Structural basis of actin recognition and arginine ADP-ribosylation by Clostridium perfringens iota-toxin

Abstract

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.

Fig. 1.

Whole structure of actin–Ia–β TAD complex. (A) Ribbon representation of actin–Ia–β TAD. Actin (green) and Ia (N domain, marine blue; C domain, yellow) molecules and cofactors [βTAD, ATP, latrunculin A (LatA), and calcium (Ca)] are labeled. (B) Detailed stereoview of the area around βTAD (boxed in A).

Fig. 2.

Butterfly representation of recognition residues between Ia and actin. Roman numerals (I–V) show the five binding loops in Ia. The actin-recognition residues on five loops of Ia are shown as circles on the left. The catalytic residues of Ia around NAD (Tyr-251, Arg-295, Arg-352, Glu-378, and Glu-380) are shown as sticks. The Ia-recognition residues of actin are shown as circles on the right.

Fig. 3.

Comparison of the molecular recognition interface of Ia and actin-binding proteins. Actin (green), Ia (N domain, marine blue; C domain, yellow), and representative actin-binding proteins (cyan) are shown. Roman numerals (I–IV) show the actin subdomains.

Fig. 4.

Mechanism of Sn1 ADP-ribosylation based on the strain-alleviation model. (A) Actin–Ia–NAD model. The structure is the same as that for actin–Ia–βTAD except that NAD is substituted for βTAD. (B) Second cationic intermediate. The N-ribose of ADP-ribose is rotated via NP-NO5 based on NAD. The torsion angles of three residues (Arg-177 and Asp-179) were manipulated to maintain appropriate bond lengths between atoms. (C) Schematic of the Sn1 mechanism of Ia: First, nicotinamide cleavage occurs via an Sn1 reaction induced by an NMN ring-like structure; second, the first oxocarbenium cation intermediate is formed with a strained conformation; third, the second cationic intermediate is induced by alleviation of the strained conformation by NP-NO5 rotation; fourth, actin Arg-177 of Ia nucleophilically attacks the NC1.