This project utilizes NMR spectroscopy to study the molecular components of HIV and model systems. Recent studies have focused on: 1) analysis of the structure, dynamics and ligand binding behavior of HIV reverse transcriptase and its RNase H domain, 2) related NMR methodological evaluations and development that facilitate the design and interpretation of the RT studies;3) studies of model nuclease and polymerase systems, particularly DNA pol beta;structural studies of the bacterial enzyme EndA, derived from Streptococcus pneumoniae, which represents a potential drug target in pneumonia-susceptible AIDS patients. Project 1. HIV Reverse Transcriptase (RT) is a primary target for drug intervention in the treatment of AIDS. We have performed the first NMR studies of methyl-13C methionine HIV-1 RT, aimed at better understanding the conformational and dynamic characteristics of RT, both in the presence and absence of the non-nucleoside RT inhibitor (NNRTI) nevirapine. The selection of methionine as a structural probe was based both on its favorable NMR characteristics, and on the presence of two important active site methionine residues in the p66 subunit: M184 and M230. Observation of the M184 resonance is subunit dependent;in the p66 subunit the solvent-exposed residue produces a readily observed signal with a characteristic resonance shift, while in the globular p51 subunit, the M184 resonance is shifted and broadened as M184 becomes buried in the protein interior. In contrast, although structural data indicates that the environment of M230 is also strongly subunit dependent, the M230 resonances from both subunits have very similar shift and relaxation characteristics. A comparison of chemical shift and intensity data with model-based predictions for the p66 subunit gives reasonable agreement for M184, while M230, located on the -hairpin """"""""primer grip"""""""", is more mobile and solvent exposed than suggested by crystal structures of the apo enzyme which have a """"""""closed"""""""" fingers-thumb conformation. This mobility of the primer grip is presumably important for binding of non-nucleoside RT inhibitors (NNRTIs), since the NNRTI binding pocket is not observed in the absence of the inhibitors, requiring instead that the binding pocket be dynamically accessible. In the presence of the nevirapine, the resonances of M184 and M230 in the p66 subunit are both significantly perturbed, while none of the methionine resonances in the p51 subunit is sensitive to this inhibitor. Site-directed mutagenesis indicates that both M16 and M357 produce two resonances in each subunit, and for both residues, the intensity ratio of the component peaks is strongly subunit dependent. Project 2. Dimerization of the p51 subunit of HIV reverse transcriptase. The dimerization of HIV reverse transcriptase has been of interest since it is significantly influenced by non-nucleoside RT inhibitors (NNRTI) and is therefore presumably related to their mechanism of action, and since the development of dimerization inhibitors has also become an active field for drug research. Our recent study utilized methyl-13Cmethionine labeling as well as small angle X-ray scattering (SAXS) to evaluate the dimerization of the p51 subunit, and to understand how this process relates to the conformational behavior of the p51 subunit. As demonstrated recently, the methionine resonances of methyl-13Cmethionine RT show significant subunit dependence, which is generally consistent with the large structural differences of the p51 and P66 subunits. The 1H-13C HSQC spectrum of methyl-13Cmethionine-labeled p51 is qualitatively similar to that expected for a mixture of the p66 and p51 subunits, indicating that a significant fraction of the p51 adopts a """"""""p66-like"""""""" conformation. Additional NMR and SAXS studies indicate that under the conditions of our studies, p51 exists primarily as a homodimer that is a conformational heterodimer. The effects of the NNRTI nevirapine and Mg concentration on both the dimerization and the conformation also have been evaluated. Project 3. Streptococcus pneumoniae is a leading cause of invasive bacterial respiratory disease in adults and children with HIV. It has been shown that S. pneumoniae is able to evade a key defense mechanism employed in the lungs, neutrophil extracellular traps (NETs), through the use of an extracellular nuclease, EndA. This bacterial strategy is successful because the NETs are composed of antimicrobial enzymes and proteins loaded onto a DNA scaffold. The nonspecific nuclease EndA digests the DNA scaffold, allowing the S. pneumoniae to escape the NETs and invade the bloodstream and other parts of the body. In order to better understand the mechanism of action of the S. pneumoniae enzyme and to provide information of value for the development of therapeutic intervention, we have determined the structure of S. pneumoniae EndA. In general, studies of non-specific nucleases such as EndA are limited by the toxicity of the nuclease to the host cell used for expression. In the case of EndA, his limitation was overcome by expression a nearly inactive mutant, EndA H160A, which dramatically reduces the degree of cellular toxicity. Preliminary structural characterization demonstrates that EndA is closely related to other beta-beta-alpha metal family nucleases, despite very low sequence homology. Current studies are in progress to identify residues important for catalysis and substrate binding.
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