Abstract
Hsp70 and Hsp90 are families of essential molecular chaperones that ensure proper proteinfolding, maturation, and quality control. Hsp70 and Hsp90 play complementary roles, with Hsp70
acting early in the folding pathway and Hsp90 assisting in later maturation steps. The ER paralogs,
BiP (Hsp70) and Grp94 (Hsp90), collaborate to help their client proteins fold properly, but the
molecular details governing their cooperation are poorly understood. This dissertation investigates
the mechanisms by which BiP and Grp94 coordinate client protein transfer, how these mechanisms
are influenced by Hsp90 inhibitors, and how client protein orientation impacts chaperone
recognition.
In Chapter 2, we dissect the mechanism of client loading from BiP to Grp94 anddemonstrate that BiP drives Grp94 into a distinct loading conformation via a secondary interaction
interface (interface II) between the nucleotide-binding domain of BiP and the N-terminal domain
of Grp94. We show that this interface is essential for Grp94 closure and ATPase stimulation and
that only a subset of ATP-competitive Hsp90 inhibitors block this loading state due to steric
clashes in the nucleotide pocket. These findings highlight mechanistic conservation between the
ER BiP/Grp94 and cytosolic Hsp70/Hsp90 systems while revealing how specific ATP-competitive
vi
inhibitors selectively disrupt the client loading conformation by sterically interfering with thenucleotide pocket.
In Chapter 3, we leverage the intrinsic fluorescence of the Hsp90 inhibitor XL888 todevelop new biophysical assays for probing Hsp90 conformation and inhibitor binding in the
presence of biologically relevant proteins such as a cochaperone and client protein. This approach
enables competition assays and reveals differential inhibitor effects on distinct Grp94
conformational states. This research allows for structure-informed drug discovery targeting Hsp90
that is actively stabilizing client proteins.
Chapter 4 focuses on assay development to examine how BiP recognizes client proteinsand whether backbone orientation influences chaperone binding and transfer. Using engineered
proIGF2 constructs, we design fluorescence and crosslinking assays to probe client orientation and
positioning in BiP and BiP/Grp94 complexes.
Together, these studies advance our molecular understanding of how BiP and Grp94cooperate to fold client proteins, how small-molecule inhibitors perturb these processes, and how
chaperone–client interactions are influenced by protein orientation. Together, these findings offer
mechanistic insights that may inform future therapeutic strategies targeting Hsp90 and aid in
advancing experimental approaches to study ER chaperone function.