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

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

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

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

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

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

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

Minimal modeling of the Asp dyad

Adding the Thr26-Gly27 H-bond

The peptide bond dipole moment

The HIV-1 PR/Pepstatin complex

13C NMR of aspartic acids H OOO O ppm 175 ppm D 0.15 ppm

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

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

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

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

Model system calculations

Resonance de/stabilizing contributions

The reaction mechanism

CMD simulation - the system

HIV-1 PR/Substrate complex flexibility

Substrate displacements

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

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

Reaction Intermediate

Active site Flaps Cantilever Cantilever Fulcrum Drug-resistant mutants