STRUCTURAL STUDIES OF HSP90 CHAPERONE-CLIENT INTERACTIONS

HSP90 伴侣-客户相互作用的结构研究

• 批准号: 8363535
• 负责人: DANIEL T GEWIRTH
• 金额: $ 2.28万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8363535
• 关键词: Address; Adenylyl Imidodiphosphate; Cells; Client; Complex; DNA Sequence Rearrangement; Data; Data Quality; Data Set; Development; Exhibits; Freezing; Funding; GRP94; Grant; Heat-Shock Proteins 90; Home environment; L-Selenomethionine; Ligand Binding; Ligands; Maintenance; Malignant Neoplasms; Molecular; Molecular Chaperones; National Center for Research Resources; Play; Principal Investigator; Proteins; Research; Research Infrastructure; Resolution; Resources; Role; Sampling; Sodium Chloride; Solutions; Source; Specificity; Staging; Structure; Time; Travel; United States National Institutes of Health; cell growth; cellular development; cost; monorden; novel; pressure; research study; small molecule; synchrotron radiation; therapeutic target

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.

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 Hsp 90分子伴侣在细胞中客户蛋白成熟的后期阶段中发挥核心作用。 这些客户中的许多人在细胞生长和发育中调节关键检查点，并且是重要的癌症治疗靶点。 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后表现出的构象重排类型。 我们正在申请束流时间来研究热休克蛋白90家族分子伴侣与新型小分子抑制配体的相互作用。 我们先前已经确定了完整的GRP 94（内质网Hsp 90）与AMPPNP复合物的高分辨率晶体结构，分辨率为2.5 º。 然而，迄今为止，没有一个完整的hsp 90分子伴侣与抑制配体复合的结构。 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GRP 94与2种抑制性配体PU 54和Radicicicol复合结晶。 PU 54复合物的晶体已经产生了SSRL到3.5的数据集; Radicicol晶体在家庭来源中的作用为6，并且预计使用同步辐射时表现与PU 54晶体一样好。 A 22磅与原始GRP 94 +AMPPNP晶体相比，c轴增加，沿着难以得到良好的分子置换溶液，这表明在GRP 94-PU 54复合物中发生了蛋白质的构象变化。 复合物的SeMet晶体已经生产出来，我们正在请求光束时间来收集来自这些晶体的MAD数据。 在MAD实验的同时，我们也在从这些样本中获取更高质量的数据。 在SSRL从PU 54晶体收集的早期数据很差，总体R合并为14%。 这些晶体是从高盐中生长出来的，我们怀疑大部分差的衍射质量是由于冷冻困难。 为了克服这一点，我们于2010年4月前往康奈尔大学，而国际象棋正在进行维护，在高压条件下冷冻PU 54和Radicicicol的天然和SeMet晶体。 这些样本当前存储在LN 2中并等待射束时间。 PU 54和Radicicol晶体均于2009年底生产。