Aids Related Nmr Research

National Institute Of Environmental Health Sciences

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Abstract

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 HIV reverse transcriptase and its RNase H domain, 2) related NMR methodological evaluations and development that facilitates the design and interpretation of the RT studies;3) studies of model nuclease and polymerase systems, particularly DNA pol beta. 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. The approach of selective labeling of RT with methionine described above has proven useful for the investigation of this large and complex molecule, but also provides less information than a more general labeling approach would yield. We have been interested in optimizing the information available from such studies. During the past year, we have performed DFT calculations on model systems in order to evaluate the conformational dependence of 3JCSCC, 3JCSCH, and the isotropic shielding of the 13C methyl group. Results have been compared with experimental data reported in the literature, as well as data obtained on methyl-13Cmethionine and on the model compounds t-butyl-methyl sulfide and t-butyl methyl ether. These studies indicate that relative to oxygen, the presence of the sulfur atom in the coupling pathway results in a significantly smaller coupling constant, 3JCSCC /3JCOCC is about .76. In addition it is demonstrated that the 3JCSCH coupling constant depends primarily on the subtended CSCH dihedral angle, and secondarily on the CSCC dihedral angle. Comparison of theoretical shielding calculations with the experimental shift range for methionine CΗ3 in the TALOS data base supports the conclusion that the intra-residue conformationally-dependent shift perturbation is the dominant determinant of the methionine methyl 13C shift. Analysis of calmodulin data based on these calculations indicates that a few residues adopt non-standard gauche rotamers characterized by very large chi3 values. The utility of the methionine methyl 13C shift as a basis for estimating the gauche/trans ratio for chi3 has been evaluated, and supports our conclusion that Met16 of HIV reverse transcriptase is primarily in the trans conformation. Project 4. DNA polymerase beta has been considered as a model for the behavior of HIV reverse transcriptase. We have previously demonstrated that conformational activation of Pol beta can be readily monitored by the methionine resonances which show characteristic perturbations upon the binding of gapped DNA substrates as well as on the formation of an abortive ternary complex. During the past year, we have extended our studies of cancer associated Pol beta mutants in order to better understand the phenotypic consequences of these mutations.