title: 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.
authors.affiliation: Institute for Health Sciences and Faculty of Pharmaceutical Sciences, Tokushima Bunri University, 180 Yamashiro-cho, Tokushima 770-8514, Japan. tsuge@tokushima.bunri-u.ac.jp
authors.forename: Hideaki
authors.initials: H
authors.lastname: Tsuge
authors.forename: Masahiro
authors.initials: M
authors.lastname: Nagahama
authors.forename: Masataka
authors.initials: M
authors.lastname: Oda
authors.forename: Shinobu
authors.initials: S
authors.lastname: Iwamoto
authors.forename: Hiroko
authors.initials: H
authors.lastname: Utsunomiya
authors.forename: Victor E
authors.initials: VE
authors.lastname: Marquez
authors.forename: Nobuhiko
authors.initials: N
authors.lastname: Katunuma
authors.forename: Mugio
authors.initials: M
authors.lastname: Nishizawa
authors.forename: Jun
authors.initials: J
authors.lastname: Sakurai
mesh_terms: D036002:ADP Ribose Transferases / Q000737:chemistry* / Q000235:genetics; D000199:Actins / Q000737:chemistry*; D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000737:chemistry*; D000818:Animals; D001120:Arginine / Q000737:chemistry*; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics; D018360:Crystallography, X-Ray; D006868:Hydrolysis; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011817:Rabbits; D012266:Ribose / Q000737:chemistry*; D013844:Thiazoles / Q000737:chemistry
chemical_list: D000199:Actins; D001427:Bacterial Toxins; D013844:Thiazoles; C050737:iota toxin, Clostridium perfringens; C085445:beta-methylene thiazole-4-carboxamide adenine dinucleotide; D000244:Adenosine Diphosphate; D012266:Ribose; D001120:Arginine; D036002:ADP Ribose Transferases