BiP is the only member of the Hsp70 chaperone family in the human endoplasmic reticulum (1). Its chaperone activity is driven by ATP binding and hydrolysis that trigger the conformational change regulating the docking and undocking of its nucleotide-binding domain (NBD) and substrate-binding domain (SBD). To achieve its many functions, BiP is regulated by several co-chaperones including the nucleotide-exchange factor (NEF) and J-domain protein (JDP). Although structure of intermediate states of BiP have been determined by x-ray crystallography (2) and specific segments of its functional cycle have been explored by NMR (3, 4) and FRET studies (5), characterization of the entire mechanism driving BiP function requires studies at the atomic level under working conditions in presence of its co-chaperone network.
Here, we report a study by real-time methyl NMR, under physiological conditions, of the BiP functional cycle in presence of its major cochaperones (JDP and NEF) and a native client protein. We demonstrate that the co-chaperones speed up the BiP functional cycle by tuning the equilibrium between its docked and undocked conformation, via regulating the allosteric communication between NBD and SBD. Furthermore, we reveal how the dynamic network of co-chaperones and client protein regulate BiP activity, providing for the first time at atomic resolution a time-resolved description of the BiP functional cycle. This study opens up new perspectives to understand how dysfunction in the regulation of BiP by its co-chaperones is linked to a broad range of BiP-related diseases such as cancer, cardiovascular and neurodegenerative disease.
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