Abstract
Actin is an essential element of both innate and adaptive immune systems and can aid in motility and translocation of bacterial pathogens, making it an attractive target for bacterial toxins. Pathogenic Vibrio and Aeromonas genera deliver actin cross-linking domain (ACD) toxin into the cytoplasm of the host cell to poison actin regulation and promptly induce cell rounding. At early stages of toxicity, ACD covalently cross-links actin monomers into oligomers (AOs) that bind through multivalent interactions and potently inhibit several families of actin assembly proteins. At advanced toxicity stages, we found that the terminal protomers of linear AOs can get linked together by ACD to produce cyclic AOs. When tested against formins and Ena/VASP, linear and cyclic AOs exhibit similar inhibitory potential, which for the cyclic AOs is reduced in the presence of profilin. In coarse-grained molecular dynamics simulations, profilin and WH2-motif binding sites on actin subunits remain exposed in modeled AOs of both geometries. We speculate, therefore, that the reduced toxicity of cyclic AOs is due to their reduced configurational entropy. A characteristic feature of cyclic AOs is that, in contrast to the linear forms, they cannot be straightened to form filaments (e.g., through stabilization by cofilin), which makes them less susceptible to neutralization by the host cell.
Highlights
While analyzing western blot images of actin cross-linked in living cells at advanced stages of actin cross-linking domain (ACD) toxicity, we discovered an unusual cross-linking array (“anomalous” oligomers) that overlaps with the regular pattern of actin oligomers (AOs)
Results in accumulation of oligomers of various lengths. These actin oligomers (AOs) normally appear on SDS-polyacrylamide gels as a series of bands, which progressively decrease in intensity at higher molecular weights
Despite evidence that linear AOs can integrate into F-actin with support from phalloidin or cofilin, the ACD-cross-linked residues are not in proximity; the side-chain nitrogen of K50 and the oxygen of E270 are separated by 18.5 Å in the cryo-electron microscopy structure of phalloidin-stabilized actin [16] (PDB: 6T1Y)
Summary
Spire-mediated nucleation, when present in nanomolar concentrations [5,6] All these actin assembly factors contain multiple sites that can bind either (i) directly to actin (e.g., through short ~17-amino acid WASP-homology motifs 2 (WH2-domains) [12], or (ii) via polyproline (PP)-rich motifs to actin-profilin complexes [13]. We used electron microscopy, polymerization assays with and without filament stabilizers, and mass spectrometry to establish that the unusual pattern is due to cyclic forms of AOs. Coarse-grained molecular dynamics (MD) simulations predicted the most populated structures for both AO types and confirmed that cross-linked geometries typically expose binding sites for profilin (~65–75% exposure) and for the WH2-domain (~96–99% exposure). In contrast to the linear AOs, the cyclic oligomers cannot be incorporated into filaments by cofilin or the actin-stabilizing drug phalloidin, suggesting their higher resistance to neutralization and possibly higher potential to disrupt the actin cytoskeleton
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