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

Abstract

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 Met47 and Gly48 in the DNAse-I-binding loop, cleavage of F-actin between Ser234 and Ser235 in subdomain 4 has recently been reported [Vahdat, Miller, Phillips, Muhlrad and Reisler (1995) FEBS Lett. 365, 149-151]. Here we show that actually a larger segment, comprising residues 227-235, is removed and the bond between Leu67 and Lys68 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 227-235 and trypsin sites between Lys373 and Cys374 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.

Identification of cation-binding sites on actin that drive polymerization and modulate bending stiffness

“…The intercept value of K polym extrapolated to 1M cation is related to the intrinsic binding free energy of an actin subunit and associated cation with a filament end (27,28). These values are not identical for all cations evaluated, suggesting that filaments assembled with different cationic species have variable thermodynamic stabilities and salt-dependent conformational distribution(s) (6,7,20,29,30).…”

“…The actin C c and (monomer and filament) conformation depend on the nucleotide-associated divalent cation (Ca 2þ or Mg 2þ ) as well as the type and concentration of ions in solution (6,7,(13)(14)(15), a behavior shared among characterized actins and their bacterial homologs (16). However, it is not firmly established if these salt effects on actin filament assembly and mechanics originate from nonspecific ion effects (e.g., electrostatic screening, counterion condensation, etc.)…”

“…The effects of solution ionic conditions on the assembly and stability of actin filaments have been investigated for several decades (6)(7)(8)(9)(10)(11)(12). The actin C c and (monomer and filament) conformation depend on the nucleotide-associated divalent cation (Ca 2þ or Mg 2þ ) as well as the type and concentration of ions in solution (6,7,(13)(14)(15), a behavior shared among characterized actins and their bacterial homologs (16).…”

Modulation of actin structure and function by phosphorylation of Tyr-53 and profilin binding

“…The susceptibility of the D-loop to subtilisin cleavage between residues Met-47 and Gly-48 has been commonly used to monitor conformational changes in the D-loop resulting from factors such as the type of nucleotide and divalent cation bound to actin (32,33), and the binding of actin-depolymerizing factor (ADF)/ cofilin to the filament (34). We found that phosphorylation of Tyr-53 protects the D-loop from subtilisin cleavage, as shown by a Ϸ50% reduction in the initial rate of cleavage of the D-loop in pY53-actin compared to unphosphorylated actin (Fig.…”

The DNase-I Binding Loop of Actin May Play a Role in the Regulation of Actin-Myosin Interaction by Tropomyosin/Troponin

“…Therefore, a slow degradation of the alanine-and serine-rich segment 227-235 in subdomain 4 (56, 57) results in fragmentation of the F-actin subunits into the N-terminal 30-kDa peptide (residues 1-227) and 16-kDa C-terminal peptide (residues 235-375). The faint 33-kDa band is a product of subtilisin cleavage between Leu 67 and Lys 68 (57). Digestion of subtilisin-modified F-actin (Fig.…”