Ligand interaction and regulation in eukaryotic Hsp90 homologs

The hsp90 chaperones, including cytosolic Hsp90 and endoplasmic reticulum resident GRP94 are a ubiquitous class of molecular chaperones. The main cellular role of the hsp90 chaperones is to regulate the conformational maturation of their clients. Notably, the clients of Hsp90 include a myriad of cell-cycle proteins, signaling kinases, and nuclear hormone receptors while GRP94, a resident of the endoplasmic reticulum lumen, has a separate pool of clients including a number of cell surface receptors and secreted proteins. As the pool of known hsp90 clients grows so does the array of cellular processes that require an hsp90 chaperone for proper function. In a way the hsp90 chaperones can be viewed as a nexus of protein biology by acting as a regulator of maturation for many proteins.;The research presented here had two major goals. The first of these was to understand the consequences of ligand binding on the molecular mechanism of hsp90 chaperones, and the second was the development of a high affinity GRP94 specific inhibitor.;Toward progress on the first goal we have solved 29 novel structures of the amino-terminal ligand binding domains of GRP94, yeast Hsp82, or human Hsp90alpha either un-liganded or in complex with the native substrate, product, or an inhibitor. The major realizations that came out of this work was that the amino-terminal domain of these hsp90 chaperones is highly flexible and responsive to ligand. Of particular importance, binding of the native substrate, ATP, to GRP94 elicits a conformation dissimilar from the un-liganded or inhibitor bound forms of GRP94 and the ATP bound form of yeast Hsp82, potentially suggesting different mechanisms for the ATP-dependent function of hsp90 paralogs.;The second goal was a collaborative pursuit wherein novel hsp90 inhibitors provided by synthetic organic chemists were co-crystallized in complex with GRP94, yeast Hsp82, and/or human Hsp90alpha. The structures of these complexes allowed a detailed examination of inhibitor binding. These structures coupled with quantum chemical calculations and experimental inhibition assays defined the mode of inhibition. Rational development of second generation inhibitors was made possible by this detailed understanding of the inhibitor binding pocket and the ability to accurately predict the effect of modifications to inhibitor scaffolds.