High-Resolution Protein and Nucleic Acid Footprinting

虽然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实现。使用蛋白质足迹法和质谱法的组合，我们已经成功地确定磷酸吡哆醛对完整的HIV-1整合酶的结合位点。用（+）链DNA合成的多嘌呤段引物进行的NMR研究已经证明了PPT-U3连接处糖环构象的变化，表明这可能是HIV-1 RT的RNA酶H结构域对其识别的重要决定因素。最后，核苷类似物的靶向插入具有与Ty 3RT的DNA聚合酶结构域相互作用的核酸底物的限定区域。研究调查了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”突出了与模板-引物双链体的各个碱基接触的Ty 3拇指亚结构域的残基，说明了核苷类似物干扰策略的重要性。

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.