Identification and Characterization of Zinc Binding Sites in Protein Kinase C

Abstract

Metal ion coordination in the regulatory domain of protein kinase C (PKC) is suggested by the conservation of six cysteines and two histidines in two homologous regions found therein. By monitoring x-ray fluorescence from a purified sample of rat PKC beta I overexpressed in insect cells, direct evidence has been obtained that PKC beta I tightly binds four zinc ions (Zn2+) per molecule. Extended x-ray absorption fine structure (EXAFS) data are best fit by an average Zn2+ coordination of one nitrogen and three sulfur atoms. Of the plausible Zn2+ coordination models, only those featuring nonbridged Zn2+ sites accommodate the EXAFS data and all of the conserved potential ligands.