The dimerization interface of the metastasis-associated protein S100A4 (Mts1) - In vivo and in vitro studies

The S100 calcium-binding proteins are implicated in signal transduction, mo tility, and cytoskeletal dynamics. The three-dimensional structure of sever al 5100 proteins revealed that the proteins form non-covalent dimers. Howev er, the mechanism of the S100 dimerization is still obscure. In this study we characterized the dimerization of S100A4 (also named Mts1) in vitro and in vivo. Analytical ultracentrifugation revealed that apoS100A4 was present in solution as a mixture of monomers and dimers in a rapidly reversible eq uilibrium (K-d = 4 +/- 2 muM) The binding of calcium promoted dimerization. Replacement of Tyr-75 by Phe resulted in the stabilization of the dimer. H elix IV is known to form the major part of the dimerization interface in ho mologous S100 proteins. By using the yeast two-hybrid system we showed that only a few residues of helix TV, namely Phe-72, Tyr-75, Phe-78, and Leu-79 , are essential for dimerization in vivo. A homology model demonstrated tha t these residues form a hydrophobic cluster on helix TV. Their role is to s tabilize the structure of individual subunits rather than provide specific interactions across the dimerization surface. Our mutation data showed that the specificity at the dimerization surface is not particularly stringent, which is consistent with recent: data indicating that S100 proteins can fo rm heterodimers.