OVEREXPRESSION AND PURIFICATION OF HUMAN CALCINEURIN-ALPHA FROM ESCHERICHIA-COLI AND ASSESSMENT OF CATALYTIC FUNCTIONS OF RESIDUES SURROUNDING THE BINUCLEAR METAL CENTER

Calcineurin is an important signal-transducing enzyme in many cell typ es including T lymphocytes and is a common target for the immunosuppre ssants cyclosporin A and FK506. The crystal structures of both calcine urin [Griffith et al. (1995) Cell 82, 507-522; Kissinger et al. (1995) Nature 378, 641-644] and a related enzyme, protein phosphatase-l [Gol dberg et al. (1995) Nature 376, 745-753], revealed that this class of serine/threonine phosphatases contain in their putative active sites a binuclear metal center formed by an Asn, two Asp, and three His resid ues. In addition, one His and two Arg residues lie in close vicinity o f the binuclear metal centers. The importance of the binuclear metal c enter and its surrounding residues in catalysis by calcineurin has not been investigated experimentally. Herein, we report an efficient bact erial expression and purification system for human calcineurin or. Usi ng this system, a systematic alanine-scan mutagenesis on the residues surrounding the putative active site was performed. It was found that an intact binuclear metal center is essential for the catalytic activi ty of the enzyme. In addition, His151, Arg122, and Arg254 also exhibit ed either a loss or a dramatic decrease in catalytic activity upon mut ation into alanines. Interestingly, the Arg254Ala mutant retained a sm all but significant amount of catalytic activity toward the small subs trate p-nitrophenyl phosphate, but is completely inactive toward a pho sphopeptide substrate, suggesting that this arginine may be involved i n the binding of phosphoprotein substrates as well as in catalysis. As all the residues in the putative active site are conserved between di fferent eukaryotic serine/threonine phosphatases, these results should apply to all members of this family of protein phosphatases.