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。结合蛋白质足迹和质谱学，我们已经成功地确定了完整的HIV-1整合酶上磷酸吡哆醛的结合部位。用(+)链DNA合成的多聚尿路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，这可能是其被HIV-1逆转录酶(RT)的RNaseH结构域识别的重要决定因素。最后，核苷类似物的定向插入定义了与Ty3 RT的DNA聚合酶区域相互作用的核酸底物区域。这项研究的延伸是研究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拇指亚域与模板-引物双链单个碱基接触的残基，说明了核苷类似物干扰策略的重要性。选择性2‘羟基酰化分析引物延伸分析(SHAPE)通过未配对构型的核糖2’OH对酰化的敏感性来检查RNA的二级结构。我们最初的SHAPE研究集中在野生型和突变型HIV-1Rev反应元件(RREs)上，随后检查了小鼠LTR的最小转运元件-逆转录转座子MU.S.。同时，我们扩展了这项技术，开发了用质谱仪(ShaMS)检查短RNA/DNA杂交物的结构的方法，并通过反义干扰形状(ai-SHAPE)定义了三级相互作用。虽然对调控RNA的结构分析将继续进行，但我们认识到SHAPE不是一项独立的技术。因此，将辅之以(A)核磁共振光谱学，在RNA大小允许的情况下，(B)小角X射线散射，和(C)与CCR化学生物实验室合作制备的“线状嵌入剂”的靶向Fe-EDTA足迹。本部门的一个长期目标是在病毒RNA基因组的背景下检查RNA结构，并将研究增加体内足迹形状敏感性的新方法。[对应于2011年10月艾滋病毒耐药性项目现场访问报告中的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]