Point mutations at K50 and E270 residues abolish cross-linking of actin. (A) Purified S. cerevisiae wild-type actin, K50C/C374A, E270D/C374A mutant actins (10 μM), and the mixture of two mutants (5 μM each) were cross-linked at 1:250 mole ratio of ACD to actin in the presence of 1 mM MgCl₂ and 15 μM Kabiramide C (to prevent polymerization) for the time spans indicated at the bottom of the SDS/PAGE gel. Numbers on the left side of the gel indicate the number of actin molecules corresponding to a given protein band. (B) HeLa cells were transiently transfected to express wild-type and mutant Myc-tagged β-actin as indicated at top. All cells were treated with rtxA⁺ V. cholerae KFV119 for 90 min before cells were collected for western blotting using α-myc antibody.

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.

Mapping the cross-linked sites on actin. (A) Actin monomers (m) and ACD cross-linked dimers (d) were subjected to limited proteolysis by GluC, trypsin, and subtilisin. The expected molecular mass of proteolytic peptides and the soybean trypsin inhibitor (in blue) are indicated. (B) Summary of the results from (A) with an indication of cleavage sites, the location of the covalent cross-link bond (yellow bar), and the masses of proteolytic peptides. Red and blue rectangles represent two protomers in the ACD cross-linked actin dimer. (C) Ribbon representation of two crystal structures of the ACD cross-linked actin dimer (AD) in complex with GS1 and with both GS1 and DNaseI (GS1 and DNaseI are not shown) and the recent Holmes model of actin filament (20). Both dimers are oriented in a way that displays better the proximity of the cross-liked peptides. Subdomains 1 to 4 of each protomer are colored blue, yellow, red, and green, respectively. Dotted black line indicates disordered residues 40–49. The cross-linked peptides 48–61 and 257–284, mapped by limited proteolysis and mass spectrometry of the biotin-labeled cross-linked peptide, are shown in black. Taking peptide 48–61 as the only constraint for the localization of one of the cross-linked residues, K50 and E270 were identified as the only two residues within 15-Å radius from each other in both crystal structures as detailed in text. (D) Tryptic peptides of cross-linked actin dimer were analyzed by ESI-FTMS. The 1095.9 (M + 5H)⁵⁺ peak in MS spectra, corresponding to the cross-linked peptide, was fragmented in the FT-ICR cell. The most intense of the 104 ions that could be matched to the sequence are annotated in blue (listed in Tables S1 and S2). The masses from resulting high resolution MS/MS spectrum were assigned to peptide fragments by using MS2Assign software. The inset shows the base line separation of the quintuply charged remainder of the precursor. (E) Schematic representation of the cross-linked peptides with the number of ions found for each bond. Of 47 bonds, product ions defining 43 were found. Importantly, 10 ions can be found related to a fragmentation next to the amino acids involved in the cross-linking. This identifies unambiguously the site of cross-linking.

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 MgCl₂ 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).