Aids Related Nmr Research

National Institute Of Environmental Health Sciences

Investigators

Linked publications, trials & patents

Abstract

This project utilizes NMR spectroscopy to study the molecular components of HIV and model systems. Recent studies have focused on: 1) analysis of the solution conformation and dynamics of the ribonuclease H (RNase H) domain of HIV reverse transcriptase, and the characterization of substrate-induced active site formation; 2) understanding the protein-mediated interactions of RT substrates and inhibitors; 3) understanding the complex structural maturation process that converts the initial p66 protein into the p66/p66' homodimer, and finally the p66/p51 heterodimer; 4) understanding the basis for the subunit-specific elimination of one RH domain 5) identification of potential targets for development of drugs that would interfere with the structural maturation process. Project 1. During the past year we have continued our studies of the maturation pathway of HIV-1 reverse transcriptase. In order to achieve economy of the coding sequence requirements, RT utilizes a metamorphic polymerase domain that is able to adopt two alternate structures and thus fulfill two different functions. Both forms are present in the active form of reverse transcriptase, the p66/p51 heterodimer. Since the active fold of the polymerase domain is much less stable than the inactive fold, ligands and mutations that stabilize the active fold also contribute to dimer stability. In the past year, we demonstrated that the basis for dimer stabilization by the Gly190Trp mutation is similar to the effect of adding an NNRTI such as efavirenz. Gly190 is located in the non-nucleoside reverse transcriptase inhibitor (NNRTI) binding pocket. The much larger Trp sidechain is able to occupy this same binding pocket, as is apparent from an overlay of the efavirenz complex with the p66(G190W)/p51 structure. Since the NNRTI binding pocket is only present in the actively-folded polymerase domain, both NNRTIs and the Gly190Trp mutation are accommodated preferentially in the actively folded polymerase, stabilizing this form of the protein and hence stabilizing the heterodimer. Thus, an NNRTI such as efavirenz and the NNRTI binding pocket mutation Gly190Trp exert similar effects on the structural equilibrium and have similar effects on dimer stability. Project 2. Recent structural characterizations of the p51 and p66 monomers has established an important starting point for understanding the maturation pathway of the HIV-1 reverse transcriptase p66/p51 heterodimer. This process requires a metamorphic transition of the polymerase domain leading to formation of a p66/p66' homodimer that exists as a structural heterodimer. In order to better understand the drivers for this metamorphic transition, we have performed NMR studies of 15N-labeled RT216 - a construct that includes the fingers and most of the palm domains. These studies are consistent with the conclusion that the p66 monomer exists as a spring-loaded complex. Initial dissociation of the fingers/palm:connection complex allows the fingers/palm to adopt an alternate, more stable structure, reducing the rate of re-association and facilitating subsequent maturation steps. One of the drivers for an initial extension of the fingers/palm domains is identified as a straightening of helix E relative to its conformation in the monomer by eliminating a bend of 50 near residue Phe160. NMR and CD data also are consistent with the conclusion that a hydrophobic surface of palm domain that becomes exposed after the initial dissociation, as well as the intrinsic conformational preferences of the palm domain C-terminal segment, facilitate formation of the beta12-beta13-beta14 beta-sheet structure that is unique to the active polymerase subunit. Recently reported NMR studies of U-2H,15Nlabeled p66 and p51 indicated that these proteins can form symmetric homodimers. However, we found that the spectra reported are also consistent with those expected for the p66 and p51 monomers. NMR studies of U-2H,15Np66PL lacking disordered residues 219-230 yielded very similar spectra to those that had been reported, consistent with the conclusion that the reported spectra that were attributed to homodimers were dominated by resonances of the smaller and more flexible monomer species. These results confirm the conclusion that the p51 and p66 homodimers exist as structural heterodimers. Project 3. Previously reported studies of HIV-infected cells have indicated that treatment with the non-nucleoside RT inhibitor efavirenz can lead to premature activation of HIV-1 protease, premature processing of the gag-pol polyprotein, and reduced viral infectivity. This result is presumably a consequence of efavirenz-induced RT dimerization and cooperative polyprotein dimerization, such that formation of the RT homodimer also induces dimerization of other proteins including HIV protease and HIV integrase. In order to better evaluate this cooperative dimerization, we have worked with Lalith Perera and Juno Krahn on molecular modeling the protease-reverse transcriptase diprotein. We showed that a construct of the protease-reverse transcriptase dimer can be formed and that it is stable over simulation periods up to 250 ns. Cooperative dimerization is undoubtedly important in understanding the RT maturation process in the virion.