Abstract
Protein folding is critical to cellular homeostasis, and molecular chaperones such as BiP, the endoplasmic reticulum (ER) specific Hsp70, are critical to protein folding. BiP works in coordination with ERdj3, an ER-specific Hsp40 chaperone containing a conserved J-domain (JD). The JD stimulates BiP ATP hydrolysis and the other part of ERdj3 can bind unfolded proteins, but a more detailed mechanistic understanding is needed to understand the functional benefit of the joint action of ERdj3 and BiP. In this study, we use biochemical and biophysical assays to investigate JD’s influence on the structure and activity of BiP. Our major finding is identifying a conformational intermediate of BiP during its ATPase cycle and determining that this intermediate is likely in the ADP-bound state. Förster resonance energy transfer (FRET) measurements with client peptides show that the client-bound state of BiP has a similar FRET efficiency to the proposed intermediate. Combining our data with structures of other Hsp70 chaperones, we propose that the BiP intermediate has a closed lid similar to existing structures of ADP-bound Hsp70s, and the intermediate is stabilized by both client peptides and the JD. Our data also indicates that another ADP-bound state of BiP with an open-lid conformation is in equilibrium with the BiP intermediate, and the lid-open state is minimally influenced by both client peptides and the JD. These findings allow us to construct a structural and mechanistic framework for the ATP-driven conformational cycle of BiP that integrates the influence of the JD and client peptides