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PDBsum entry 3cjb
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Structural protein
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PDB id
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3cjb
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References listed in PDB file
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Key reference
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Title
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Connecting actin monomers by iso-Peptide bond is a toxicity mechanism of the vibrio cholerae martx toxin.
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Authors
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D.S.Kudryashov,
Z.A.Durer,
A.J.Ytterberg,
M.R.Sawaya,
I.Pashkov,
K.Prochazkova,
T.O.Yeates,
R.R.Loo,
J.A.Loo,
K.J.Satchell,
E.Reisler.
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Ref.
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Proc Natl Acad Sci U S A, 2008,
105,
18537-18542.
[DOI no: ]
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PubMed id
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Abstract
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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.
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Figure 3.
Inhibition of actin polymerization by the ACD induced
cross-linking. (A–C) Polymerization and cross-linking of 10
μM rabbit skeletal actin in the presence or absence of ACD were
initiated simultaneously by adding 1.0 mM MgCl[2] and 50 mM KCl
and monitored by light scattering (A), sedimentation assay (B),
and electron microscopy (C). Compared with polymerization of
actin in the absence of ACD, the polymerization was strongly
inhibited at 1:1000 mole ratio of ACD to actin and it was
blocked completely at 1:100 mole ratio to actin. ACD
cross-linked actin oligomers form aggregates (C), which do not
pellet during ultracentrifugation (B). In all cases, s and p
designate supernatant and pellet fractions, respectively.
(D–F) The increase in light scattering upon addition of 15 μM
phalloidin (red trace) or 10 μM cofilin (blue trace) indicates
that the polymerization of ACD cross-linked actin oligomers (at
1:100 mole ratio of ACD to actin) can be rescued by these
agents. To estimate the extent of polymerization and the
appearance of the filaments, samples from (D) were analyzed by a
sedimentation assay (E) and electron microscopy (F).
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Figure 4.
Mechanism of the actin cytoskeleton disruption by MARTX[Vc].
Upon transport through the cytoplasmic membrane of the host
cell, the cysteine protease domain (CPD) of MARTX[Vc] cleaves
and releases into the cytoplasm functional domains, the
Rho-inactivation domain (RID) and the actin cross-linking domain
(ACD). RID shifts equilibrium from F- to G-actin by affecting
Rho signaling via an unknown mechanism. ACD uses the enriched
G-actin pool, maintained by thymosin β4 and profilin, as a
substrate for covalent cross-linking dependent on the hydrolysis
of ATP. In the resulting oligomers of actin, residue K50 of each
actin protomer is connected via an iso-peptide bond with E270 of
an adjacent protomer in a conformation incompatible with
polymerization. This results in irreversible disruption of the
actin cytoskeleton. The white background highlights the
mechanism of action elucidated for ACD in the present study.
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