Structural Investigation Of The Interaction Between The Tandem Sh3Domains Of C-Cbl-associated Protein (CAP) And Vinculin

As a vinculin-binding protein, CAP belongs to the vinexin family and has a unique structure consisting of a sorbin homology (SoHo) domain in the N terminus and three consectutive SH3domains in the C terminus. CAP impairs focal adhesion turnover and negatively regulates cell migration. Zhang et al. demonstrated that the designated mutants of CAP that disrupt its interaction with vinculin do not exhibit these abilities. The srtuctural mechanism of the interaciton between CAP and vinculin is not clear.Vinculin is comprised of three seven-helical bundle domains (D1, D2, and D3) and a four-helical bundle (D4) domain that is connected to a five-helical bundle (D5or vinculin tail) domain via a proline-rich hinge region, and the D1-D4domains form the vinculin head (Vh) domain. The first and second SH3domains of CAP that bind to the proline-rich region of vinculin have been reported to be responsible for the localization of CAP in cell-EXM adhesions.Although the structures of complexes consisting of a single SH3domain bound with proline-rich motifs have been previouly repoted, there is very limited knowledge about the structural information regarding multiple SH3domains complexed with their binding partners. In this study, we determined the solution structure of the tandem SH3domains of CAP using nuclar magnetic resonance (NMR) spectroscopy. Our structure and NMR relaxation data show that there is no fixed relative orientation between the SH3a and SH3b domains. NMR chemical shift perturbation and X-ray crystallography were further used to investigate the interaction interface of the tandem SH3domains of CAP with the proline-rich region of vinculin. Moreover, small angle X-ray scattering (SAXS) and negative staining EM were employed to analyze the complex structrue of the tandem SH3domains of CAP and vinculin. Through molecular dynamics (MD) simulations, we generated a structural model of the tandem SH3domains of CAP complexed with vinculin.Vinculin undergoes a conformational changes when localizing to focal adhesions. To investigate the influence of CAP to the activation of vinculin, we analyzed the complex structure of CAP and full-length vinculin. There is a conformational change in the D4domain observed in our SAXS results. Furthermore, we employed co-sedimentation assays to examine whether the binding of the tandem SH3domains of CAP to vinculin influences the activation of vinculin. Unfortunately, our co-sedimentation assays results indicate that the increase in tandem SH3domains causes almost no changes to the amount of vinculin bound with F-actin, suggesting that the conformational change of the D4domain may not disrupt its interaction with the D5domain of vinculin to facilitate the interaction between vinculin and F-actin.