Abstract
Hsp90 is a highly conserved family of dimeric molecular chaperones whose function depends on ATP binding and hydrolysis. Metazoans express three classes of Hsp90: cytosol specific (e.g. Hsp82 in yeast); mitochondria specific (Trap1); and endoplasmic reticulum specific (Grp94). Although these three Hsp90 classes all share a common dependence on ATP, there is conflicting structural information about their ATP-driven conformational cycle. For example, non-hydrolysable ATP (AMPPNP) causes Trap1 and Hsp82 to undergo an open to closed conformational change, however Trap1 has an asymmetric closed structure whereas Hsp82 is symmetric. The Hsp82 structure was determined with a symmetrically bound inhibiting cochaperone, suggesting the possibility that the symmetric closed state is inactive. Adding further confusion, a structure of Grp94 has been reported with AMPPNP but in an open conformation, in marked contrast to the results from other Hsp90 homologs. These divergent structural conclusions make it difficult to propose a common mechanism. Here we revisit previous structural work on Grp94. We find that a kinetic trapping strategy can be used to accumulate a uniform closed population of Grp94. Small angle x-ray scattering measurements and computational modeling reveals that the closed state of Grp94 is well described by a symmetric conformation. To critically evaluate this structural conclusion, we constructed a model of closed symmetric Grp94 and used this model to rationally design ATPase activating mutations that remove hydrophobic surface area selectively from the closed state. Similarly, based on our closed state model, we identified a salt bridge mutation that also activates Grp94. Our results clarify conflicting structural conclusions concerning Grp94, and provide a rational engineering strategy for increasing the activity of any Hsp90.