Effects of the type of divalent cation, Ca2+ or Mg2+, bound at the high-affinity site and of the ionic composition of the solution on the structure of F-actin

F-actins containing either Ca2+ or Mg2+ at the single high-affinity site for a divalent cation differ in their dynamic properties [Carlier (1991) J. Biol. Chem. 266, 1-4]. In an attempt to obtain information on the structural basis of this difference, we probed the conformation of specific sites in the subunits of Mg- and Ca-F-actin with limited proteolysis by subtilisin and trypsin. The influence of the kind of polymerizing salt was also investigated. At high proteinase concentrations required for digestion of actin in the polymer form, subtilisin gives a complex fragmentation pattern. In addition to the earlier known cleavage between Met(47) and Gly(48) in the DNAse-I-binding loop, cleavage of F-actin between Ser(234) and Ser(235) in subdomain 4 has recently been reported [Vahdat, Miller, Phillips, Muhlrad and Reisler (1995) FEES Lett. 365, 149-151]. Here we show that actually a larger segment, comprising residues 227-235, is removed and the bond between Leu(67) and Lys(68) in subdomain 2 is split in both G- and F-actin, and that the differences in the fragmentation patterns of the G- and F-forms are accounted for by the protection of the bond 47-48 in F-actin. The subtilisin and trypsin cleavage sites in segment 61-69, subtilisin sites in segment 237-235 and trypsin sites between Lys373 and Cys(374) were less accessible in Mg-F-actin than in Ca-F-actin. These are intramolecular effects, as similar changes were observed on Ca2+/Mg2+ replacement in G-actin. The cation-dependent effects, in particular those on segment 61-69, were however less pronounced in F-actin than in G-actin. The results suggest that substitution of Mg2+ for Ca2+, and KCl-induced polymerization of CaATP-G-actin, bring about a similar change in the conformation of subdomain 2 of the monomer. The presence of Mg2+ at the high-affinity site also resulted in an increased protection of the bond 47-48. This latter appears to be an intermolecular effect because it is specific for F-actin. The susceptibility to subtilisin and trypsin was also strongly influenced by the kind and concentration of polymerizing salt. The digestion patterns suggest that the exposure and/or flexibility of the regions containing the cleavage sites diminish with enhancement of the ionic strength of the solution. The results are discussed in terms of the current models of F-actin.