Enzymatic and Structural Studies of the Molecular Chaperone Hsp90
- Author(s): Cunningham, Christian Nathaniel
- Advisor(s): Agard, David A
- et al.
Heat Shock Protein 90 (Hsp90) is an essential molecular chaperone that makes up 1-2% of all cytosolic proteins under normal conditions and increases to 4-6% under stress. Unlike other members of the chaperone family, Hsp90 interacts in the later stages of protein folding promoting subtle rearrangements and client protein activation. Although no substrates have been confirmed to date for the bacterial homolog, the eukaryotic homologs have been shown to interact with a wide variety of proteins including kinases, steroid hormone receptors, nitric oxide synthase, and telomerase. Given the critical nature of the proteins listed, Hsp90 has been the focus of several cancer studies and become an attractive target for therapeutics. Crystal structures of the bacterial and yeast Hsp90 homologs provided the first major insight into the role of nucleotide and the dramatic conformational changes that Hsp90 undergoes during its nucleotide cycle. Electron microscopy and SAXS studies further examined these conformational changes and revealed that unlike other chaperones, Hsp90 does not undergo discrete nucleotide driven conformational changes but rather displays a dynamic conformational equilibrium of multiple states that is then shifted towards one state or another by nucleotide. Using ATP hydrolysis and mutational analysis I investigated the interplay between ATP binding and hydrolysis on the closed conformation of Hsp90. Residues of both monomers were shown to participate in a cross-monomer hydrophobic interaction network that synergistically stabilizes a hydrolysis competent conformation providing the first explanation for structural cooperativity and the importance of dimer formation. While investigating these interactions I was also able to clarify the role of the Middle Domain arginine by showing that it is directly involved in stabilizing the closed state of Hsp90 and not involved in the catalysis of ATP as previously thought. All of these studies were built upon the successes of solving crystal structures of Hsp90 homologs in the apo and nucleotide bound states. A large portion of my work was also focused on utilizing other homologs, nucleotide analogs, small molecules, co-chaperones and substrates to attempt to crystallize a novel conformation of Hsp90 that might reveal new insights into its chaperoning mechanism.