Mechanism by which the Grp94 molecular chaperone folds insulin-like growth factors - Project Summary The endoplasmic reticulum (ER) chaperone Grp94 is required for the correct folding and secretion of insulin-like growth factors but the underlying mechanism is not understood. The overall goal of the NIGMS funded research in my lab has been discovering how Grp94 assists the folding of insulin-like growth factor (IGF) proteins. My lab discovered that Grp94 directly operates with a second chaperone, BiP, and we are dissecting the functional mechanism of the combined BiP/Grp94 system. When BiP brings an IGF “client” to Grp94 the system is tasked with an assessment – should the client be transferred to Grp94 or retained on BiP? This will dictate different folding and degradation outcomes for the client because BiP and Grp94 interact with different pro- folding and pro-degradation components of the ER quality control system. The broad goals over the next five years are determining how, and under what conditions, does the client get transferred from BiP to Grp94, and what benefit for the client is achieved from such a transfer. We have discovered that the client-binding domain of BiP (the “SBD”) can physically dock close to the client-binding region of Grp94. This proximity suggests that the docked BiP SBD may directly guide a client protein onto Grp94. However, client loading also requires Grp94 to undergo large-scale conformational changes to reach a partially-closed state and BiP must remain bound during this process. One goal is to test whether the client stabilizes the initial BiP/Grp94/client ternary complex to provide Grp94 sufficient time to adopt the partially- closed transfer state. A second goal is to test whether the BiP SBD must be docked to guide the client between the dimer arms of Grp94. In performing a joint function together, we propose BiP and Grp94 can perform mechanical unfolding work on a bound client protein by BiP and Grp94 cyclically binding and unbinding each other without letting go of the client. We will test this novel model of chaperone action with a variety of biophysical measurements and single molecule analysis. Grp94 and BiP are members of the Hsp90 and Hsp70 family of chaperones, respectively. Hsp90 members have long been a drug target, with many small molecule ATP-competitive inhibitors developed as anti- cancer drugs. However, it has been difficult to determine how these inhibitors work when Hsp90 is operating in concert with Hsp70. One of our goals is to use the BiP/Grp94 system to better understand how these inhibitors disrupt chaperone function.
