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. Conformational selection mechanism of dimer formation. The dimerization of HIV reverse transcriptase (RT) 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-13C methionine 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. More recently, we have expanded these studies to investigate the p66 subunit and utilized methyl-13Cisoelucine labeling as well as methionine labeling. Since many of the resonances show significant subunit dependence, it becomes possible to study dimerization as well as conformational behavior. Recent studies provide interesting clues regarding the nature of the p66 homodimer and the basis for the asymmetric proteolytic processing of one of the p66 subunits to yield the p66/p51 heterodimer. Project 3. Conformational monitoring of RT with a methionine probe. The relative orientation of the fingers and thumb subdomains of HIV reverse transcriptase is highly variable. The apo enzyme shows a significant preference for a closed conformation in which the tips of the two domains are nearly in contact. In contrast, RT complexes with primter/template substrates or with non-nucleoside RT inhibitors exhibit an open conformation in which the primter/template DNA or the NNRTI occupies the region between the thumb and fingers subdomains. Consistent with these conformational effects, the binding constant of inhibitors such as nevirapine with RT in the open conformation is several orders of magnitude tighter than with RT in the closed conformation. In additional to crystallographic determinations, ESR of spin-labeled RT has been used to monitor the conformational state. However, this approach involves fairly significant modifications of the structure. We have developed an NMR approach in which residues near the tips of the fingers or thumb domains are substituted with methyl-13Cmethionine, providing a reasonably sensitive NMR label for the relative fingers/thumb orientation. The methionine label provides a useful probe of NNRTI binding, since the shift is dependent on the open/closed conformation ratio, which is in turn altered by the bound inhibitor. Using this approach, we were able to evaluate the binding constant of nevirapine (NVP). Further, we observed an unanticipated conformational antagonism between nevirapine and Mg-ATP. Thus, addition of the Mg-ATP reversed most of the conformational effect of the nevirapine. Also of interest, the shift of residue M230, which is located on the primer grip near the NNRTI binding site, exhibits a large shift upon NVP binding. This shift is altered upon the addition of Mg-ATP, indicating that the Mg-ATP alters the NVP binding site, although the detailed nature of this alteration remains unknown. Further studies of the effects of other ligands on the fingers/thumb orientation are in progress. Project 4. 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. Our structural determination of EndA was recently completed. The EndA structure reveals a DRGH (Asp-Arg-Gly-His) motif-containing beta-beta-alpha-metal finger catalytic core augmented by an interesting finger-loop interruption of the active site alpha-helix. We also evaluated DNA binding versus catalytic functionality using structure-based alanine substitution mutagenesis. Three mutants, H154A, Q186A and Q192A, exhibited decreased nuclease activity that appears to be in- dependent of substrate binding. Glu205 was found to be crucial for catalysis, while residues Arg127/ Lys128 and Arg209/Lys210 contribute to substrate binding. The structural and activity data provide a molecular foundation for development of specific antibiotic inhibitors for EndA.
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