Mechanistic Insights of Client Handoff Between ER Chaperones BiP and Grp94

BiP and Grp94 are the endoplasmic reticulum (ER) paralogs of the Hsp70 and Hsp90 chaperone families, which function as a coordinated pair to assist client protein folding. BiP can deliver unfolded client proteins to Grp94, and the current model proposes a sequential transfer of clients through delivery, loading, and trapping steps. Two recently published structures – the client loading step of cytosolic Hsp70/Hsp90, and a lower-resolution structure of BiP/Grp94 – raise questions regarding the location and interactions of the Hsp70 substrate binding domain (SBD) within the loading state, as the Hsp70SBD bound to the client is unresolved in each structure. Additionally, they each propose that a second “scaffolding” Hsp70, with no client bound, is required for client delivery and loading. Here, I confirm previous results showing that the BiP_SBD forms a stabilizing contact with the Grp94 dimer on the same arm to which BiP is bound. I identify three residues within the Grp94 cleft important for client anchoring. Additionally, beyond determining compatibility with the semi-closed loading complex, Hsp90 inhibitors appear to allosterically modulate client mobility during the client transfer process. I also demonstrate that high ATP concentrations maintain BiP in a lid-open state incompatible with binding to Grp94, and client binding stimulates hydrolysis and triggers a lid-closed conformation that supports Grp94 association. These results suggest that a scaffolding BiP is unlikely to associate with Grp94 prior to client binding, challenging the two-BiP model. Together, these findings provide new insight into the mechanistic and structural determinants that coordinate the client handoff process from BiP to Grp94.