Recognition of RhoA by Clostridium botulinum C3 exoenzyme

Abstract

The C3-like ADP-ribosyltransferases exhibit a very confined substrate specificity compared with other Rhomodifying bacterial toxins; they selectively modify the RhoA, -B, and -C isoforms but not other members of the Rho or Ras subfamilies. In this study, the amino acid residues involved in the RhoA substrate recognition by C3 from Clostridium botulinum are identified by applying mutational analyses of the nonsubstrate Rac. First, the minimum domain responsible for the recognition by C3 was identified as the N-terminal 90 residues. Second, the combination of the N-terminal basic amino acids ((Rho)Arg5-Lys6), the acid residues (Rho)Glu47 and (Rho)Glu54 only slightly increases ADP-ribosylation but fully restores the binding of the respective mutant Rac to C3. Third, the residues (Rho)Glu40 and (Rho)Val43 also participate in binding to C3 but they are mainly involved in the correct formation of the ternary complex between Rho, C3, and NAD+. Thus, these six residues (Arg5, Lys6, Glu40, Val43, Glu47, and Glu54) distributed over the N-terminal part of Rho are involved in the correct binding of Rho to C3. Mutant Rac harboring these residues shows a kinetic property with regard to ADP-ribosylation, which is identical with that of RhoA. Differences in the conformation of Rho given by the nucleotide occupancy have only minor effects on ADP-ribosylation.