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.
Project description: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 A. 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 alpha4 and strand beta6 (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.
Project description:Besides botulinum C2 toxin, Clostridium botulinum type C produces another ADP-ribosyltransferase, which we termed 'C3'. ADP-ribosyltransferase C3 has a molecular mass of 25 kDa and modifies 21-24 kDa protein(s) in platelet and brain membranes. C3 was about 1000 times more potent than botulinum C1 toxin in ADP-ribosylation of membrane proteins. C3-catalysed ADP-ribosylation of the 21-24 kDa protein(s) was decreased by stable guanosine triphosphates, with the potency order GTP[S] much greater than p[NH]ppG greater than p[CH2]ppG. GTP[S] inhibited the ADP-ribosylation caused by C3 by maximally 70-80%, with half-maximal and maximal effects occurring at 0.3 and 10 microM-GTP[S] respectively. The concomitant addition of GTP decreased the inhibitory effect of GTP[S]. GTP[S]-induced inhibition of ADP-ribosylation was resistant to washing of pretreated platelet membranes. The data suggest that the novel botulinum ADP-ribosyltransferase C3 modifies eukaryotic 21-24 kDa guanine nucleotide-binding protein(s).
Project description:C3-like exoenzymes are ADP-ribosyltransferases that specifically modify some Rho GTPase proteins, leading to their sequestration in the cytoplasm, and thus inhibiting their regulatory activity on the actin cytoskeleton. This modification process goes through three sequential steps involving NAD-hydrolysis, Rho recognition, and binding, leading to Rho ADP-ribosylation. Independently, three distinct residues within the ARTT loop of the C3 exoenzymes are critical for each of these steps. Supporting the critical role of the ARTT loop, we have shown previously that it adopts a distinct conformation upon NAD binding. Here, we present seven wild-type and ARTT loop-mutant structures of C3 exoenzyme of Clostridium botulinum free and bound to its true substrate, NAD, and to its NAD-hydrolysis product, nicotinamide. Altogether, these structures expand our understanding of the conformational diversity of the C3 exoenzyme, mainly within the ARTT loop.
Project description:C3 protein toxins produced by Clostridium (C.) botulinum and C. limosum are mono-ADP-ribosyltransferases, which specifically modify the GTPases Rho A/B/C in the cytosol of monocytic cells, thereby inhibiting Rho-mediated signal transduction in monocytes, macrophages, and osteoclasts. C3 toxins are selectively taken up into the cytosol of monocytic cells by endocytosis and translocate from acidic endosomes into the cytosol. The C3-catalyzed ADP-ribosylation of Rho proteins inhibits essential functions of these immune cells, such as migration and phagocytosis. Here, we demonstrate that C3 toxins enter and intoxicate dendritic cells in a time- and concentration-dependent manner. Both immature and mature human dendritic cells efficiently internalize C3 exoenzymes. These findings could also be extended to the chimeric fusion toxin C2IN-C3lim. Moreover, stimulated emission depletion (STED) microscopy revealed the localization of the internalized C3 protein in endosomes and emphasized its potential use as a carrier to deliver foreign proteins into dendritic cells. In contrast, the enzyme C2I from the binary C. botulinum C2 toxin was not taken up into dendritic cells, indicating the specific uptake of C3 toxins. Taken together, we identified human dendritic cells as novel target cells for clostridial C3 toxins and demonstrated the specific uptake of these toxins via endosomal vesicles.
Project description:C3 exoenzyme is a mono-ADP-ribosyltransferase (ART) that catalyzes transfer of an ADP-ribose moiety from NAD(+) to Rho GTPases. C3 has long been used to study the diverse regulatory functions of Rho GTPases. How C3 recognizes its substrate and how ADP-ribosylation proceeds are still poorly understood. Crystal structures of C3-RhoA complex reveal that C3 recognizes RhoA via the switch I, switch II, and interswitch regions. In C3-RhoA(GTP) and C3-RhoA(GDP), switch I and II adopt the GDP and GTP conformations, respectively, which explains why C3 can ADP-ribosylate both nucleotide forms. Based on structural information, we successfully changed Cdc42 to an active substrate with combined mutations in the C3-Rho GTPase interface. Moreover, the structure reflects the close relationship among Gln-183 in the QXE motif (C3), a modified Asn-41 residue (RhoA) and NC1 of NAD(H), which suggests that C3 is the prototype ART. These structures show directly for the first time that the ARTT loop is the key to target protein recognition, and they also serve to bridge the gaps among independent studies of Rho GTPases and C3.
