Abstract
Hsp90 is a conserved and ubiquitously expressed ATP-dependent molecular chaperone, responsible for the proper folding and maturation of its client proteins. Many Hsp90 clients are oncogenic and require Hsp90 for their stability. As a result, ATP-competitive Hsp90 inhibitors have been extensively investigated as anti-cancer drugs. My thesis focuses on Hsp90 inhibitors, and factors that affect their efficacy in vitro that are relevant in vivo. An abundance of high affinity Hsp90 inhibitors based off of highly diverse structural scaffolds have been developed. This diversity in commercially available inhibitors in combination with structural data of these inhibitors bound to Hsp90 provides an opportunity to study the structural and energetic basis of their high affinity. Chapter 2 examines the structural factors of inhibitor binding affinity. I discover a dominant energetic contribution from a hydrogen-bonding hot-spot centered on a conserved aspartate on Hsp90. This interaction is pH-sensitive for inhibitors of the resorcinol family, where the hydrogen-bonding hydroxyl group can titrate. While Hsp90 inhibitors have large impacts on the conformational cycle of Hsp90 and the stability of clients in vivo, they have minimal effect on the ability of Hsp90 to interact with and refold clients in vitro. Indeed, Hsp90 has a minimal capacity to fold proteins alone but can effectively chaperone clients in combination with Hsp70, another chaperone protein. Hsp70 and Hsp90 is a highly conserved proteostasis system. However, on a structural and mechanistic level, little is known about how Hsp90 inhibitors influence the Hsp70/Hsp90 system. Despite the strong motivation to understand how Hsp90 inhibitors work, dissecting how they influence the cytosol-specific Hsp70/Hsp90 system has proven technically challenging.
Chapter 3 describes how the endoplasmic reticulum (ER) specific Hsp70/Hsp90 system (BiP/Grp94) is an ideal system for studying how these chaperones work together. The ER system is simpler than the cytosolic system which has HOP, a bridging protein, but still has the conserved Hsp70/Hsp90 core. BiP and Grp94 interact in the absence of client protein and in a nucleotide-dependent manner. Binding of BiP stabilizes a conformation that is an intermediate between the open and closed conformation of Grp94, termed C’. Through stabilizing this intermediate BiP acts as a closure accelerating co-chaperone of Grp94. While I contributed to these findings, work within Chapter 3 was primarily performed by Ming Sun and Bin Huang.
The findings within Chapter 3 provide a foundation for Chapter 4, where I examine how inhibitors influence the BiP/Grp94 system. In ATPase experiments BiP confers enhanced inhibitor resistance to Grp94. However, the extent of this resistance varies between inhibitors. Hsp90 inhibitors also have varied effects on structure of Grp94 in the context of the BiP/Grp94 system, either allowing Grp94 to populate the C’ state or causing Grp94 to populate a novel conformation. These inhibitor-specific effects are hidden in experiments on Grp94 alone. Additionally, I discovered the commercial inhibitor XL888 is naturally fluorescent and is well suited for evaluating inhibitors binding under the complicated conditions with Grp94 and BiP under ATP turnover conditions.