High-Resolution Protein and Nucleic Acid Footprinting

尽管HIV-1整合酶仍然是开发小分子药物的优先目标， 拮抗剂，完整分子的高分辨率结构仍然难以捉摸。使用 结合蛋白质足迹和质谱，我们已经成功地定义了 完整HIV-1整合酶上磷酸吡哆醛的结合位点。NMR研究， 多嘌呤道引物的（+）链DNA合成已经证明了糖环的变化 PPT-U3连接处的构象，这表明这可能是PPT-U3连接的重要决定因素。 通过HIV-1逆转录酶（RT）的RNase H结构域识别。最后，针对性 核苷类似物的插入具有限定的核酸底物区域， 与Ty 3 RT的DNA聚合酶结构域相互作用。 Ty 3 RT突变体是否能够逆转类似物诱导的DNA合成抑制。 这种生物化学互补突出了Ty 3拇指亚结构域的残基， 模板-引物双链体的单个碱基，说明了核苷的重要性。 模拟干扰策略。[对应于2007年4月网站上的Le Grice项目2 艾滋病毒耐药性项目访问报告]

Although HIV-1 integrase remains a priority target for development of small-molecule antagonists, a high-resolution structure of the intact molecule remains elusive. Using a combination of protein footprinting and mass spectrometry, we have been successful in defining the binding site for pyridoxal phosphate on intact HIV-1 integrase. NMR studies with the polypurine tract primer of (+) strand DNA synthesis have demonstrated a change in sugar ring conformation at the PPT-U3 junction, suggesting this may be an important determinant for its recognition by the RNase H domain of HIV-1 reverse transcriptase (RT). Finally, targeted insertion of nucleoside analogs has defined regions of the nucleic acid substrate that interact with the DNA polymerase domain of Ty3 RT. An extension of this study investigated whether Ty3 RT mutants were capable of reversing analog-induced inhibition of DNA synthesis. Such biochemical complementation highlighted residues of the Ty3 thumb subdomain that contact individual bases of the template-primer duplex, illustrating the important of nucleoside analog interference strategies. [Corresponds to Le Grice Project 2 in the April 2007 site visit report of the HIV Drug Resistance Program]