Watoc luca-de-vico-21-july-2011


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Watoc luca-de-vico-21-july-2011

  1. 1. A Computational Enzyme Activity Design of HIV-1 Protease<br />Luca De Vico<br />WATOC 2011<br />Santiago de Compostela, Spain<br />
  2. 2. What the project is about<br />Modify a protease in order to cleave another desired sequence<br />New enzymatic activity towards a specific sequence<br />Rational redesign of the enzyme<br />Valuable tool for biological applications<br />Extended to any sequence<br />Experimental and computational collaboration<br />
  3. 3. Enzyme redesign: what is needed<br />- A target sequence to cleave<br />- A template protease: HIV-1 protease<br />✂<br />Pro<br />His<br />Leu<br />Ser<br />Phe<br />Met<br />Ala<br />Ile<br />Pro<br />Pro<br />
  4. 4. Enzyme redesign: what is needed<br /><ul><li> A computational tool capable of handling HIV-1 protease</li></ul>PyRosetta 1.1 based peptide-docking protocol(Chaudhury and Gray, Structure, 2009)<br />tailored for HIV-1 protease<br />discern cleavability<br />predict the specificity determining residues<br />
  5. 5. Enzyme redesign: what is needed<br />X-ray structure of wild type protease docked with one of its natural substrates: 1KJ7<br />The protocol has to produce:<br />- reasonable structures with many different substrate peptides bound<br />- generate mutants with lower binding energy<br />
  6. 6. Our PyRosetta optimization protocol<br />Substrate peptide sequence<br />Starting structure<br />x6<br />f, ψ, χ perturbations<br />Energy minimization<br />(David-Fletcher-Powell)<br />x8<br />x4<br />Out of 500 decoy structures the lower in energy is chosen.<br />x6<br />MC criterion<br />Side-chain packing<br />MC criterion<br />ca. 24 hours on 5 cpus<br />Output decoy<br />
  7. 7. The perturbation and minimization are on:<br />Backbone<br />Side chains<br />Substrate<br />peptide<br />Protease<br />cavity<br />Residues inside 5 Å radius from the peptide<br />Any residue reported as active in Chaudhury et al.<br />Their ±1 sequence neighbors <br />Both chains have the same residues as “movable”<br />
  8. 8. Refinement of the PyRosetta results<br /><ul><li>FMO MP2/6-31G*//PyRosetta + PCM single point energy calculations (GAMESS)
  9. 9. FMO inputs from FRAGIT
  10. 10. ca. 8 hours on 64 cpus per structure
  11. 11. Used to compute qualitative binding energies</li></ul>Ebinding = Ecomplex – Eapo – Epeptide<br />
  12. 12. Qualitative binding energies<br />FMO MP2/PCM/6-31G* (kcal/mol), Wild Type protease<br />Average binding energy <br />Natural Target Cleavable Peptides<br />-60.9<br />Min value<br />-79.2<br />Max value<br />-41.1<br />Standard deviation<br />11.0<br />Average binding energy <br />Known<br />NON-CleavablePeptides<br />-13.7<br />Min value<br />-68.3<br />Max value<br />61.0<br />Standard deviation<br />28.1<br />Target peptide sequence binding energy: -24.1<br />
  13. 13. Enzyme redesign: mutation protocol<br />Among the cleavable peptides sequences, the closest to the target is mutated into the target sequence, one amino acid at the time<br />Cleavable start<br />✂<br />✂<br />Val<br />Ser<br />Phe<br />Asn<br />Phe<br />Met<br />Ala<br />Ile<br />Leu<br />Thr<br />Pro<br />His<br />Leu<br />Ser<br />Phe<br />Pro<br />Gln<br />Ile<br />Pro<br />Pro<br />Target<br />
  14. 14. Enzyme redesign: mutation protocol<br /><ul><li>f, ψ, χ perturbations include all amino acid side chains in the Dunbrackrotamer library set
