Abstract
All Hsp90 chaperone proteins undergo an ATP-dependent conformational cycle that is essential for their activity. Unlike other Hsp90 paralogs, the endoplasmic reticulum Hsp90 paralog Grp94 builds up a steady-state closed state population under ATP turnover conditions, enabling study of both opening and closure rates. Introducing the opening-rate accelerating R102A mutation, previously partially characterized in the yeast cytosolic Hsp90 paralog Hsp82, into Grp94 allows me to characterize the effect of increasing the rate of ATP hydrolysis in an Hsp90 system. Grp94 has been previously observed to enter two different closed states observable via smFRET, a long-lived closed state followed by a short-lived higher-FRET closed state referred to as the C’ state, which is present immediately prior to reopening. I observed that the R102A mutant appears to close and open directly into the C’ state. This supports a previously proposed hypothesis that the C’ state is entered after hydrolysis of bound molecules of ATP. I also characterized ATP-concentration dependent substrate inhibition of Grp94. After finding that Mg2+ inhibits Grp94 in a concentration-dependent manner, and subsequently controlling for Mg2+ concentration, I was able to use the increased ATPase rate of R102A to characterize the ATP-concentration dependent substrate inhibition of Grp94. ATPase data were fit to a simple model where binding of a second molecule of ATP leads to a lower ATPase rate. Addition of the closure-accelerating nucleotide binding domain of the cochaperone BiP decreased the degree of substrate inhibition observed, and abolished inhibition completely in the context of R102A and K364A mutants, indicating that increased ATP concentrations inhibit Grp94 dimer closure. Taken together, these results illustrate how ATP is not only necessary for Grp94 activity, but also slows conformational changes through multiple independent mechanisms.