Adenosine 5′-triphosphate-dependent vitamin D sterol binding to heat shock protein-70 chaperones

Chaperone proteins in the heat shock protein-70 family possess endogenous ATP binding and ATPase activity and interact with intracellular protein substrates in an ATP-dependent manner; the hydrolysis of ATP to ADP results in an increase in the affinity of the chaperone for protein substrates. Heat shock protein-70s can also specifically interact with 25-hydroxylated vitamin D metabolites. Using constitutively expressed heat shock protein-70 (hsc70) as chaperone, here we demonstrate that vitamin D metabolite binding to hsc70 is also ATP dependent. Transient overexpression of an hsc70green fluorescent protein chimeric construct in primate kidney cells resulted in a 6-fold increase in specific, extractable 25-hydroxyvitamin D-3 binding. When ATPase capability of hsc70 was disabled, this increase was completely blocked. In solution, the binding of 25-hydroxylated vitamin D metabolites to hsc70 was significantly increased (P < 0.01) in the presence of ATP and a nonmetabolizable ATP analog. The ATP-directed increase in specific binding resulted from an increase in the abundance of relatively high-affinity hormone-binding sites (K-d, similar to 0.24 nM). These results suggest that ATP hydrolysis to ADP would favor the release of vitamin D from a donor hsc70 molecule at a time when an hsc70-bound acceptor protein substrate is anchored to the chaperone with relative avidity. We theorize that the endogenous ATPase activity of hsc70 promotes the transfer of vitamin D sterols to other intracellular vitamin D binding proteins, such as the vitamin D receptor and vitamin D hydroxylases, to which hsc70 is known to bind.