Making Ends Meet: The Importance of the N- and C-Termini for the Structure, Stability, and Function of the Third SH3 Domain of CIN85

SH3 domains are common structure, interaction, and regulation modules found in more than 200 human proteins. In this report, we studied the third SH3 domain from the human CIN85 adaptor protein, which plays an important role in both receptor tyrosine kinase downregulation and phosphatidylinositol 3 kinase inhibition. The structure of this domain includes an additional 90° kink after the last canonical β-strand and features unusual interactions between the termini well outside the boundaries of the standard SH3 domain definition. The extended portions of the domain are well-structured and held together entirely by side chain−side chain interactions. Extensive expression screening showed that these additional contacts provide significantly increased stability to the domain. A similar 90° kink is found in only one other SH3 domain structure, while side chain contacts linking the termini have never been described before. As a result of the increased size of CIN85 SH3 domain C, the proximal proline rich region is positioned such that a possible intramolecular interaction is structurally inhibited. Using the key interactions of the termini as the basis for sequence analysis allowed the identification of several SH3 domains with flanking sequences that could adopt similar structures. This work illustrates the importance of careful experimental analysis of domain boundaries even for a well-characterized fold such as the SH3 domain.