  15. 15. Only the protease 6 specificity determining residues are allowed to mutate (ca. 2000 total rotamers per perturbation)
  16. 16. ca. 40 hours on 5 cpus per cycle</li></li></ul><li>Suggested mutant sequences<br />FMO MP2/PCM/6-31G* binding energies (kcal/mol) with the target sequence<br />Homodimer<br />Heterodimer<br />Mutant 1<br />-17.7<br />Mutant 1<br />-6.5<br />Mutant 2<br />-29.6<br />Mutant 2<br />-12.9<br />Mutant 3<br />-13.6<br />Mutant 3<br />-7.6<br />Wild type protease binding energy: -24.1<br />
  17. 17. Outlook Acknowledgement<br /><ul><li>Transition state for the cleavage reaction, both in wild type and mutated protease
  18. 18. Evaluation of energy barrier for qualitative differences between wild type and mutated protease
  19. 19. Comparison with on-going experimental data
  20. 20. Dep. Chemistry: Jan Jensen, Casper Steinmann
  21. 21. Dep. Biology: JakobWinther, Martin Willemöes, Helen Webb
  22. 22. Funding provided by the Danish Research Council for Technology and Production Sciences (FTP)
  23. 23. Danish Center for Scientific Computing at Copenhagen University (DCSC.KU)</li></li></ul><li>References<br />PYROSETTA - Chaudhury S.; Gray, J. J. ‘Identification of Structural Mechanisms of HIV-1 Protease Specificity Using Computational Peptide Docking: Implications for Drug Resistance’, Structure (2009) 17, (12), 1636-1648<br />PYROSETTA - Chaudhury, S.; Lyskov, S.; Gray, J. J. ‘PyRosetta: a script-based interface for implementing molecular modeling algorithms using Rosetta’, Bioinformatics (2010) 26, 689-691<br />GAMESS - Schmidt, M. W.; Baldridge, K. K.; Boatz, J. A.; Elbert, S. T.; Gordon, M. S.; Jensen, J. H.; Koseki, S.; Matsunaga, N.; Nguyen, K. A.; Su, S.; Windus, T. L.; Dupuis, M.; Montgomery J. A. ‘General Atomic and Molecular Electronic Structure System’, J. Comput. Chem. (1993) 14, 1347-1363<br />FMO - Fedorov, D. G.; Kitaura, K. ‘Extending the Power of Quantum Chemistry to Large Systems with the Fragment Molecular Orbital Method’, J. Phys. Chem. A (2007) 11, 6904-6914<br />PCM – Tomasi, J.; Mennucci, B.; Cammi, R. ‘Quantum Mechanical Continuum Solvation Models’, Chem. Rev. (2005) 105, 2999-3093<br />FRAGIT – Steinmann, C.; Ibsen, : W.; Hansen, A. S.; Jensen, J. H. ‘FragIt: A tool to Prepare Proteins for Fragment Based Quantum Calculations’, In Preparation<br />
  24. 24. Extra<br />
  25. 25. Cleavable sequences<br />WT HIV-1 protease recognized sequences<br />
  26. 26. Example of optimization convergence<br />MA-CA<br />Each optimization cycle requires ca. 24 hours on 5 cpus<br />
  27. 27. FMO MP2/PCM/6-31G* binding energies of cleavable peptides<br />Kcal/mol<br />Differences between the binding energies of WT HIV-1 protease and its natural substrates are expected and will be experimentally checked<br />
  28. 28. Enzyme redesign<br />Among the experimentally verified cleavable peptides, the closest to the target sequence is chosen<br />Target sequence<br />TF-PR cleavage sequence, candidate starting sequence<br />Specificity most involved peptide residues<br />The candidate substrate sequence is mutated into the target sequence one amino acid at the time<br />
  29. 29. Enzyme redesign<br />The candidate substrate sequence is mutated into the target sequence one amino acid at the time<br />
  30. 30. Specificity determining residues<br />P3’<br />V82<br />G48’<br />L76<br />P4’<br />high<br />P1’<br />I47’<br />medium<br />P2<br />P2’<br />D30’<br />low<br />P1<br />P3<br />P4<br />I84’<br />