRKADEDSIRKQQVSDSTKNGDGTKRPFRQNTHGIQMTSI KKRRSPDDELLYLPVRGRETYEMLLKIKESLELMQYLPQHTIETYRQQQQ QQHQHLLQKQTSIQSPSSYGNSSPPLNKMNSMNKLPSVSQLINPQQRNAL TPTTIPDGMGANIPMMGTHMPMAGDMNGLSPTQALPPPLSMPSTSHCTPP PPYPTDCSIVSFLARLGCSSCLDYFTTQGLTTIYQIEHYSMDDLASLKIP EQFRHAIWKGILDHRQLHEFSSPSHLLRTPSSASTVSVGSSETRGERVID AVRFTLRQTISFPPRDEWNDFNFDMDARRNKQQRIKEEGE P63 sequence Structure! EEC syndrome

37. Arginine Serine Mutation R  S Loss of negative charge Loss of interaction with the DNA

38. Homology Modeling… What? Prediction of an unknown structure based on an homologous and known structure Why? To answer biological and medical questions when the “real” structure is unknown When? A template with enough identity must be available How? 8 Steps  Use the models for mutant analysis, experimental design and understanding of the protein in general To conclude….