Rho GTPase Recognition by C3 Exoenzyme Based on C3-RhoA Complex Structure [Enzymology]

June 11th, 2015 by Toda, A., Tsurumura, T., Yoshida, T., Tsumori, Y., Tsuge, H.

C3 exoenzyme is a mono-ADP-ribosyltransferase (ART) that catalyzes transfer of an ADP-ribose moiety from NAD+ to Rho GTPases. C3 has long been used to study the diverse regulatory functions of Rho GTPases. How C3 recognizes its substrate and ADP-ribosylation proceeds are still poorly understood. Crystal structures of C3-RhoA complex reveal that C3 recognizes RhoA via switch I, switch II and interswitch regions. In C3-RhoA(GTP) and C3-RhoA(GDP), switch I and II adopt the GDP and GTP conformations, respectively, which explains why C3 can ADP-ribosylate both nucleotide forms. Based on structural information, we successfully changed Cdc42 to active substrate with combined mutations in the C3-Rho GTPase interface. Moreover, the structure reflects the close relationship among Gln183 in the QXE-motif (C3), a modified Asn41 residue (RhoA) and NC1 of NAD(H), which suggests C3 is the prototype ART. The structures show directly for the first time that the ARTT-loop is the key to target protein recognition and also serve to bridge the gaps among independent studies of Rho GTPases and C3.