STRUCTURAL STUDIES OF HSP90 CHAPERONE-CLIENT INTERACTIONS

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 HSP90分子伴侣在细胞内客户蛋白成熟的后期阶段起着核心作用。这些客户中的许多人控制着细胞生长和发育的关键关卡，是癌症治疗的重要目标。目前该领域最前沿的问题集中在开发高特异性的抑制性配体和了解配体结合时表现出的构象重排类型。 我们正在请求束流时间来研究HSP90分子伴侣家族与新的小分子抑制配体的相互作用。我们以前已经测定了完整的GRP94(内质网Hsp90)与AMPPNP络合物的高分辨晶体结构，分辨率为2.5？然而，到目前为止，还没有一个完整的HSP90伴侣的结构与抑制配体的复合体。为了解决我们理解中的这一根本差距，我们现在已经将完整的GRP94结晶在带有两个抑制配体PU54和自由基的复合体中。PU54络合物的晶体在SSRL到3.5？产生了数据集；自由基醇晶体在国内来源衍射率为6？，预计性能与使用同步辐射的PU54晶体差不多。与原来的GRP94+AMPPNP晶体相比，c轴增加了22？，并且很难获得良好的分子置换溶液，这表明GRP94-PU54复合体中的蛋白质已经发生了构象变化。该复合体的SEMET晶体已经产生，我们正在请求光束时间来从这些晶体中收集MAD数据。 在进行MAD实验的同时，我们也在从这些样本中寻求更高质量的数据。从SSRL的PU54晶体收集的早期数据很差，总体Rmerge为14%。这些晶体是从高盐中生长出来的，我们怀疑很大程度上是由于冷冻困难造成的衍射质量较差。为了克服这一点，我们于2010年4月前往康奈尔，当时国际象棋正在进行维护，在高压条件下冷冻PU54和自由基的本地和赛梅特晶体。这些样品目前储存在液氮中，等待束流时间。PU54和自由基酚晶体都是在2009年底生产的。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Hsp90 chaperones play central roles in the later stages of client protein maturation in the cell. Many of these clients moderate key checkpoints in cellular growth and development, and are significant cancer therapeutic targets. Questions at the forefront of the field currently center on the development of inhibitory ligands of high specificity and understanding the types of conformational rearrangements exhibited upon ligand binding. We are requesting beam time to study the interaction of the hsp90 family of molecular chaperones with novel small molecule inhibitory ligands. We have previously determined high resolution crystal structures of intact GRP94 (the endoplasmic recticulum Hsp90) in complex with AMPPNP to 2.5 ¿ resolution. To date, however, there is no structure of an intact hsp90 chaperone in complex with an inhibitory ligand. To address this fundamental gap in our understanding, we have now crystallized intact GRP94 in complex with 2 inhibitory ligands, PU54 and Radicicol. Crystals of the PU54 complex have yielded data sets at SSRL to 3.5 ¿ ; the Radicicol crystals diffract to 6 ¿ at home sources and are expected to perform about as well as the PU54 crystals using synchrotron radiation. A 22 ¿ increase in the c-axis, compared to the original GRP94+AMPPNP crystals, along with difficulties in getting a good molecular replacement solution suggest that a conformational change in the protein has occurred in the GRP94-PU54 complex. SeMet crystals of the complex have been produced, and we are requesting beam time to collect MAD data from these crystals. In parallel with the MAD experiments, we are also pursuing better quality data from these samples. The earlier data collected from the PU54 crystals at SSRL was poor, with overall Rmerges of 14%. The crystals were grown out of high salt, and we suspect that much of the poor diffraction quality was due to freezing difficulties. To overcome this, we traveled to Cornell in April 2010, while CHESS was down for maintenance, to freeze native and SeMet crystals of the PU54 and Radicicol under conditions of high pressure. These samples are currently stored in LN2 and await beam time. Both the PU54 and Radicicol crystals were produced in late 2009.