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. Project 1. In contrast with the extensive literature on proteinase inhibition, very little information has been available in the area of nuclease inhibition. We recently determined the structure of NucA, a cyanobacterial, non-specific nuclease that in many respects serves as an analog of the RNase H domain of HIV reverse transcriptase. In particular, both the reverese transcriptase RNase H domain and NucA are sequence-independent, degrade double stranded substrates, and require Mg2+ for activity. Hence, a determination of the molecular basis for the inhibition of NucA by NuiA could provide useful insights for the development of inhibitors of HIV RNase H activity. During the past year, we determined the structure of the complex formed between NucA and its specific inhibitor, NuiA. NucA inhibition by NuiA involves an unusual divalent metal ion bridge that connects the nuclease with its inhibitor. The C-terminal Thr135 hydroxyl oxygen of NuiA is directly coordinated with the catalytic Mg2+ of the nuclease active site, and the NuiA Glu24 residue also extends into the active site, mimicking the charge of a scissile phosphate. NuiA residues Asp75 and Trp76 form a second interaction site, contributing to the strength and specificity of the interaction. Project 2. We have continued studies of the base excision repair enzymes, DNA polymerase beta and DNA polymerase lambda, as useful model systems for understanding the behavior of HIV reverse transcriptase. During the past year, we have assigned ~ 85 % of the backbone resonances of DNA pol beta in a triply labeled sample of the enzyme complexed with double hairping DNA to mimic a gapped DNA substrate. These studies demonstrate substantial internal motion of the enzyme, primarily invoving the DNA binding residues. During the past year, we completed studies of the response of the [methyl-13C]methionine-labeled Pol ? 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. In order to better understand the lyase reaction catalyzed by these enzymes, we developed new approaches involving the use of [6-13C] and [5-13C] labeled lysine. This approach has allowed us to determine the pK value for the catalytically important K72 (pol beta) and K312 (pol lambda) residues, providing insight into the catalytic mechanism.