Abstract
S100 family proteins are characterized by short individual N and C termini and a conserved central part, harboring two Ca(2+)-binding EF-hands, one of them highly conserved among EF-hand family proteins and the other characteristic for S100 proteins. In addition to Ca(2+), several members of the S100 protein family, including S100A2, bind Zn(2+). Two regions in the amino acid sequences 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 Zn(2+)-binding due to the presence of histidine and/or cysteine residues. Human S100A2 contains four cysteine residues, each of them located at positions that may be important for Zn(2+) binding. We have now constructed and purified 10 cysteine-deficient mutants of human S100A2 by site-directed mutagenesis and investigated the contribution of the individual cysteine residues to Zn(2+) binding. Here we show that Cys(1(3)) (the number in parentheses indicating the position in the sequence of S100A2) is the crucial determinant for Zn(2+) binding in association with conformational changes as determined by internal tyrosine fluorescence. Solid phase Zn(2+) binding assays also revealed that the C-terminal residues Cys(3(87)) and Cys(4(94)) mediated a second type of Zn(2+) binding, not associated with detectable conformational changes in the molecule. Cys(2(22)), by contrast, which is located within the first EF hand motif affected neither Ca(2+) nor Zn(2+) binding, and a Cys "null" mutant was entirely incapable of ligating Zn(2+). These results provide new information about the mechanism and the site(s) of zinc binding in S100A2.
Highlights
The S100 family of small cation-binding proteins currently comprises 16 closely related members and two proteins containing the S100 sequence as part of a fusion protein
The addition of Zn2ϩ to the wild type S100A2 molecule in the presence or absence of Ca2ϩ led to an Conformational effects were determined by monitoring the changes of internal tyrosine fluorescence
Zn2ϩ binding to the ”N-terminal“ site is closely linked to conformational alterations in the dimeric S100A2 molecule, and the affinity for Zn2ϩ increases in response to Ca2ϩ the accessibility of the cysteine residue Cys1(3) itself may not be altered upon Ca2ϩ binding
Summary
The S100 family of small cation-binding proteins currently comprises 16 closely related members and two proteins containing the S100 sequence as part of a fusion protein These and 13 of the S100 genes are clustered in the epidermal differentiation complex located on human chromosome 1 (1). Brodersen and colleagues reported the crystal structures of the human S100A7 (psoriasin) dimer, both in the Zn2ϩ-loaded and the free form and determined two zinc binding sites per dimer (11) From their data, these authors further delineated two regions, one located in the first, variable EF-hand of one monomer and the second residing in the C terminus of the other monomer, which could function as potential ligands for zinc ions. The first EF-hand of S100 proteins has only low affinity for Ca2ϩ, is 2 amino acid residues longer than canonical EF-hands and, in the present case, contains a cysteine flanked by a highly conserved histidine residue. In the absence of a conserved sequence motif that would provide all of the four necessary ligand amino acids for the Zn2ϩ ion, we hypothesize an involvement of the
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