HUMAN HSP70 MOLECULAR CHAPERONE BINDS 2 CALCIUM-IONS WITHIN THE ATPASE DOMAIN

Background: The 70 kDa heat shock proteins (Hsp70) are a family of mol ecular chaperones, which promote protein folding and participate in ma ny cellular functions. The Hsp70 chaperones are composed of two major domains. The N-terminal ATPase domain binds to and hydrolyzes ATP, whe reas the C-terminal domain is required for polypeptide binding. Cooper ation of both domains is needed for protein folding, The crystal struc ture of bovine Hsc70 ATPase domain (bATPase) has been determined and, more recently, the crystal structure of the peptide-binding domain of a related chaperone, DnaK, in complex with peptide substrate has been obtained. The molecular chaperone activity and conformational switch a re functionally linked with ATP hydrolysis. A high-resolution structur e of the ATPase domain is required to provide an understanding of the mechanism of ATP hydrolysis and how it affects communication between C - and N-terminal domains. Results: The crystal structure of the human Hsp70 ATPase domain (hATPase) has been determined and refined at 1.84 Angstrom, using synchrotron radiation at 120K. Two calcium sites were identified: the first calcium binds within the catalytic pocket, bridg ing ADP and inorganic phosphate, and the second calcium is tightly coo rdinated on the protein surface by Glu231, Asp232 and the carbonyl of His227, Overall, the structure of hATPase is similar to bATPase. Diffe rences between them are found in the loops, the sites of amino acid su bstitution and the calcium-binding sites. Human Hsp70 chaperone is pho sphorylated in vitro in the presence of divalent ions, calcium being t he most effective. Conclusions: The structural similarity of hATPase a nd bATPase and the sequence similarity within the Hsp70 chaperone fami ly suggest a universal mechanism of ATP hydrolysis among all Hsp70 mol ecular chaperones. Two calcium ions have been found in the hATPase str ucture. One corresponds to the magnesium site in bATPase and appears t o be important for ATP hydrolysis and in vitro phosphorylation, Local changes in protein structure as a result of calcium binding may facili tate phosphorylation, A small, but significant, movement of metal ions and sidechains could position catalytically important threonine resid ues for phosphorylation. The second calcium site represents a new calc ium-binding motif that can play a role in the stabilization of protein structure. We discuss how the information about catalytic events in t he active site could be transmitted to the peptide-binding domain.