Biochemical and structural studies on the high affinity of Hsp70 for ADP

The molecular chaperone 70-kDa heat shock protein (Hsp70) is driven by ATP hydrolysis and ADP-ATP exchange. ADP dissociation from Hsp70 is reportedly slow in the presence of inorganic phosphate (P-i). In this study, we investigated the interaction of Hsp70 and its nucleotide-binding domain (NBD) with ADP in detail, by isothermal titration calorimetry measurements and found that Mg2+ ion dramatically elevates the affinity of Hsp70 for ADP. On the other hand, P-i increased the affinity in the presence of Mg2+ ion, but not in its absence. Thus, P-i enhances the effect of the Mg2+ ion on the ADP binding. Next, we determined the crystal structures of the ADP-bound NBD with and without Mg2+ ion. As compared with the Mg2+ ion-free structure, the ADP- and Mg2+ ion-bound NBD contains one Mg2+ ion, which is coordinated with the beta-phosphate group of ADP and associates with Asp10, Glu175, and Asp199, through four water molecules. The Mg2+ ion is also coordinated with one P-i molecule, which interacts with Lys71, Glu175, and Thr204. In fact, the mutations of Asp10 and Asp199 reduced the affinity of the NBD for ADP, in both the presence and the absence of P-i. Therefore, the Mg2+ ion-mediated network, including the P-i and water molecules, increases the affinity of Hsp70 for ADP, and thus the dissociation of ADP is slow. In ADP-ATP exchange, the slow ADP dissociation might be rate-limiting. However, the nucleotide-exchange factors actually enhance ADP release by disrupting the Mg2+ ion-mediated network.