1. Ab initio MD studies of HIV-1 Protease Candidate: Stefano Piana Agostinetti Supervisor: Paolo Carloni

2. Outline Biochemistry of the enzyme HIV-1 protease (HIV-1 PR) Results –The Active site conformational flexibility in the free enzyme –NMR signal calculations in the HIV-1 PR/Pepstatin adduct –Interplay between global protein motions and the reaction mechanism of HIV-1 PR Conclusions

3. The HIV-1 Protease Active Site Flap Flap FulcrumFulcrum Cantilever Cantilever

4. HIV-PR is required for viral maturation Immature non-infective viral particles HIV-1 PR Infective Viruses

5. HIV-1 PR cleaves polypeptide chains Polypeptide chain (Substrate) Flap Flap Active site

6. The HIV-1 Protease cleavage site Asp25 Asp25’ Gly27Gly27’Thr26 Thr26’

7. The Asp Dyad protonation state Asp25 Asp25’ Asp25 Asp25’ Asp25 Asp25’ Unstable 0.0 kcal/Mol 2.0 kcal/Mol

8. Minimal modeling of the Asp dyad

9. Adding the Thr26-Gly27 H-bond

10. The peptide bond dipole moment

11. The HIV-1 PR/Pepstatin complex

12. 13C NMR of aspartic acids H OOO O - 180 ppm 175 ppm D 0.15 ppm

13. 13C NMR of the Asp dyad in the HIV-1 PR/Pepstatin complex H OO OO- 178 ppm 172 ppm Isotopic substitution

14. Ab initio calculations of the 13C NMR chemical shift of the Asp dyad 176 ppm

15. Ab initio calculations of the 13C NMR chemical shift of the Asp dyad 179 ppm 175 ppm

16. Resonance stabilization 175 ppm 180 ppm MBO ratio: 1.00 MBO ratio: 1.68

17. Model system calculations

18. Resonance de/stabilizing contributions

19. The reaction mechanism

20. CMD simulation - the system

21. HIV-1 PR/Substrate complex flexibility

26. Substrate displacements

27. Model complexes to study the reaction profile > 50 kcal/Mol 20 kcal/Mol 50 kcal/Mol

28. Transition states 50 kcal/Mol Single proton transfer 20 kcal/Mol Concerted double proton transfer

29. Reaction Intermediate

30. Active site Flaps Cantilever Cantilever Fulcrum Drug-resistant mutants