Abstract
Structural and functional analysis of Hsp70 and Hsp90 chaperones has advanced significantly over the last decade. However, within the cell Hsp70 and Hsp90 often work together, and little is known about the molecular mechanisms that allow these two chaperones to productively coordinate their activities. Much of my thesis is focused on discoveries that reveal a high level of regulation in how Hsp70 and Hsp90 work together. I specifically focus on the endoplasmic reticulum (ER)-specific Hsp70/Hsp90 homologs, BiP and Grp94. Chapter 2 describes the discovery that BiP and Grp94 directly interact even in the absence of a client protein, and that their interaction is nucleotide specific. BiP and Grp94 associate only when BiP is in the ADP conformation. Because BiP traps client proteins while in the ADP conformation, this observation provides a mechanism for the delivery of client proteins from BiP to Grp94.
Chapter 3 describes how BiP acts as a potent regulator of the structure and ATPase activity of Grp94. Thus, I have found that BiP acts both as a chaperone and also as a co-chaperone of Grp94. BiP exerts a strong influence over Grp94 by targeting the open configuration of Grp94 and then driving Grp94 into the hydrolytically active conformation. The acceleration of Grp94 closure is achieved by BiP stabilizing a specific conformational intermediate that can be populated en-route to the closed state.
Chapter 4 is focused only on the Grp94 conformational cycle, which proceeds slowly (less than one cycle per minute) in the absence of stimulating factors such as BiP. The slow rate of the Grp94 conformational cycle imposes a practical challenge in some mechanistic experiments, and it would be helpful to have mutations that enhance the cycle rate. Chapter 4 describes the identification of a group of mutations that activate Grp94. For the most active Grp94 double mutation, the arm closure rate is forty-fold faster than the ATP hydrolysis rate, demonstrating that Grp94 ATPase activity can be decoupled from arm-closure kinetics.