Crystal structure of the C3bot–RalA complex reveals a novel type of action of a bacterial exoenzyme

Abstract

C3 exoenzymes from bacterial pathogens ADP-ribosylate and inactivate low-molecular-mass GTPases of the Rho subfamily. Ral, a Ras subfamily GTPase, binds the C3 exoenzymes from Clostridium botulinum and C. limosum with high affinity without being a substrate for ADP ribosylation. In the complex, the ADP-ribosyltransferase activity of C3 is blocked, while binding of NAD and NAD-glycohydrolase activity remain. Here we report the crystal structure of C3 from C. botulinum in a complex with GDP-bound RalA at 1.8 A resolution. C3 binds RalA with a helix-loop-helix motif that is adjacent to the active site. A quaternary complex with NAD suggests a mode for ADP-ribosyltransferase inhibition. Interaction of C3 with RalA occurs at a unique interface formed by the switch-II region, helix alpha3 and the P loop of the GTPase. C3-binding stabilizes the GDP-bound conformation of RalA and blocks nucleotide release. Our data indicate that C. botulinum exoenzyme C3 is a single-domain toxin with bifunctional properties targeting Rho GTPases by ADP ribosylation and Ral by a guanine nucleotide dissociation inhibitor-like effect, which blocks nucleotide exchange.

C3 exoenzymes, novel insights into structure and action of Rho-ADP-ribosylating toxins

“…In contrast, Ras, which is the closest homolog to Ral, does not interact with C3 (Pautsch et al 2005). However, ADP-ribosylation of RhoA by C3cer and C3stau2 was less or not influenced in the presence of Ral.…”

“…6). The biological relevance of the interface deduced by Pautsch et al was then verified by mutational analysis (Pautsch et al 2005). The sequence of the C3 helix-loop-helix motif is less conserved in C3cer and the C3stau isoforms, explaining the specificity of Ral for the two other toxins.…”

“…In addition, C3-like ADP-ribosyltransferases interact non-enzymatically with Ral without ADP-ribosylating the GTPase (Wilde et al 2002a;Pautsch et al 2005). Ral belongs to the Ras branch of low molecular GTPases and is involved in transcriptional activation (Hernandez-Munoz et al 2000), Ras-mediated cell transformation Urano et al 1996), vesicle trafficking (Shen et al 2001) and cytoskeletal rearrangements (Jullien-Flores et al 1995;Ohta et al 1999).…”

“…The molecular basis of the C3bot-RalA interaction was recently identified by two groups (Holbourn et al 2005;Pautsch et al 2005). The structure of a stochiometric RalA (GDP)-C3bot complex with an apparent K D value of ∼60 nM was determined by X-ray crystallography (Pautsch et al 2005).…”

“…The structure of a stochiometric RalA (GDP)-C3bot complex with an apparent K D value of ∼60 nM was determined by X-ray crystallography (Pautsch et al 2005). In the structure of this complex C3bot inserts a previously unrecognized helix-loop-helix motif into a deep pocket formed predominantly by the switch II region and helix α3 of RalA (Fig.…”