Abstract
We have solved the crystal structures of Clostridium botulinum C3 exoenzyme free and complexed to NAD in the same crystal form, at 2.7 and 1.95 A, respectively. The asymmetric unit contains four molecules, which, in the free form, share the same conformation. Upon NAD binding, C3 underwent various conformational changes, whose amplitudes were differentially limited in the four molecules of the crystal unit. A major rearrangement concerns the loop that contains the functionally important ARTT motif (ADP-ribosyltransferase toxin turn-turn). The ARTT loop undergoes an ample swinging motion to adopt a conformation that covers the nicotinamide moiety of NAD. In particular, Gln-212, which belongs to the ARTT motif, flips over from a solvent-exposed environment to a buried conformation in the NAD binding pocket. Mutational experiments showed that Gln-212 is neither involved in NAD binding nor in the NAD-glycohydrolase activity of C3, whereas it plays a critical role in the ADP-ribosyl transfer to the substrate Rho. We observed additional NAD-induced movements, including a crab-claw motion of a subdomain that closes the NAD binding pocket. The data emphasized a remarkable NAD-induced plasticity of the C3 binding pocket and suggest that the NAD-induced ARTT loop conformation may be favored by the C3-NAD complex to bind to the substrate Rho. Our structural observations, together with a number of mutational experiments suggest that the mechanisms of Rho ADP-ribosylation by C3-NAD may be more complex than initially anticipated.
Highlights
Many bacterial toxins ADP-ribosylate nucleotide-binding proteins that are involved in essential cell functions
To investigate the role of Ser-174 in the catalytic mechanism, we prepared the S174A mutant and tested its ADP-ribosyltransferase activity. This mutant retains the wild-type ADP-ribosyltransferase activity (Fig. 6b), suggesting that Ser-174 plays no major role in C. botulinum C3 exoenzyme activity. This structural study has shown that binding of NAD to the C3 exoenzyme has induced various conformational changes, FIG. 6
The first one concerns the functionally important ARTT loop [14], which undergoes an ample motion that occurs only in the absence of local crystal-packing constraints. Such a movement is not unique to C3 because upon NAD analogs binding to P. aeruginosa exotoxin A the region equivalent to the ARTT loop undergoes a conformational change [22]
Summary
Protein Expression—The mature form of C. botulinum C3 exoenzyme (residues 41–251) was subcloned into the pET-28a vector, expressed in the BL21 (DE3) strain of Escherichia coli, and purified to homogeneity in one step by cation exchange chromatography (CM-Sepharose Fast Flow). To measure ADP-ribosyltransferase activity of C3 wild-type or its mutants, 0.1 nmol of the exoenzyme diluted in 10 l of Tris 50 mM, pH 7.5, containing 100 mM NaCl, 10 mM MgCl2, 20 mM dithiothreitol and 1.3 M [32P]NAD was incubated for 1 h at 37 °C with 0.05 nmol of RhoA. This mixture was loaded on a 12% SDS polyacrylamide gel, and radioactive RhoA was directly detected and quantified on the dried gel by an imaging system (Storm 840, Molecular Dynamics). The data were fitted using the steady state affinity (Req versus C) option available within BIAevaluation 3 software
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