PDBsum entry 3cjb

Structural protein

PDB id: 3cjb

Contents

Protein chains

360 a.a.

125 a.a. *

Ligands

ATP

Metals

_CA ×2

Waters ×2

* Residue conservation analysis

PDB id:

3cjb

Name:

Structural protein

Title:

Actin dimer cross-linked by v. Cholerae martx toxin and complexed with gelsolin-segment 1

Structure:

Actin, alpha skeletal muscle. Chain: a. Synonym: alpha-actin-1. Gelsolin. Chain: g. Fragment: segment 1. Synonym: actin-depolymerizing factor, adf, brevin, agel. Engineered: yes

Source:

Oryctolagus cuniculus. Rabbit. Organism_taxid: 9986. Other_details: skeletal muscle. Homo sapiens. Human. Organism_taxid: 9606. Gene: gsn. Expressed in: escherichia coli bl21(de3).

Resolution:

3.21Å R-factor: 0.246 R-free: 0.269

Authors:

M.R.Sawaya,D.S.Kudryashov,I.Pashkov,E.Reisler,T.O.Yeates

Key ref:

D.S.Kudryashov et al. (2008). Connecting actin monomers by iso-peptide bond is a toxicity mechanism of the Vibrio cholerae MARTX toxin. Proc Natl Acad Sci U S A, 105, 18537-18542. PubMed id: 19015515 DOI: 10.1073/pnas.0808082105

Date:

12-Mar-08 Release date: 25-Mar-08

Protein chain

P68135  (ACTS_RABIT) -  Actin, alpha skeletal muscle from Oryctolagus cuniculus

Seq: 377 a.a.

Struc: 360 a.a.

Protein chain

P06396  (GELS_HUMAN) -  Gelsolin from Homo sapiens

Seq: 782 a.a.

Struc: 125 a.a.

Enzyme reactions

• Enzyme class 1: Chain A: E.C.3.6.4.- - ?????
• Enzyme class 2: Chain G: E.C.?

Note, where more than one E.C. class is given (as above), each may correspond to a different protein domain or, in the case of polyprotein precursors, to a different mature protein.

DOI no: 10.1073/pnas.0808082105

Proc Natl Acad Sci U S A 105:18537-18542 (2008)

PubMed id: 19015515

Connecting actin monomers by iso-peptide bond is a toxicity mechanism of the Vibrio cholerae MARTX toxin.

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.

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.

Selected figure(s)

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).

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.

Figures were selected by an automated process.