Project description:Clostridium botulinum exoenzyme C3 is the prototype of C3-like ADP-ribosyltransferases that modify the GTPases RhoA, B, and C. C3 catalyzes the transfer of an ADP-ribose moiety from the co-substrate nicotinamide adenine dinucleotide (NAD) to asparagine-41 of Rho-GTPases. Although C3 does not possess cell-binding/-translocation domains, C3 is able to efficiently enter intact cells, including neuronal and macrophage-like cells. Conventionally, the detection of C3 uptake into cells is carried out via the gel-shift assay of modified RhoA. Since this gel-shift assay does not always provide clear, evaluable results an additional method to confirm the ADP-ribosylation of RhoA is necessary. Therefore, a new monoclonal antibody has been generated that specifically detects ADP-ribosylated RhoA/B, but not RhoC, in Western blot and immunohistochemical assay. The scFv antibody fragment was selected by phage display using the human naive antibody gene libraries HAL9/10. Subsequently, the antibody was produced as scFv-Fc and was found to be as sensitive as a commercially available RhoA antibody providing reproducible and specific results. We demonstrate that this specific antibody can be successfully applied for the analysis of ADP-ribosylated RhoA/B in C3-treated Chinese hamster ovary (CHO) and HT22 cells. Moreover, ADP-ribosylation of RhoA was detected within 10 min in C3-treated CHO wild-type cells, indicative of C3 cell entry.
Project description:BACKGROUND: Cholix toxin is an ADP-ribosyltransferase found in non-O1/non-O139 strains of Vibrio cholera. The catalytic fragment of cholix toxin was characterized as a diphthamide dependent ADP-ribosyltransferase. RESULTS: Our studies on the enzymatic activity of cholix toxin catalytic fragment show that the transfer of ADP-ribose to toxin takes place by a predominantly intramolecular mechanism and results in the preferential alkylation of arginine residues proximal to the NAD+ binding pocket. Multiple arginine residues, located near the catalytic site and at distal sites, can be the ADP-ribose acceptor in the auto-reaction. Kinetic studies of a model enzyme, M8, showed that a diffusible intermediate preferentially reacted with arginine residues in proximity to the NAD+ binding pocket. ADP-ribosylarginine activity of cholix toxin catalytic fragment could also modify exogenous substrates. Auto-ADP-ribosylation of cholix toxin appears to have negatively regulatory effect on ADP-ribosylation of exogenous substrate. However, at the presence of both endogenous and exogenous substrates, ADP-ribosylation of exogenous substrates occurred more efficiently than that of endogenous substrates. CONCLUSIONS: We discovered an ADP-ribosylargininyl activity of cholix toxin catalytic fragment from our studies in auto-ADP-ribosylation, which is mediated through diffusible intermediates. The lifetime of the hypothetical intermediate exceeds recorded and predicted lifetimes for the cognate oxocarbenium ion. Therefore, a diffusible strained form of NAD+ intermediate was proposed to react with arginine residues in a proximity dependent manner.
