Crystal structure and novel recognition motif of Rho ADP-ribosylating C3 exoenzyme from Clostridium botulinum: Structural insights for recognition specificity and catalysis

Clostridium botulinum C3 exoenzyme inactivates the small GTP-binding protei n family Rho by ADP-ribosylating asparagine 41, which depolymerizes the act in cytoskeleton. C3 thus represents a major family of the bacterial toxins that transfer the ADP-ribose moiety of NAD to specific amino acids in accep tor proteins to modify key biological activities in eukaryotic cells, inclu ding protein synthesis, differentiation, transformation, and intracellular signaling. The 1.7 Angstrom resolution C3 exoenzyme structure establishes t he conserved features of the core NAD-binding beta -sandwich fold with othe r ADP-ribosylating toxins despite little sequence conservation. Importantly , the central core of the C3 exoenzyme structure is distinguished by the ab sence of an active site loop observed in many other ADP-ribosylating toxins . Unlike the ADP-ribosylating toxins that possess the active site loop near the central core, the C3 exoenzyme replaces the active site loop with an a lpha -helix, alpha3. Moreover, structural and sequence similarities with th e catalytic domain of vegetative insecticidal protein 2 (VIP2), an actin AD P-ribosyltransferase, unexpectedly implicates two adjacent, protruding turn s, which join beta5 and beta6 of the toxin core fold, as a novel recognitio n specificity motif for this newly defined toxin family. Turn 1 evidently p ositions the solvent-exposed, aromatic side-chain of Phe209 to interact wit h the hydrophobic region of Rho adjacent to its GTP-binding site. Turn 2 ev idently both places the Gln212 side-chain for hydrogen bonding to recognize Rho Asn41 for nucleophilic attack on the anomeric carbon of NAD ribose and holds the key Glu214 catalytic side-chain in the adjacent catalytic pocket . This proposed bipartite ADP-ribosylating toxin turn-turn (ARTT) motif pla ces the VIP2 and C3 toxin classes into a single ARTT family characterized b y analogous target protein recognition via turn 1 aromatic and turn 2 hydro gen-bonding side-chain moieties. Turn 2 centrally anchors the catalytic Glu 214 within the ARTT motif, and furthermore distinguishes the C3 toxin class by a conserved turn 2 Gin and the VIP2 binary toxin class by a conserved t urn 2 Glu for appropriate target side-chain hydrogen-bonding recognition. T aken together, these structural results provide a molecular basis for under standing the coupled activity and recognition specificity for C3 and for th e newly defined ARTT toxin family, which acts in the depolymerization of th e actin cytoskeleton. This beta5 to beta6 region of the toxin fold represen ts an experimentally testable and potentially general recognition motif reg ion for other ADP-ribosylating toxins that have a similar beta -structure f ramework. (C) 2001 Academic Press.