UCSF

Hsp90 is a highly conserved, ATP-dependent molecular chaperone that is essential for maintaining the functions of its client proteins. It has been estimated that about 10% of the proteome of a eukaryotic cell interacts with Hsp90. A large subset of this portion consists of protein kinases and steroid hormone receptors, putting Hsp90 as the master regulator of many essential cellular functions. The mechanism of how Hsp90 uses ATP hydrolysis to carry out its function remains unclear. Structural studies of Hsp90 revealed that Hsp90 is a V-shaped homodimer with each protomer composed of three well-folded domains: an ATP-binding N-terminal Domain (NTD), a Middle Domain (MD), and a C-terminal dimerization Domain (CTD). Efficient ATP hydrolysis by Hsp90 requires that the dimer forms a closed state where NTDs are dimerized, forming a closed ”clamp” conformation that is stabilized by ATP binding. The kinetics of forming this stably closed state is not driven by ATP binding, as there are other rate-limiting steps that need to occur within the protein to allow the NTDs to be dimerized. So how does Hsp90 actually use the energy from ATP to remodel its client protein? the focus of this thesis examines the other possibility of this happening during ATP hydrolysis. In the first chapter, I followed up an observation made by a previous graduate student Laura Lavery. She observed that the ATP-bound closed state of a mitochondrial Hsp90 (TRAP1) is asymmetric. The asymmetry is most prominent at the juncture between the MD:CTD interface—one protomer is buckled while the other remains straight, resembling the same conformation previously observed in the symmetric closed ”clamp” state. This buckling happens precisely where conserved binding sites have been mapped for client proteins. This suggests that if conformational changes were to occur due to ATP hydrolysis, subsequent rearrangements of the asymmetric MD:CTD interfaces back to the previously observed symmetric closed state

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could be used to drive client protein remodeling. Using a combination of biophysical methods (crystallography, Double Electron-Electron Resonance (DEER), and FRET), we observed that TRAP1 hydrolyzes the 2 bound ATPs sequentially. The buckled protomer hydrolyzes the first ATP, which is then followed by a flip in the asymmetry (the buckled conformation becomes straight and vice versa, on each side), which primes the second ATP for hydrolysis by a buckled protomer. In this model, the MD:CTD interface is guaranteed to undergo remodeling with each ATP hydrolysis and would make efficient use of energy from ATP. The implications for this asymmetric ATP hydrolysis mechanism may also be relevant to other Hsp90s. While we have not observed any other asymmetric Hsp90 structures by itself, several functional Hsp90 complexes seen so far seem to have asymmetric composition/arrangements of their components. In the second chapter, we explore how TRAP1 ATPase activity can be modulated by different divalent cations as co-factors. Despite having two ATP binding sites, the ATPase activity of most Hsp90 homologs appears to be non-cooperative (each site behaves independently from one another). However, we saw that ATPase activity of TRAP1 can be cooperative in presence of calcium, and the activity in presence of magnesium appears to be bi-phasic, with higher activity at low ATP concentrations. This unique behavior of TRAP1 may yet be another adaptation of the Hsp90 machine that has evolved within the mitochondrial matrix environment. Using crystallography, we also discovered that calcium binds to the NTD of TRAP1 unlike previously observed chaperone/calcium/ATP complexes. While the exact biological role for this phenomena is not yet clear, these findings provide a clear molecular basis for the regulation of TRAP1 by calcium. Taken together, the work described in this thesis provide insights into the mechanism of ATP hydrolysis by Hsp90, and a potential role that TRAP1 plays in calcium/magnesium-regulated mitochondrial physiology.