Structural Analysis and Client Binding Studies of the ER-Specific Molecular Chaperones BiP and Grp94
Creators: name: Deans, Erin Elizabeth
Publisher: name: Brandeis University
Year: 2024
Subjects: BiP
Client
Grp94
Hsp70
Hsp90
proIGF2

description: Hsp70 and Hsp90 are families of ATP-dependent chaperones that are part of a quality control network that maintains cellular homeostasis by assisting protein folding. Hsp90s provide folding assistance for many oncogenic proteins, and numerous Hsp90-specific inhibitors have been developed as potential cancer treatments. Hsp90s have minimal capacity to fold client proteins (“clients”) alone and instead often work in coordination with Hsp70s. Recent evidence suggests that the combined Hsp70/Hsp90 system may be an important biological target of Hsp90 inhibitors. However, the structural and mechanistic basis of Hsp70 and Hsp90 coordinated function, and how Hsp90 inhibitors influence this function, remains an active area of research. My thesis focuses on the ER-specific Hsp70/Hsp90 pair, BiP/Grp94, and how these chaperones bind clients and work together.Hsp70s are able to differentiate between oligomeric and monomeric states of certain clients by binding oligomers with higher affinity than monomers. In contrast, other Hsp70 clients exhibit a negligible difference in affinity between oligomers and monomers. Up until now, it was not known why Hsp70s only bind certain oligomers with high affinity and what the driving force behind this high affinity is. In Chapter 2, I discuss a novel electrostatic steering mechanism by which negatively-charged BiP binds oligomers of a positively-charged client, proIGF2, with high affinity compared to monomeric peptides. Based on results in the literature, this electrostatic mechanism could be widespread for the Hsp70 family.
description: Recently, a structure of the cytosol-specific Hsp70/Hsp90 pair bound to a monomeric client was solved (the “loading structure”), which provides new structural insights into cytosolic Hsp70/Hsp90 coordinated function. However, it is unclear if these insights are relevant to the BiP/Grp94 system. In Chapter 3 I examine three questions raised by the loading structure using BiP, Grp94, and proIGF2.
description: Previous findings from the Street Lab provide the foundation for Chapter 4, where it was found that BiP radically alters the impact of ATP-competitive inhibitors on the conformation of Grp94. Specifically, some inhibitors are compatible with a BiP-stabilized closure intermediate of Grp94 (the “C’-state”) while others are not. In Chapter 4, I combine structural data, FRET data, and mutational analysis to discover the basis for this surprising effect. I mutated residues in the Grp94 nucleotide pocket based on candidate structures, one of which being the loading structure. Mutation of a phenylalanine based on the loading structure indicates this residue undergoes a rotamer change in the C’ state. Some inhibitors can accommodate this rotamer change while others cannot, explaining the observed conformational specificity of certain Hsp90 inhibitors when bound to Grp94.
description: The findings in Chapters 3 and 4 show examples in which specific insights from the cytosolic Hsp70/Hsp90 system are applicable to BiP/Grp94. This opens the door for the BiP/Grp94 system to be utilized for structural and mechanistic analysis of Hsp90 inhibitor action on client proteins.