Characterization of ATP hydrolysis in the Hsp70 BiP nucleotide binding domain

The 70 kDa heat shock protein (Hsp70) family of molecular chaperones is crucial for protein biogenesis and homeostasis in all kingdoms of life. Hsp70 activity is driven by ATP hydrolysis in the nucleotide binding domain (NBD). Here, we report an experimental setup to resolve the functional cycle of Hsp70 in unprecedented spatial and temporal resolution. The method combines high-resolution NMR spectroscopy with embedded kinetic measurements to simultaneously resolve kinetic rates and structural information of the individual states of an Hsp70 functional cycle. We benchmark the method on the example of the NBD of the human Hsp70 chaperone BiP. Precision measurements connect the ATP hydrolysis rate ( k _cat ) and the ADP lifetime ( k _off ) to conventional bulk experiments and thus reveal that ADP-Pi release and not ATP hydrolysis is the limiting step of the cycle. Unlike commonly thought, the phosphate generated from ATP hydrolysis locks the ADP-Pi into the NBD, and thus decouples the ADP release rate from the effect of external factors such as the bulk phosphate and calcium concentration. The method will serve as a platform for studies of the Hsp70 protein family and their co-chaperones, including full-length constructs that have key roles in biogenesis and disease.