Mapping the zinc ligands of S100A2 by site-directed mutagenesis

S100 family proteins are characterized by short individual N and C termini and a conserved central part, harboring two Ca2+-binding EF-hands, one of t hem highly conserved among EF-hand family proteins and the other characteri stic for S100 proteins. In addition to Ca2+ several members of the S100 pro tein family, including S100A2, bind Zn2+. Two regions in the amino acid seq uences of S100 proteins, namely the helices of the N-terminal EF-hand motif and the very C-terminal loop are believed to be involved in Zn2+-binding d ue to the presence of histidine and/or cysteine residues, Human S100A2 cont ains four cysteine residues, each of them located at positions that may be important for Zn2+ binding. We have now constructed and purified 10 cystein e-deficient mutants of human S100A2 by site-directed mutagenesis and invest igated the contribution of the individual cysteine residues to Zn2+ binding , Here we show that Cys(1(3)) (the number in parentheses indicating the pos ition in the sequence of S100A2) is the crucial determinant for Zn2+ bindin g in association with conformational changes as determined by internal tyro sine fluorescence. Solid phase Zn2+ binding assays also revealed that the C -terminal residues Cys(3(S7)) and Cys(4(94)) mediated a second type of Zn2 binding, not associated with detectable conformational changes in the mole cule. Cys(2(22)), by contrast, which is located within the first EF hand mo tif affected neither Ca2+ nor Zn2+ binding, and a Cys "null" mutant was ent irely incapable of ligating Zn2+, These results provide new information abo ut the mechanism and the site(s) of zinc binding in S100A2.