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Research Database PMU-SQQUID

Mapping the zinc ligands of S100A2 by site-directed mutagenesis.
Stradal, TB; Troxler, H; Heizmann, CW; Gimona, M;
J Biol Chem. 2000; 275(18): 13219-13227.
Originalarbeiten (Zeitschrift)

PMU-Authors

Gimona Mario

Abstract

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 them highly conserved among EF-hand family proteins and the other characteristic for S100 proteins. In addition to Ca2+ several members of the S100 protein family, including S100A2, bind Zn2+. 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 Zn2+-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 Zn2+ 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 Zn2+ 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 Zn2+ binding in association with conformational changes as determined by internal tyrosine 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 molecule. Cys(2(22)), by contrast, which is located within the first EF hand motif affected neither Ca2+ nor Zn2+ binding, and a Cys "null" mutant was entirely incapable of ligating Zn2+, These results provide new information about the mechanism and the site(s) of zinc binding in S100A2.


Useful keywords (using NLM MeSH Indexing)

Binding Sites

Chemotactic Factors/chemistry*

Chemotactic Factors/genetics

Humans

Ligands

Mutagenesis, Site-Directed

Recombinant Proteins/chemistry

Recombinant Proteins/genetics

S100 Proteins/chemistry*

S100 Proteins/genetics

Zinc/chemistry*