ABSTRACT p130Cas (Crk-associated substrate) is a docking protein that is involved in assembly of focal adhesions and concomitant cellular signaling. It plays a role in physiological regulation of cell adhesion, migration, survival and proliferation, as well as in oncogenic transformation. The molecule consists of multiple protein-protein interaction motifs including a serine-rich region that is positioned between Crk and Src-binding sites. This study reports the first structure of a functional domain of Cas. The solution structure of the serine-rich region has been determined by nuclear magnetic resonance spectroscopy (NMR) demonstrating that this is a stable domain that folds as a four-helix bundle, a protein-interaction motif. The serine-rich region bears strong structural similarity to four-helix bundles found in other adhesion components like focal adhesion kinase, a-catenin, or vinculin. Potential sites for phosphorylation and interaction with the 14-3-3 family of cellular regulators are identified in the domain, and characterized by site-directed mutagenesis and binding assays. Mapping the degree of amino acid conservation onto the molecular surface revealed a patch of invariant residues near the C-terminus of the bundle, that may represent a new site for protein interaction.