Aids Related NMR Research

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This project utilizes NMR spectroscopy to study the molecular components of HIV and model systems. The primary research areas are: 1) analysis of the structure, dynamics and ligand binding behavior of the Ribonuclease H domain of HIV reverse transcriptase, and 2) studies of model nuclease and poymerase systems. During the past year, we have cloned and experessed the isotopically labeled, U-[13C,15N] RNase H domain of reverse transcriptase derived from HIV-2. It is anticipated that a detailed structural and dynamic comparison of the RNase H domains derived from HIV-1 and HIV-2 can provide insight into the solution behavior of this domain which can be used for drug development. We have also studied the interaction of this domain with hydrophobic molecules in order to identify potential ligand binding sites. As part of the second objective noted above, we have been interested in studies of model nuclease-inhibitor systems as a basis for understanding the principles involved in nuclease inhibition. We had previously determined the solution structure of NuiA, an inhibitor of Nuclease A (NucA) from Anabaena. During the past year, we have determined the structure of NucA by X-ray crystallography. Although initially planned as an NMR study, the screening of solution conditions for improved solubility led to the unexpected crystallization of this enzyme, which was exploited to obtain the crystal structure. The structure of NucA, which shows considerable structural homology with the Serratia nuclease derived from Serratia marcescens while sharing only 22% sequence identity, nevertheless is a monomer rather than a dimer, and also revealed an unanticipated additional binding site for divalent metal ions located ~ 26 ? from the active enzyme site. The availability of the enzyme structure provides insight into the catalytic mechansim, and studies are currently in progress to obtain the structure of the NucA-NuiA, nuclease-inhibitor complex. In addition to this work, we have a continuing interest in the DNA repair enzyme Pol ? as a model system for HIV reverse transcriptase. We have previously demonstrated that NMR studies of [methyl-13C]methionine labeled Pol ? provides a useful probe for studying the response of the enzyme to nucleotide substrates. During the past year, we studied the response of the enzyme to a DNA analog in which a thymidine isostere - difluorotoluene - was introduced into the templating base position. Upon addition of dATP to form an abortive ternary complex, the enzyme fails to show the normal conformational activation that would be observed if thymine were in the templating base position. This observation explains the failure of the enzyme to proceed with the polymerization reaction under these conditions.