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捉摸。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研究表明，在PPT-U3连接处糖环构象发生了变化，这表明这可能是HIV-1逆转录酶（RT）的RNA酶H结构域对其识别的重要决定因素。最后，有针对性地插入核苷类似物已定义的区域的核酸底物，与DNA聚合酶域的Ty 3 RT相互作用。本研究的延伸研究是否Ty 3 RT突变体能够逆转类似物诱导的抑制DNA合成。这种生物化学互补突出了Ty 3拇指亚结构域的残基，其接触模板-引物双链体的各个碱基，说明了核苷类似物干扰策略的重要性。通过引物延伸分析的选择性2'羟基酰化（SHAPE）通过未配对构型中的核糖2' OH对酰化的敏感性来检查RNA二级结构。我们最初的SHAPE研究主要集中在野生型和突变型HIV-1 Rev反应元件（RRE）上，随后研究了鼠LTR-逆转录转座子MusD的最小转运元件。与此同时，我们通过开发方法来扩展这项技术，通过质谱法（SHAMS）检查短RNA/DNA杂交体的结构，并通过反义干扰SHAPE（ai-SHAPE）定义三级相互作用。虽然调控RNA的结构分析将继续进行，但我们认识到SHAPE不是一种独立的技术。因此，化学-酶足迹法将与（a）NMR光谱法（在RNA大小允许的情况下）、（B）小角度X射线散射和（c）与CCR化学生物学实验室合作制备的具有“穿线嵌入剂”的靶向Fe-EDTA足迹法进行补充。我们部门的长期目标是在病毒RNA基因组的背景下检查RNA结构，并将研究增加体内足迹的SHAPE敏感性的新方法。[对应于艾滋病毒耐药性计划2011年10月现场访问报告中的Le Grice项目1]

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 importance of nucleoside analog interference strategies. Selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) examines RNA secondary structure via sensitivity of the ribose 2' OH in an unpaired configuration to acylation. Our initial SHAPE studies focused on wild-type and mutant HIV-1 Rev response elements (RREs) and subsequently examined the minimal transport element of the murine LTR-retrotransposon MusD. In parallel, we expanded this technology by developing methods to examine the structure of short RNA/DNA hybrids by mass spectrometry (SHAMS) and defining tertiary interactions via antisense-interfered SHAPE (ai-SHAPE). While structural analysis of regulatory RNAs will continue, we recognize that SHAPE is not a stand-alone technique. Chemo-enzymatic footprinting will therefore be complemented with (a) NMR spectroscopy, where the size of the RNA permits, (b) small-angle X-ray scattering, and (c) targeted Fe-EDTA-footprinting with "threading intercalators," prepared in collaboration with the CCR Chemical Biology Laboratory. A long-term goal of our Section is to examine RNA structure in the context of the viral RNA genome, and novel approaches to increase SHAPE sensitivity for in vivo footprinting will be investigated. [Corresponds to Le Grice Project 1 in the October 2011 site visit report of the HIV Drug Resistance Program]