The actin/actin interactions involving the N‐terminus of the DNase‐I‐binding loop are crucial for stabilization of the actin filament

Abstract

Actin can be specifically cleaved between residues 42 and 43 with a novel protease from Escherichia coli A2 strain (ECP) [Khaitlina, S. Y., Collins, J. H., Kuznetsova, I. M., Pershina, V. P., Synakevich, I. G., Turoverov, K. K. & Usmanova, A. M. (1991) FEBS Lett. 279, 49-51]. The resulting C-terminal and N-terminal fragments remained associated to one another in the presence of either Ca" or Mg". The protease-treated actin was, however, neither able to spontaneously assemble into filaments nor to copolymerize with intact actin unless its tightly bound Caz+ was replaced with Mg". Substitution of Mg2+ for the bound Ca2+ was also necessary to partially restore the ability of the protease-treated actin to inhibit the DNase I activity.The critical concentration for KC1-induced polymerization of ECP-treated ATP-Mg-G-actin, determined by measuring the fluorescence of pyrenyl label, was approximately threefold higher than that for actin cleaved between residues 47 and 48 using subtilisin, and 36-fold higher than the critical concentration for polymerization of intact actin under the same conditions. Morphologically, the filaments of ECP-treated actin were indistinguishable from those of intact actin. Comparison of the fluorescence spectra of pyrenyl-labelled actins and chemical cross-linking with N, Nf-l,2-phenylenebismaleimide have, however, revealed structural differences between the filaments assembled from ECP-treated actin and those of intact as well as subtilisin-treated actin. Moreover, the filaments of ECP-treated actin were easily disrupted by centrifugal forces or shearing stress unless they were stabilized by phalloidin. The results are consistent with the direct participation of the region around residues 42 and 43 in the monomer/monomer interactions as predicted from the atomic model of F-actin [Holmes, K. C., Popp, D., Gebhard, W. & Kabsch, W. (1990) Nature 347, [44][45][46][47][48][49] and suggest that the interactions involving this region are of primary importance for stabilization of the actin filament. The mechanism of the regulation of actin polymerization by the tightly bound divalent cation is also discussed.The atomic model of F-actin presented by Holmes et al.[ 11 has resolved the earlier controversies about the monomer orientation in the F-actin filament and, for the first time, provided precise information on the intermonomer contact sites. This model is, however, based on the monomer structure in G-actidDNase I complex in which certain regions of the actin molecule could be distorted by the interaction with DNase I (21. Moreover, it is well documented that the monomer undergoes conformational changes at various stages of the polymerization process [3], although so far very little is known of the exact nature of these changes.According to the model of Holmes et al., the N-terminal segment (residues 40-45) of a surface loop 39-51 on the top of subdomain 2 of the monomer [2] is involved in extenCorrespondence to H.

Conformational changes in subdomain I of actin induced by proteolytic cleavage within the DNase I‐binding loop: energy transfer from tryptophan to AEDANS

“…The cleavage products were analysed by SDS-PAGE [7]. Integrity of the cleaved molecules with which the C-terminal core and N-terminal 8 kDa fragments remain associated as long as the cleaved actin contains a tightly bound cation [2,8] was proved by the presence of both fragments in the samples obtained after a cycle of polymerization-depolymerization (Fig. 1).…”

Phosphorylation of actin Tyr-53 inhibits filament nucleation and elongation and destabilizes filaments

“…Several studies have shown that proteolytic cleavage (15,16) of the DNase I-binding loop or chemical modification of neighboring residues Lys-61 (17)(18)(19) and Tyr-69 (20) reduce actin's affinity for DNase I and inhibit polymerization, which is reversed by phalloidin. More directly relevant to this article, chemical modification of Tyr-53 by reaction with diazonium tetrazole also blocks polymerization (21), which is not reversed by phalloidin (20).…”

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

“…The fungal toxin phalloidin and actinbinding protein cofilin can rescue via different mechanisms the polymerization of actins whose self-assembly was impaired by mutations, limited proteolysis, or chemical modifications (23)(24)(25)(26). Therefore, we used these two actin-interacting partners to test the rescue of filament assembly for ACD cross-linked actin.…”