Localization of the tightly bound divalent‐cation‐dependent and nucleotide‐dependent conformation changes in G‐actin using limited proteolytic digestion

Abstract

Using proteolytic susceptibility as a probe, we have identified four regions of the actin polypeptide chain where structural rearrangements, dependent on the nature of the tightly bound metal ion and/or nucleotide, take place. Replacement of the tightly bound Ca2+ by Mg2+ in ATP-actin strongly affected the regions around Arg26 and Lys68, as judged from nearly complete inhibition of tryptic cleavages of the polypeptide chain at these residues. It also significantly diminished the rates of splitting by trypsin of the peptide bonds involving carbonyl groups of Arg372 and of Lys373 in the C-terminal segment. Conversion of ATP-actin to ADP-actin (with Mg2+ as the tightly bound cation) abolished the protective effect of Mg2+ on specific tryptic cleavage and, in contrast, largely inhibited proteolysis at specific sites for subtilisin and for a novel protease from Escherichia coli A2 strain within a surface loop of residues 39-51. We also examined the effect of proteolytic cleavage or chemical modification at certain sites on the kinetics of proteolysis at other sites of the molecule. These experiments demonstrated structural relationships between loop 39 -51 and regions involving Lys61 and Lys68. It is suggested that the conformational transitions reflected in the observed changes in proteolytic susceptibility may underlie the known influence of the nature of the tightly bound cation and nucleotide on the kinetics of actin polymerization and stability of the polymer.The ability of monomeric actin to polymerize into helical filaments is a fundamental property of this protein. It is well established that polymerization is strongly influenced by the kind of divalent cation and nucleotide occupying the single high-affinity site for each of these ligands. Actin containing bound Mg2+ (Mg-actin) has a lower critical concentration and polymerizes faster than actin with bound Ca2+ (Ca-actin). Similarly, actin prepared by procedures commonly in use, which contains ATP at the high-affinity site for nucleotide (ATP-actin), has a lower critical concentration and polymerizes faster than ADP-actin. These differences themselves, as well as a number of other observations, are indicative of nucleotide-dependent and metal-ion-dependent alterations in the monomer conformation (for reviews, see [l, 21). Little information, however, is available on the location of these changes in the actin polypeptide chain. Most extensively examined were the alterations in the environment of Cys374 monitored as changes in the fluorescence of actin labelled at this residue with N-iodoacetyl-K-(5-sulfo-l-naphthyl)-

Conformational and Dynamic Differences between Actin Filaments Polymerized from ATP- or ADP-Actin Monomers

“…The Ca 2ϩ bound by the actin monomers was replaced by Mg 2ϩ in these samples by adding EGTA and MgCl 2 at final concentrations of 0.2 and 0.1 mM, respectively. The samples were incubated for 10 -15 min at room temperature (33). To obtain filaments from ATP-monomeric actin the samples were polymerized with 2 mM MgCl 2 and 100 mM KCl (final concentrations) after the cation exchange.…”

Mechanisms of leiomodin 2-mediated regulation of actin filament in muscle cells

“…4 C and D). Hydrolysis of ATP into ADP and inorganic phosphate in actin (LRR) and actin(W) and the subsequent release of inorganic phosphate may change the conformation of actin subdomain 2 (37,38), result in a partial dissociation of Lmod2 from the nucleus, thereby releasing the steric clash to allow fast growth at the barbed end (Fig. 5C, Left).…”

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.…”