Structural organization of the guanine nucleotide exchange factor C3G

Abstract

C3G is a guanine nucleotide exchange factor (GEF) that activates the small GTPases Rap1 and R-Ras. C3G is involved in multiple cellular functions including adhesion, migration, cytoskeletal remodeling, cell proliferation, differentiation, transformation, and apoptosis. C3G (120 kDa) has a tripartite structure. The N-terminal region (N-C3G) mediates binding to E-cadherin at nascent adherent junctions. The structure of N-C3G is unknown and bears no similarity to other proteins. The central region contains five Pro-rich sequence motifs that mediate the interaction with protein that contain SH3 domains, such as Crk, p130Cas, Grb2, Hck and c-Abl. The central region also mediates binding to the TC-PTP phosphatase and to ß-catenin, which do not contain SH3 domains. The C-terminal catalytic region consists of a REM (Ras exchange motif) and a Cdc25H domain. Deletion of the N-terminal half of C3G increases the GEF activity, suggesting that this N-terminal half acts as a regulatory element. Yet, the mechanisms of autoinhibition of C3G remain unknown. Here we have identified and characterized an intramolecular interaction between the N-C3G and the catalytic region. We have identified residues important for maintaining the close conformation. This head-tail interaction in C3G resembles autoinhibitory conformations of other GEFs of the Cdc25H family, such as Sos1, Epac2, and RasGRP1. Finally, using recombinant N-C3G fragments we show that this region has a high content of α-helical structure. Sequence analysis suggests the presence of amphipathic helices that could fold in a helical bundle.