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) analysis of the conformational processes involved in going from p66 and other early forms of the polyprotein to formation of the mature heterodimer structure. Project 1. HIV-1 reverse transcriptase (RT) contains a C-terminal ribonuclease H domain (RH) on its p66 subunit that can be expressed as a stable, although inactive protein. Recent crystallographic studies of several ribonuclease H enzymes derived from other organisms demonstrate that substrate binding plays a major role in the creation of the active site. In the absence of substrate, the C-terminal helix E of the RT RNase H domain is dynamic, characterized by severe exchange broadening of its backbone amide resonances, so that the solution characterization of this region of the protein has been limited. NMR studies of 13C-labeled RH as a function of experimental conditions revealed that the delta1 methyl resonance of Ile556, located in a short, random coil segment following helix E, experiences a large 13C shift corresponding to a conformational change of this residue that results from packing of helix E against the central beta-sheet. This shift provides a useful basis for monitoring the effects of various ligands on active site formation. Using this approach, the fractional probability for formation and correct orientation of helix E can be determined, so that the effects of various experimental conditions and ligands on helix stabilization could be evaluated. It was thus possible to identify new ligands that support stabilization of helix E. Additionally, we found that the RNase H complexes formed with one or both divalent ions can be individually observed and characterized using diamagnetic Zn2+ as a substitute for Mg2+. Zn2+ has been shown to support RNase H activity at much lower concentrations than Mg2+, although with significantly reduced activity. It was found that ordering of helix E results specifically from the interaction of the divalent ion with the lower affinity A binding site, which involves binding to Asp549 on helix E. Project 2. Non-nucleoside reverse transcriptase inhibitors (NNRTIs) play a central role in the treatment of AIDS, but their mechanisms of action are incompletely understood. The interaction of the NNRTI nevirapine (NVP) with HIV-1 reverse transcriptase (RT) is characterized by a preference for the open orientation of the fingers/thumb subdomains, and a reported variation of three orders of magnitude between the binding affinity of NVP for RT in the presence or absence of primer/template DNA. In order to investigate the relationship between conformation and ligand binding, we evaluated the use of methionine NMR probes positioned near the tip of the fingers or thumb subdomains and thus expected to be sensitive to changes in local environment dependent on the fractions of open and closed RT. In the presence of the nevirapine, the resonances of active site residues M184 and M230 on the p66 subunit are both significantly perturbed, while none of the methionine resonances in the p51 subunit is sensitive to this inhibitor. Comparisons of the NMR spectra of three conservative mutations, I63M, L74M, and L289M, indicated that M63 showed the greatest shift sensitivity to the addition of nevirapine. The exchange kinetics of the M63 resonance are fast on the chemical shift time scale, but become slow in the presence of nevirapine due to the slow binding of RT with the inhibitor. Ratios of the open/closed M63 resonance were used to evaluate the NVP dissociation constant. Addition of MgATP destabilizes NVP binding, altering the exchange behavior of the M184 and M63 resonances affected by NVP from slow to fast/intermediate on the shift timescale. The fraction of RT in the open conformation, as judged by the M63 resonance shift, is significantly reduced in the presence of both ligands. Although there is presumably some displacement of bound NVP in the presence of MgATP, the shift behavior of the M230 resonance indicates the presence of an RT-MgATP-NVP ternary complex. Project 3. Conformational maturation of HIV reverse transcriptase. HIV reverse transcriptase undergoes a conformational maturation process in the virion that results in cleavage from the other protein components of the pol polyprotein, as well as selective cleavage of one of the two RNase H domains on the p66/p66'homodimer to produce the mature p66/p51'heterodimer. We have been investigating this process using selectively labeled p66. During the past year, we developed a conformational restriction mutation that allows us to selectively label p66 in the closed conformation. This allows the selective labeling of either of the subunits of the p66/p66'homodimer. Characterizing the intermediate conformational states of RT provides a basis for targeting the maturation process rather than the mature enzyme, so that the drug does not have to compete with the RT substrates for binding.
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