Aids Related Nmr Research

National Institute Of Environmental Health Sciences

Investigators

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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. In addition, we evaluate other potential targets that may be both interesting and amenable to the research tools, primarily NMR spectroscopy, in which the group has expertise. A recent convergence of interests has led to studies of the role that the protein CYREN, cell cycle regulator of non-homologous end joining, that has been shown to be involved in modulating enzymes involved in the final integration step in which viral DNA is inserted into the host chromosomal dNA. Project 1. Recent studies of the structural maturation of HIV-1 reverse transcriptase (RT) have analyzed the metamorphic transition of the RT polymerase domain, which is the first step in this pathway. Although the end points of the transformation are fairly well defined from structural studies, the drivers for these conformational changes are less obvious. We recently performed studies of a truncated construct of the fingers/palm subdomains, since this region of the protein exhibits some of the important characteristics needed to understand the process, particularly expansion of the angle between the fingers and palm, and refolding of the palm domain C-terminal residues to form a short, amphiphilic beta-sheet. NMR studies of U-2H,13C,15N-labeled RT216 show that one of the drivers for the conformational change that occurs subsequent to dissociation of the connection domain is an elimination of the sharp kink near Phe160 in helixE. It was hypothesized that formation of the palm domain C-terminal beta-sheet is driven largely by the intrinsic conformational preferences of the C-terminal residues, that become expressed after connection domain dissociation. In this case, dissociation of the connection domain exposes the surface of the palm domain beta sheet involving beta6-beta9-beta10. The C-terminal hydrophobic beta sheet: beta11-beta-12-beta13 then forms a hydrophobic sandwich with the larger beta sheet. A circular dichroism analysis of the isolated palm domain C-terminal peptide indicates that it largely adopts the beta sheet structure even in the absence of the remaining palm domain structure. These results indicate that during the maturation process, removal of the inter-domain interactions allows the intra-domain interactions to become domainant, and these favor the alternate structure seen in the active, p66 subunit. Project 2. RT is currently targeted with two classes of clinically approved drugs: nucleoside RT inhibitors (NRTI) and non-nucleoside inhibitors (NNRTI). The mechanism of NRTI inhibition is transparent, as these drugs become phosphorylated and compete with the dNTP substrates, generally being incorporated at the primer terminus. Alternatively, the NNRTI inhibitors bind to a pocket that is not present in the unliganded enzyme. This remarkable observation apparently indicates that the structure of the apo-enzyme is dynamic, with the binding pocket appearing only intermittently. Since NNRTI binding is strongly correlated with a large separation of the fingers and thumb subdomains, it is likely that the fraction of RT molecules exhibiting an NNRTI binding pocket is closely correlated with the fraction of molecules that adopt the open conformation. This has been studied both experimentally and theoretically, and is found to exhibit a strong temperature dependence, such that the fraction of molecules in the open conformation exhibits an inverse temperature dependence. A truncated construct of the N-terminal fingers/palm subdomains has been studied using multiple techniques. Unfortunately, this truncated construct lacks most of the NNRTI binding pocket. Based on our recent studies, it is likely that a longer construct can be studied that includes the full NNRTI binding pocket. We have performed initial studies on this construct, and find that it is readily expressed. Analysis of this construct should facilitate a better understanding of the dynamic factors that influence NNRTI binding pocket formation, and provide a useful model for further understanding of the basis of NNRTI-RT inhibition. Further, since this region of the enzyme undergoes extensive refolding as part of the metamorphic transition that may be subject to interference with appropriately designed molecules. Project 3. During the past year, we became aware of an interesting convergence between our studies of DNA repair and HIV. Viral dsDNA formed by action of reverse transcriptase on viral RNA, is inserted into host chromosomal DNA by HIV integrase. However, the integrase-catalyzed step is necessary but not sufficient for completion of the viral life cycle. Insertion is completed by additional enzymatic steps that utilize the host genome non-homologous end-joining (NHEJ) DNA repair system. Recently, it has been shown that this repair pathway does not function uniformly during the cell cycle, but is inhibited during the S and G2 phases. This strategy allows the host cell to rely more heavily on the more accurate homologous recombination pathway during these phases. The inhibitory effect has been attributed to the action of a small protein, the cell cycle regulator of non-homologous end joining (CYREN). Other studies have demonstrated that CYREN also referred to as modulator of retrovirus infection (MRI), influences the viral dsDNA integration step, consistent with its role in controlling flux through the NHEJ pathway. The control of NHEJ flux by CYREN/MRI rests in part on the presence of a Ku binding motif (KBM) in CYREN that competes with the Ku binding motif present in other proteins, including the double strand break scaffold protein APLF, recently studied in our group. We are currently working toward understanding the structural basis underlying the interactions of CYREN/MRI with other repair enzymes.