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逆转录酶（RT）的RNase H结构域识别它的一个重要决定因素。最后，核苷类似物的靶向插入确定了与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。通过引物延伸（SHAPE）分析选择性2‘羟基酰化（Selective 2’ hydroxyl酰化），通过非配对构型的核糖2' OH对酰化的敏感性来检测RNA二级结构。我们最初的SHAPE研究集中在野生型和突变型HIV-1 Rev应答元件（RREs）上，随后研究了小鼠ltr -反转录转座子MusD的最小运输元件。与此同时，我们通过开发质谱仪（SHAMS）检测短RNA/DNA杂交体结构的方法以及通过反义干扰形状（ai-SHAPE）定义三级相互作用来扩展该技术。虽然对调控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，化学酶足迹将辅以(a)核磁共振波谱，在RNA大小允许的情况下，(b)小角度x射线散射，以及(c)与CCR化学生物学实验室合作制备的“穿线插入器”靶向fe - edta足迹。本部分的长期目标是在病毒RNA基因组的背景下检查RNA结构，并将研究提高体内足迹的SHAPE敏感性的新方法。[对应2011年10月HIV耐药项目实地考察报告中的Le Grice Project 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]