Project description:The ADP-ribosylating toxins (ADPRTs) produced by pathogenic bacteria modify intracellular protein and affect eukaryotic cell function. Actin-specific ADPRTs (including Clostridium perfringens iota-toxin and Clostridium botulinum C2 toxin) ADP-ribosylate G-actin at Arg-177, leading to disorganization of the cytoskeleton and cell death. Although the structures of many actin-specific ADPRTs are available, the mechanisms underlying actin recognition and selective ADP-ribosylation of Arg-177 remain unknown. Here we report the crystal structure of actin-Ia in complex with the nonhydrolyzable NAD analog betaTAD at 2.8 A resolution. The structure indicates that Ia recognizes actin via five loops around NAD: loop I (Tyr-60-Tyr-62 in the N domain), loop II (active-site loop), loop III, loop IV (PN loop), and loop V (ADP-ribosylating turn-turn loop). We used site-directed mutagenesis to confirm that loop I on the N domain and loop II are essential for the ADP-ribosyltransferase activity. Furthermore, we revealed that Glu-378 on the EXE loop is in close proximity to Arg-177 in actin, and we proposed that the ADP-ribosylation of Arg-177 proceeds by an SN1 reaction via first an oxocarbenium ion intermediate and second a cationic intermediate by alleviating the strained conformation of the first oxocarbenium ion. Our results suggest a common reaction mechanism for ADPRTs. Moreover, the structure might be of use in rational drug design to block toxin-substrate recognition.
Project description:ADP-ribosylation of proteins is emerging as an important regulatory mechanism. Depending on the family member, ADP-ribosyltransferases either conjugate a single ADP-ribose to a target or generate ADP-ribose chains. Here we characterize Parp9, a mono-ADP-ribosyltransferase reported to be enzymatically inactive. Parp9 undergoes heterodimerization with Dtx3L, a histone E3 ligase involved in DNA damage repair. We show that the Dtx3L/Parp9 heterodimer mediates NAD+-dependent mono-ADP-ribosylation of ubiquitin, exclusively in the context of ubiquitin processing by E1 and E2 enzymes. Dtx3L/Parp9 ADP-ribosylates the carboxyl group of Ub Gly76. Because Gly76 is normally used for Ub conjugation to substrates, ADP-ribosylation of the Ub carboxyl terminus precludes ubiquitylation. Parp9 ADP-ribosylation activity therefore restrains the E3 function of Dtx3L. Mutation of the NAD+ binding site in Parp9 increases the DNA repair activity of the heterodimer. Moreover, poly(ADP-ribose) binding to the Parp9 macrodomains increases E3 activity. Dtx3L heterodimerization with Parp9 enables NAD+ and poly(ADP-ribose) regulation of E3 activity.
Project description:NAD+ glycohydrolase (NADase) and non-enzymic ADP-ribosylation have been thought to be involved in the regulation of mitochondrial Ca2+ fluxes. In this study it was found that several conditions (5 mM nicotinamide, 5 mM 3-aminobenzamide, 2 mM EDTA, 1 mM ATP, 10 mM dithiothreitol) known to strongly inhibit the NADase decreased ADP-ribosylation in bovine liver mitochondrial membranes with [32P]NAD+ as substrate to only a limited extent, if at all. The reaction led to the specific modification of two proteins with apparent molecular masses of approx. 26 and 53 kDa. An excess of added free ADP-ribose diminished the incorporation of label from [32P]NAD+ only slightly. Dithiothreitol inactivated the NADase, whereas ADP-ribosylation was unaffected. At low concentrations (25 microM) ADP-ribosylation was efficient with NAD+, but not ADP-ribose, as substrate. Under these conditions mitochondrial ADP-ribosylation seems to occur as an enzymic reaction rather than a non-enzymic transfer of ADP-ribose previously liberated from NAD+ by NAD+ glycohydrolase. The chemical stability of the protein-ADP-ribose bonds in the mitochondrial membranes indicated that cysteine residues are the predominant acceptors. Moreover, yeast aldehyde dehydrogenase, known to be a substrate for thiol-associated ADP-ribosylation, was efficiently ADP-ribosylated by using the mitochondrial activity and NAD+ as substrate. The modification of a cysteine residue in the aldehyde dehydrogenase was verified by the observation that pretreatment of this acceptor protein with N-ethylmaleimide substantially decreased its modification. It is therefore concluded that bovine liver mitochondria contain a cysteine-specific ADP-ribosyltransferase.