Abstract
ABSTRACT
Structural and Functional studies of the Hsp90 molecular chaperone
A dissertation presented to the Faculty of the
Graduate School of Arts and Sciences of Brandeis University
Waltham, Massachusetts
By Yi Jin
Hsp90 and Hsp70 are ATP-dependent molecular chaperones that are responsible for efficient protein (“client”) folding and the regulation of cellular homeostasis. Many Hsp90 clients are oncogenic, which has led to a decades-long effort to develop Hsp90 inhibitors that disrupt the ATP-driven cycle of Hsp90 and consequently destabilize oncogenic Hsp90 clients. Because of the clinical importance of Hsp90 inhibitors, numerous labs have been attempting to understand the Hsp90 conformational cycle on a detailed level. The Hsp90 ATPase cycle is characterized by an open-to-closed conformational transition that involves major local and global structural rearrangements. These numerous changes make it challenging to identify key residues that control the Hsp90 conformational cycle and to study this cycle on a molecular and energetic level. In Chapter 2 of my dissertation, I describe a single residue in Hsp90 that confers a striking pH-dependent ATPase activity. Using this unique feature, I generated a quantitative and predictive model that explains how this residue affects Hsp90 conformation, Hsp90 affinity for ATP, and ATPase rate.
In contrast to the extensive studies on the Hsp90 conformation cycle, much less is known about how Hsp90 influences client folding. The Hsp90 field has focused primarily on kinase and transcription factor clients, large proteins that have not proven amenable to detailed analysis of folding and misfolding. In Chapter 3, I establish insulin like growth factor II (IGF2) as a tractable system to study the chaperoning function of the endoplasmic reticulum Hsp90 and Hsp70 pair (Grp94 and BiP). IGF2 is a small disulfide-linked protein with a disordered C-terminal peptide extension. I find that IGF2 readily forms oligomers due to this disordered region. While BiP and Grp94 have only a modest influence on the folding of IGF2, I find that these chaperones have a much greater influence on regulating the oligomerization of IGF2.
The finding that BiP and Grp94 influence IGF2 oligomerization was unexpected. Therefore, I also examined a second client protein system In Chapter 4. Specifically, the E.coli L2 ribosomal protein is known to bind tightly to the bacterial Hsp90 homolog. Like IGF2, L2 contains a region that can fold as well as disordered C-terminal peptide extension. While it is unclear whether L2 oligomerization plays an important role in how HtpG recognizes this client, I have identified regions on both HtpG and L2 that mediate their interaction.