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 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.
Stradal
T B
TB
Institute of Molecular Biology, Department of Cell Biology, Austrian Academy of Sciences, Billrothstrasse 11, A-5020 Salzburg, Austria.
Troxler
H
H
Heizmann
C W
CW
Gimona
M
M
D001665:Binding Sites; D002630:Chemotactic Factors / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D009418:S100 Proteins / Q000737:chemistry* / Q000235:genetics; D015032:Zinc / Q000737:chemistry*
D002630:Chemotactic Factors; D008024:Ligands; D011994:Recombinant Proteins; D009418:S100 Proteins; C098997:S100A2 protein, human; D015032:Zinc