High-Resolution Protein and Nucleic Acid Footprinting

尽管HIV-1整合酶仍然是开发小分子拮抗剂的优先目标，但完整分子的高分辨率结构仍然难以捉摸。利用蛋白质足迹和质谱的结合，我们已经成功地确定了磷酸吡哆醛在完整的HIV-1整合酶上的结合位点。用（+）链DNA合成的多嘌呤引物进行的核磁共振研究表明，PPT-U3连接处的糖环构象发生了变化，这可能是HIV-1 rt的RNase H结构域识别它的一个重要决定因素。核苷类似物的靶向插入定义了与Ty3 RT的DNA聚合酶结构域相互作用的核酸底物区域。本研究的延伸研究了Ty3 RT突变体是否能够逆转类似物诱导的DNA合成抑制。这种生化互补突出了Ty3拇指子结构域的残基，这些残基与模板-引物双工的单个碱基接触，说明了核苷类似物干扰策略的重要性。[对应于2007年4月艾滋病毒耐药性项目实地考察报告中的Le Grice项目2]

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 RT. Finally, targeted insertion of nucleoside analogs has defined regions the nucleic acid substrate which 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.]