title: Connecting actin monomers by iso-peptide bond is a toxicity mechanism of the Vibrio cholerae MARTX toxin.
abstract: The Gram-negative bacterium Vibrio cholerae is the causative agent of a severe diarrheal disease that afflicts three to five million persons annually, causing up to 200,000 deaths. Nearly all V. cholerae strains produce a large multifunctional-autoprocessing RTX toxin (MARTX(Vc)), which contributes significantly to the pathogenesis of cholera in model systems. The actin cross-linking domain (ACD) of MARTX(Vc) directly catalyzes a covalent cross-linking of monomeric G-actin into oligomeric chains and causes cell rounding, but the nature of the cross-linked bond and the mechanism of the actin cytoskeleton disruption remained elusive. To elucidate the mechanism of ACD action and effect on actin, we identified the covalent cross-link bond between actin protomers using limited proteolysis, X-ray crystallography, and mass spectrometry. We report here that ACD catalyzes the formation of an intermolecular iso-peptide bond between residues E270 and K50 located in the hydrophobic and the DNaseI-binding loops of actin, respectively. Mutagenesis studies confirm that no other residues on actin can be cross-linked by ACD both in vitro and in vivo. This cross-linking locks actin protomers into an orientation different from that of F-actin, resulting in strong inhibition of actin polymerization. This report describes a microbial toxin mechanism acting via iso-peptide bond cross-linking between host proteins and is, to the best of our knowledge, the only known example of a peptide linkage between nonterminal glutamate and lysine side chains.
authors.affiliation: Department of Chemistry and Biochemistry and Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA.
authors.forename: Dmitri S
authors.initials: DS
authors.lastname: Kudryashov
authors.forename: Zeynep A Oztug
authors.initials: ZA
authors.lastname: Durer
authors.forename: A Jimmy
authors.initials: AJ
authors.lastname: Ytterberg
authors.forename: Michael R
authors.initials: MR
authors.lastname: Sawaya
authors.forename: Inna
authors.initials: I
authors.lastname: Pashkov
authors.forename: Katerina
authors.initials: K
authors.lastname: Prochazkova
authors.forename: Todd O
authors.initials: TO
authors.lastname: Yeates
authors.forename: Rachel R Ogorzalek
authors.initials: RR
authors.lastname: Loo
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Loo
authors.forename: Karla J Fullner
authors.initials: KJ
authors.lastname: Satchell
authors.forename: Emil
authors.initials: E
authors.lastname: Reisler
mesh_terms: D000199:Actins / Q000737:chemistry*; D000818:Animals; D001427:Bacterial Toxins / Q000633:toxicity*; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D013058:Mass Spectrometry; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry*; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D014734:Vibrio cholerae / Q000737:chemistry*
chemical_list: D000199:Actins; D001427:Bacterial Toxins; D010455:Peptides