1. Applications of Homology Modeling Hanka Venselaar

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

3. MSQSTQTNEFLSPEVFQHIWDFLEQPICSVQPIDLNFVDEPSEDGATNKI EISMDCIRMQDSDLSDMWPQYTNLGLLNSMDQQIQNGSSSTSPYNTDHAQ NSVTAPSPYAQPSSTFDALSPSPAIPSNTDYPGPHSFDVSFQQSSTAKSA TWTYSTELKKLYCQIAKTCPIQIKVMTPPPQGAVIRAMPVYKKAEHVTEV VKRCPNHELSREFNEGQIAPPSHLIRVEGNSHAQYVEDPITGRQSVLVPY EPPQVGTEFTTVLYNFMCNSSCVGGMNRRPILIIVTLETRDGQVLGRRCF EARICACPGRDRKADEDSIRKQQVSDSTKNGDGTKRPFRQNTHGIQMTSI KKRRSPDDELLYLPVRGRETYEMLLKIKESLELMQYLPQHTIETYRQQQQ QQHQHLLQKQTSIQSPSSYGNSSPPLNKMNSMNKLPSVSQLINPQQRNAL TPTTIPDGMGANIPMMGTHMPMAGDMNGLSPTQALPPPLSMPSTSHCTPP PPYPTDCSIVSFLARLGCSSCLDYFTTQGLTTIYQIEHYSMDDLASLKIP EQFRHAIWKGILDHRQLHEFSSPSHLLRTPSSASTVSVGSSETRGERVID AVRFTLRQTISFPPRDEWNDFNFDMDARRNKQQRIKEEGE Sequence: EEC syndrome No structure: EEC syndrome

4. 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 Use one structure to predict another

5. Homology modeling Example: by 80 residues  30% identity sufficient # residues % identity * * Actually, modelling is possible, but we cannot get an alignment… O

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

7. The 8 steps of Homology modeling

8. 1: Template recognition and initial alignment

9. BLAST your sequence against PDB Best hit  normally template Initial alignment  NSDSECPLSHDGYCLHDGVC || || | ||||| ||| NSYPGCPSSYDGYCLNGGVC

10. 1: Template recognition and initial alignment 2: Alignment correction

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

12. 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

13. 1: Template recognition and initial alignment 2: Alignment correction 3: Backbone generation

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

15. 1: Template recognition and initial alignment 2: Alignment correction 3: Backbone generation 4: Loop modeling

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

17. 1: Template recognition and initial alignment 2: Alignment correction 3: Backbone generation 4: Loop modeling 5: Sidechain modeling

18. 5: Side-chain modeling Several options Libraries of preferred rotamers based upon backbone conformation

19. 1: Template recognition and initial alignment 2: Alignment correction 3: Backbone generation 4: Loop modeling 5: Sidechain modeling 6: Model optimization

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

21. 1: Template recognition and initial alignment 2: Alignment correction 3: Backbone generation 4: Loop modeling 5: Sidechain modeling 6: Model optimization 7: Model validation

22. 7: Model Validation Second opinion by PDBreport /WHATIF Errors in active site?  new alignment/ template No errors?  Model!

23. 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

24. 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

25. 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

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

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

28. Another real world example: Mutation analysis HFE

29. HFE – complex: HFE β2-microglobulin  Facilitates trafficking of HFE to the cellmembrane Transferrin receptor (dimer)  binds iron/transferrin complex -Signaling and regulation of iron in bloodstream. -Expressed in liver and colon. -Mutations cause iron deposition disease “Hereditary Hemachromatosis“

30. Hereditary Hemachromatosis  3 occuring mutations C280Y D41H L161P C280Y D41H L161P

31. Mutation C260Y Loss of cystein bridge Disturbing of β2-microglobulin binding domain No trafficking to membrane

32. Mutation H41D Introduction of additional negative charge Disturbing of hydrogen bridges Loss of stability in this area

33. Mutation L161P Loss hydrophobic interactions Major disturbance of the helix Less interaction of the helix with the transferrin receptor

34. Seriousness of mutation D41H L161P C260Y Seriousness of the disease D41H L161P C260Y  Conclusion: the seriousness of the mutation is related to the seriousness of the disease and can be explained by analyzing the mutations with the 3D structure.

35. 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 Real world examples: mutations in EEC syndrome and HFE can be explained To conclude….