Mechanism of ATP hydrolysis in the Hsp70 BiP nucleotide-binding domain

The 70 kDa heat shock protein (Hsp70) family of molecular chaperones ensures protein biogenesis and homeostasis, driven by ATP hydrolysis. Here, we introduce in-cyclo NMR, an experimental setup that combines high-resolution NMR spectroscopy with an ATP recovery and a phosphate removal system. In-cyclo NMR simultaneously resolves kinetic rates and structural information along functional cycles of ATP-driven molecular machines. We benchmark the method on the nucleotide binding domain (NBD) of the human Hsp70 chaperone BiP. The protein cycles through ATP binding, hydrolysis, and two parallel pathways of product release. We determine the kinetic rates of all eleven underlying elementary reactions and show these to match independent measurements. The two product release pathways regulate the cycle duration dependent on the products concentration. Under physiological conditions, they are both used. The in-cyclo NMR method will serve as a platform for studies of ATP-driven functional cycles at a remarkable level of detail.