THE ACTIN ACTIN INTERACTIONS INVOLVING THE N-TERMINUS OF THE DNASE-I-BINDING LOOP ARE CRUCIAL FOR STABILIZATION OF THE ACTIN FILAMENT

Actin can be specifically cleaved between residues 42 and 43 with a no vel protease from Escherichia coli A2 strain (ECP) [Khaitlina, S. Y., Collins, J. H., Kuznetsova, 1. 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 Ca2+ or Mg2+. The protease-treat ed actin was, however, neither able to spontaneously assemble into fil aments nor to copolymerize with intact actin unless its tightly bound Ca2+ was replaced with Mg2+. Substitution of Mg2+ for the bound Ca2+ w as also necessary to partially restore the ability of the protease-tre ated actin to inhibit the DNase I activity. The critical concentration for KCl-induced polymerization of ECP-treated ATP-Mg-G-actin, determi ned by measuring the fluorescence of pyrenyl label, was approximately threefold higher than that for actin cleaved between residues 47 and 4 8 using subtilisin, and 36-fold higher than the critical concentration for polymerization of intact actin under the same conditions. Morphol ogically, the filaments of ECP-treated actin were indistinguishable fr om those of intact actin. Comparison of the fluorescence spectra of py renyl-labelled actins and chemical cross-linking with N,N'-1,2-phenyle nebismaleimide 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-treat ed actin were easily disrupted by centrifugal forces or shearing stres s unless they were stabilized by phalloidin. The results are consisten t with the direct participation of the region around residues 42 and 4 3 in the monomer/monomer interactions as predicted from the atomic mod el of F-actin [Holmes, K. C., Popp, D., Gebhard, W. & Kabsch, W. (1990 ) Nature 347, 44-49] and suggest that the interactions involving this region are of primary importance for stabilization. of the actin filam ent. The mechanism of the regulation of actin polymerization by the ti ghtly bound divalent cation is also discussed.