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The Pre‐N Domain is a Distinct Feature of Grp94 that is Essential for Client Maturation and Regulation

The chaperone cycle of hsp90s involves a series of large conformational shifts between open and closed dimer states that are driven by the binding of ATP to the N‐terminal domain (NTD). Unlike its cytosolic counterpart, the ER hsp90 paralog, Grp94, does not possess a network of co‐chaperones to regulate dimer closure and modulate the rate of ATP hydrolysis. Although hsp90s are highly homologous with each other, regions of divergence may lead to the functional differences that are key to Grp94's adaptation to the ER. One such region, the Pre‐N domain, precedes the NTD and varies substantially in length and sequence among hsp90s, with the Grp94 Pre‐N domain (50 a.a.) dwarfing the Pre‐N domains of the other paralogs (Trap‐1: 24 aa; Hsp90a: 16 aa; Hsp90b: 11 aa). For Grp94, the Pre‐N domain slows the rate of ATP hydrolysis but the mechanism by which it does so and its role in client maturation are not understood. Here we describe crystal structures of Grp94 containing the Pre‐N domain in open and closed dimer states. Surprisingly for a significant regulatory element, the Pre‐N domain makes only minimal stabilizing contacts with the opposite NTD in the closed dimer state. In the open dimer state, however, the Pre‐N domain adopts a novel trajectory and secondary structure that may regulate the ATP‐bound conformation through allosteric auto‐inhibition of the NTD. Our functional analysis further revealed that both the length and sequence composition of the Pre‐N domain are required for client proteins to reach the cell surface. We are currently investigating the interplay between the Pre‐N domain and other ER folding factors, such as post‐translational modifications, to understand the role of the Pre‐N domain in client maturation. Taken together, these results imply a functional role for the Grp94 Pre‐N domain that mimics that of the cytoplasmic Hsp90 co‐chaperones, which are missing in the ER.