Recognition of RhoA by Clostridium botulinum C3 exoenzyme

The C3-like ADP-ribosyltransferases exhibit a very confined substrate speci ficity compared with other Rho-modifying bacterial toxins; they selectively modify the RhoA -B, and -C isoforms but not other members of the Rho or Ra s subfamilies. In this study, the amino acid residues involved in the RhoA substrate recognition by C3 from Clostridium botulinum are identified by ap plying mutational analyses of the nonsubstrate Rac. First, the minimum doma in responsible for the recognition by C3 was identified as the N-terminal 9 0 residues. Second, the combination of the N-terminal basic amino acids ((R ho)Arg(5)-Lys(6)), the acid residues (Rho)Glu(47) and (Rho)GlU(54) only sli ghtly increases ADP-ribosylation but fully restores the binding of the resp ective mutant Rac to C3. Third, the residues (Rho)GlU(40) and (Rho)Val(43) also participate in binding to C3 but they are mainly involved in the corre ct formation of the ternary complex between Rho, C3, and NAD(+). Thus, thes e six residues (Arg(5), Lys(6), Glu(40), Val(43), Glu(47), and Glu(54)) dis tributed over the N-terminal part of Rho are involved in the correct bindin g of Rho to C3. Mutant Rac harboring these residues shows a kinetic propert y with regard to ADP-ribosylation, which is identical with that of RhoA. Di fferences in the conformation of Rho given by the nucleotide occupancy have only minor effects on ADP-ribosylation.