Mechanisms of the modulation of actin-myosin interactions by A1-type myosin light chains.

Background: The interaction of N-terminal extension of the myosin A1 essential light chain (A1 ELC) with actin is receiving increasing attention as a target in utilizing synthetic A1 ELC N-terminal-derived peptides in cardiac dysfunction therapy.

Methods: To elucidate the mechanism by which these peptides regulate actin-myosin interaction, here we have investigated their effects on the myosin subfragment 1 (S1)-induced polymerization of G-actin.

Results: The MLCF and MLCS peptides spanning the 3-12 of A1 ELC sequences from fast and slow skeletal muscle, respectively, increased the rate of actin polymerization not only by S1(A2) but also the rate of S1(A1)-induced actin polymerization, suggesting that they did not interfere with the direct binding of A1 ELC with actin.

Myopathy-Sensitive G-Actin Segment 227-235 Is Involved in Salt-Induced Stabilization of Contacts within the Actin Filament.

Formation of stable actin filaments, critically important for actin functions, is determined by the ionic strength of the solution. However, not much is known about the elements of the actin fold involved in ionic-strength-dependent filament stabilization. In this work, F-actin was destabilized by Cu binding to Cys374, and the effects of solvent conditions on the dynamic properties of F-actin were correlated with the involvement of Segment 227-235 in filament stabilization.

New aspects of the spontaneous polymerization of actin in the presence of salts.

The mechanism of salt-induced actin polymerization involves the energetically unfavorable nucleation step, followed by filament elongation by the addition of monomers. The use of a bifunctional cross-linker, N,N'-(1,4-phenylene)dimaleimide, revealed rapid formation of the so-called lower dimers (LD) in which actin monomers are arranged in an antiparallel fashion. The filament elongation phase is characterized by a gradual LD decay and an increase in the yield of "upper dimers" (UD) characteristic of F-actin.

Role of actin DNase-I-binding loop in myosin subfragment 1-induced polymerization of G-actin: implications for the mechanism of polymerization.

Proteolytic cleavage of actin between Gly(42) and Val(43) within its DNase-I-binding loop (D-loop) abolishes the ability of Ca-G-actin to spontaneously polymerize in the presence of KCl. Here we show that such modified actin is assembled into filaments, albeit at a lower rate than unmodified actin, by myosin subfragment 1 (S1) carrying the A1 essential light chain but not by S1(A2). S1 titration of pyrene-G-actin showed a diminished affinity of cleaved actin for S1, but this could be compensated for by using S1 in excess.

The DNase-I binding loop of actin may play a role in the regulation of actin-myosin interaction by tropomyosin/troponin.

Various lines of evidence suggest that communication between tropomyosin and myosin in the regulation of vertebrate-striated muscle contraction involves yet unknown changes in actin conformation. Possible participation of loop 38-52 in this communication has recently been questioned based on unimpaired Ca(2+) regulation of myosin interaction, in the presence of the tropomyosin-troponin complex, with actin cleaved by subtilisin between Met(47) and Gly(48). We have compared the effects of actin cleavage by subtilisin and by protease ECP32, between Gly(42) and Val(43), on its interaction with myosin S1 in the presence and absence of tropomyosin or tropomyosin-troponin.

Role of the DNase-I-binding loop in dynamic properties of actin filament.

Effects of proteolytic modifications of the DNase-I-binding loop (residues 39-51) in subdomain 2 of actin on F-actin dynamics were investigated by measuring the rates of the polymer subunit exchange with the monomer pool at steady state and of ATP hydrolysis associated with it, and by determination of relative rate constants for monomer addition to and dissociation from the polymer ends. Cleavage of actin between Gly-42 and Val-43 by protease ECP32 resulted in enhancement of the turnover rate of polymer subunits by an order of magnitude or more, in contrast to less than a threefold increase produced by subtilisin cleavage between Met-47 and Gly-48. Probing the structure of the modified actins by limited digestion with trypsin revealed a correlation between the increased F-actin dynamics and a change in the conformation of subdomain 2, indicating a more open state of the filament subunits relative to intact F-actin.