Abstract
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 how ADP-ribosylation proceeds are still poorly understood. Crystal structures of C3-RhoA complex reveal that C3 recognizes RhoA via the 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 an active substrate with combined mutations in the C3-Rho GTPase interface. Moreover, the structure reflects the close relationship among Gln-183 in the QXE motif (C3), a modified Asn-41 residue (RhoA) and NC1 of NAD(H), which suggests that C3 is the prototype ART. These structures show directly for the first time that the ARTT loop is the key to target protein recognition, and they also serve to bridge the gaps among independent studies of Rho GTPases and C3.
Highlights
Bacterial ADP-ribosylating toxin C3 has long been used to study the diverse regulatory functions of Rho GTPases
To investigate how C3 from Bacillus cereus (C3cer) recognizes RhoA, we determined the crystal structure of apo(NADϩ-free)-C3cer complexed with human RhoA(GTP) at 1.8 Å resolution (Fig. 2A and Table 1)
Judged from the fact of ADP-ribosylation occurring in the C3-RhoA(GTP) crystal, it is important to note that the structure presented here is the active complex (Fig. 2F)
Summary
Bacterial ADP-ribosylating toxin C3 has long been used to study the diverse regulatory functions of Rho GTPases. The structure reflects the close relationship among Gln-183 in the QXE motif (C3), a modified Asn-41 residue (RhoA) and NC1 of NAD(H), which suggests that C3 is the prototype ART These structures show directly for the first time that the ARTT loop is the key to target protein recognition, and they serve to bridge the gaps among independent studies of Rho GTPases and C3. Han and Tainer [32] proposed that the bipartite ADPribosylating toxin turn-turn (ARTT) loop, which consists of turns 1 and 2, is responsible for substrate recognition and is crucial for the ARTase activity of C3-like exoenzymes and binary toxins This finding has prompted the examination of the conformation and significance of the ARTT loop within the NADϩ-bound and -free structures of C3 [33, 34]. These structures provide the first direct evidence that the ARTT loop is essential for target protein recognition and explain the recognition between C3 and RhoA and provide insight into the asparagine ART reaction
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