Connecting actin monomers by iso-peptide bond is a toxicity mechanism of the Vibrio cholerae MARTX toxin.
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.
Kudryashov
Dmitri S
DS
Department of Chemistry and Biochemistry and Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA.
Durer
Zeynep A Oztug
ZA
Ytterberg
A Jimmy
AJ
Sawaya
Michael R
MR
Pashkov
Inna
I
Prochazkova
Katerina
K
Yeates
Todd O
TO
Loo
Rachel R Ogorzalek
RR
Loo
Joseph A
JA
Satchell
Karla J Fullner
KJ
Reisler
Emil
E
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*
D000199:Actins; D001427:Bacterial Toxins; D010455:Peptides