Aids Related Nmr Research

艾滋病相关核磁共振研究

• 批准号: 8553723
• 负责人: Robert E London
• 金额: $ 67.33万
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8553723
• 关键词: Acquired Immunodeficiency Syndrome; Active Sites; Address; Affect; Affinity; Amides; Base Excision Repairs; Base Pairing; Behavior; Binding; Binding Sites; Biological Models; C-terminal; Cells; Chemicals; Complex; DBL Oncoprotein; DNA; DNA Polymerase beta; DNA Repair Enzymes; Dimerization; Diphosphates; Dissociation; Environment; Enzymes; Fingers; Genetic Transcription; Genome; HIV; HIV-1; Human; Hybrids; Hydrogen Bonding; Individual; Ions; Isoleucine; Kinetics; Label; Lead; Ligand Binding; Ligands; Lysine; Malignant Neoplasms; Measurement; Metals; Methionine; Methods; MgATP; Modeling; Molecular; Molecular Conformation; Monitor; Motion; Mutation; NMR Spectroscopy; Nevirapine; Nuclear Magnetic Resonance; Nucleosides; Nucleotides; Organism; Pharmaceutical Preparations; Play; Polymerase; Positioning Attribute; Probability; Process; Property; Protein Region; Proteins; Purines; Pyrimidine; RNA; RNA-Directed DNA Polymerase; Reaction; Relative (related person); Reporting; Research; Retroviridae; Ribonuclease H; Role; Site; Solutions; Structure; Study models; Suggestion; System; Systems Analysis; Techniques; Temperature; Thumb structure; Time; Tumor Suppression; Variant; Vertebral column; Viral; Viral Genome; Zidovudine; analog; base; beta pleated sheet; depolymerization; dimer; divalent metal; ds-DNA; enzyme activity; experience; inhibitor/antagonist; insight; interest; non-nucleoside reverse transcriptase inhibitors; novel strategies; nuclease; preference; purine; response

This project utilizes NMR spectroscopy to study the molecular components of HIV and model systems. Recent studies have focused on: 1) analysis of the solution conformation and dynamics of the ribonuclease H (RNase H) domain of HIV reverse transcriptase; 2) conformational analysis of HIV reverse transcriptase in response to various ligands and inhibitors; 3) studies of model nuclease and polymerase systems, particularly DNA pol beta. Project 1. HIV-1 reverse transcriptase (RT) contains a C-terminal ribonuclease H domain (RH) on its p66 subunit that can be expressed as a stable, although inactive protein. Recent crystallographic studies of several ribonuclease H enzymes derived from other organisms demonstrate that substrate binding plays a major role in the creation of the active site. In the absence of substrate, the C-terminal helix E of the RT RNase H domain is dynamic, characterized by severe exchange broadening of its backbone amide resonances, so that the solution characterization of this region of the protein has been limited. NMR studies of 13C-labeled RH as a function of experimental conditions revealed that the delta1 methyl resonance of Ile556, located in a short, random coil segment following helix E, experiences a large 13C shift corresponding to a conformational change of this residue that results from packing of helix E against the central beta-sheet. This shift provides a useful basis for monitoring the effects of various ligands on active site formation. Using this approach, the fractional probability for formation and correct orientation of helix E can be determined, so that the effects of various experimental conditions and ligands on helix stabilization could be evaluated. It was thus possible to identify new ligands that support stabilization of helix E. Additionally, we found that the RNase H complexes formed with one or both divalent ions can be individually observed and characterized using diamagnetic Zn2+ as a substitute for Mg2+. Zn2+ has been shown to support RNase H activity at much lower concentrations than Mg2+, although with significantly reduced activity. It was found that ordering of helix E results specifically from the interaction of the divalent ion with the lower affinity A binding site. Project 2. Non-nucleoside reverse transcriptase inhibitors (NNRTIs) play a central role in the treatment of AIDS, but their mechanisms of action are incompletely understood. The interaction of the NNRTI nevirapine (NVP) with HIV-1 reverse transcriptase (RT) is characterized by a preference for the open orientation of the fingers/thumb subdomains, and a reported variation of three orders of magnitude between the binding affinity of NVP for RT in the presence or absence of primer/template DNA. In order to investigate the relationship between conformation and ligand binding, we evaluated the use of methionine NMR probes positioned near the tip of the fingers or thumb subdomains and thus expected to be sensitive to changes in local environment dependent on the fractions of open and closed RT. In the presence of the nevirapine, the resonances of active site residues M184 and M230 on the p66 subunit are both significantly perturbed, while none of the methionine resonances in the p51 subunit is sensitive to this inhibitor. Comparisons of the NMR spectra of three conservative mutations, I63M, L74M, and L289M, indicated that M63 showed the greatest shift sensitivity to the addition of nevirapine. The exchange kinetics of the M63 resonance are fast on the chemical shift time scale, but become slow in the presence of nevirapine due to the slow binding of RT with the inhibitor. Ratios of the open/closed M63 resonance were used to evaluate the NVP dissociation constant. Addition of MgATP destabilizes NVP binding, altering the exchange behavior of the M184 and M63 resonances affected by NVP from slow to fast/intermediate on the shift timescale. The fraction of RT in the open conformation, as judged by the M63 resonance shift, is significantly reduced in the presence of both ligands. Although there is presumably some displacement of bound NVP in the presence of MgATP, the shift behavior of the M230 resonance indicates the presence of an RT-MgATP-NVP ternary complex. Project 3. DNA polymerase beta provides a useful model system for analysis of the behavior of the HIV enzyme. Binding of the catalytic divalent ion to the ternary DNA polymerase beta/gapped DNA/dNTP complex is thought to represent the final step in the assembly of the catalytic complex and is consequently a critical determinant of replicative fidelity. We have analyzed the effects of Mg2+ and Zn2+ on the conformational activation process based on NMR measurements of methyl 13Cmethionine DNA polymerase beta. Unexpectedly, both divalent metal ions were able to produce a template base-dependent conformational activation of the polymerase/one-nucleotide gapped DNA complex in the absence of a complementary incoming nucleotide, although with different temperature thresholds. This conformational activation is abolished by substituting Glu295 with lysine, thereby interrupting key hydrogen bonds necessary to stabilize the closed conformation. These and other results indicate that metal-binding can promote translocation of the primer terminus base pair into the active site, expulsion of an unpaired pyrimidine, but not purine, base from the template-binding pocket, and motions of polymerase subdomains that close the active site. We were further able to demonstrate that the predicted translocation of the primer terminus into the active site can lead to the reverse, depolymerization reaction in the presence of pyrophosphate, so that the primer terminus nucleotide is removed from the DNA, increasing the size of the nucleotide gap. This observation is of particularly relevance to the behavior of HIV reverse transcriptase, since the reverse reaction involving the pyrophosphate analog ATP is thought to represent an important mechanism for the escape of the polymerase from the chain terminating effects of AZT and other nucleoside drugs. These findings provide new insight into the relationships between conformational activation, enzyme activity and polymerase fidelity.

本项目利用核磁共振波谱技术研究HIV的分子组成和模型系统。近年来的研究主要集中在：1)HIV逆转录酶核糖核酸酶H （RNase H）结构域的溶液构象和动力学分析；2) HIV逆转录酶对各种配体和抑制剂的构象分析；3)模型核酸酶和聚合酶系统的研究，特别是DNA pol β。