Molecular recognition of an ADP-ribosylating Clostridium botulinum C3 exoenzyme by RalA GTPase

Abstract

C3 exoenzymes (members of the ADP-ribosyltranferase family) are produced by Clostridium botulinum (C3bot1 and -2), Clostridium limosum (C3lim), Bacillus cereus (C3cer), and Staphylococcus aureus (C3stau1-3). These exoenzymes lack a translocation domain but are known to specifically inactivate Rho GTPases in host target cells. Here, we report the crystal structure of C3bot1 in complex with RalA (a GTPase of the Ras subfamily) and GDP at a resolution of 2.66 Å. RalA is not ADP-ribosylated by C3 exoenzymes but inhibits ADP-ribosylation of RhoA by C3bot1, C3lim, and C3cer to different extents. The structure provides an insight into the molecular interactions between C3bot1 and RalA involving the catalytic ADP-ribosylating turn-turn (ARTT) loop from C3bot1 and helix ␣4 and strand ␤6 (which are not part of the GDP-binding pocket) from RalA. The structure also suggests a molecular explanation for the different levels of C3-exoenzyme inhibition by RalA and why RhoA does not bind C3bot1 in this manner.ADP-ribosylation ͉ protein-protein interaction ͉ x-ray crystallography B acteria produce many enzymes that show extraordinary specificity for mammalian intracellular proteins. The specificity of these bacterial enzymes has not only made them a valuable tool for elucidating the cellular functions of their targets but has also increased our understanding of protein interactions. Clostridium botulinum is no exception, producing two classes of enzymes that have very specific protein targets, the neurotoxins A-G and the ADP-ribosyltransferases C2, C3bot1, and C3bot2. C2 and C3bot are part of a larger family of ADP-ribosylating toxins (1, 2), including diphtheria toxin and cholera toxin, which cleave NAD and transfer ADP-ribose to target proteins. Although the members of this family have homologous enzymatic domains and similar active sites, these toxins ADP-ribosylate and, therefore, disable a range of cellular targets.

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

“…Another possible explanation for the inhibitory effect is that Ral prevents conformational changes in C3bot, which occur during the ADP-ribosylation. It is noteworthy that the crystal structure of the RAL-C3 complex reported by Acharya and coworkers (Holbourn et al 2005) predicts an interaction of the GTPase with the ARTT loop of C3. However, this model could not be supported by biochemical and mutational data (Pautsch et al 2005).…”

“…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).…”

Rho GTPase Recognition by C3 Exoenzyme Based on C3-RhoA Complex Structure

“…Holbourn et al (52) and Pautsch et al (53) have reported different structures for the C3-RalA complex; however, the former ␣4-ARTT model is likely to be a crystallographic artifact, based on crystallographic and mutational results (53). The model proposed by Pautsch et al (53) is that the loop between helices ␣3 and ␣4 (C3bot93-C3bot101) inserts into the groove of the switch I and II regions of RalA.…”

Novel Use of Botulinum Toxin to Ameliorate Arthrofibrosis: An Experimental Study in Rabbits

“…Although the members of this family have homologous enzymatic domains and similar active sites, these toxins ADP ribosylate and therefore, disable a range of cellular targets (Holbourn et al, 2005). Rho family GTPases controls the assembly of both cell-matrix and cell-cell adhesion complexes.…”

“…The specificity of these bacterial enzymes has not only made them a valuable tool for elucidating the cellular functions of their targets but has also increased our understanding of protein interactions (Holbourn et al, 2005). Clostridium botulinum is no exception, producing 2 classes of enzymes that have very specefic protein targets, neurotoxin A-G and the ADPribosyltrans-ferases C2, C3 bot 1, and C3 bot 2 (Holbourn et al, 2005). C2 and C3 bot are part of a larger family of ADPribosylating toxins, including diphtheria toxin and cholera toxin, which cleave NAD and transfer ADP-ribose to target proteins.…”