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.

National Institute of Health (NIH)
National Center for Research Resources (NCRR)
Biotechnology Resource Grants (P41)
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Special Emphasis Panel (ZRG1-BCMB-E (40))
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Cornell University
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