Conformational effects of cation binding to myosin and their relation to phosphorylation

Abstract

Binding profiles of calcium or magnesium ions to rabbit skeletal myosin can be determined from the change in the products of chymotryptic digestion with concentration of the ion. Qualitatively similar effects are brought about by sodium ions at much higher concentrations, and the profiles follow expectations for stoichiometric binding of the univalent ion. The affinity for sodium ions depends on the phosphorylation state of the metal-binding ("regulatory") light chain. The degree of saturation at physiological ionic strength is substantially higher for the phosphorylated form than for the unphosphorylated form. This effect can account quantitatively for the apparent difference in affinity for calcium ions between these two states, ,measured at physiological sodium ion concentration. Thus if phosphorylation of the light chain leads to a structural perturbation linked to the binding of cations, this may be presumed to come about by way of a net change in occupancy of the cation binding sites rather than through displacement of a resident ion by calcium. A similar pattern of behavior is displayed by cardiac myosin. Like calcium or magnesium, sodium ions are found to protect the proteolytically labile sites in the metal-binding light chain itself and at the head-rod junction, and they promote scission in the putative hinge region, with liberation of heavy meromyosin. The digestion pattern is, under the conditions employed here, C A T I O N B I N D I N G T O M Y O S I N V O L . 2 1 , N O . 6 , 1 9 8 2

Qualitative analysis of skeletal myosin as substrate of Ca2+-activated neutral protease: comparison of filamentous and soluble, native, and L2-deficient myosin.

“…9, A and B). Similar results had been obtained with chymotrypsin (42,43). Surprisingly, the transition from filamentous to soluble myosin did not substantially increase the extent of degradation during the…”

“…Therefore, one characteristic property of CAF was that all the preferred cleavage sites were located on the heavy chain in the vicinity of the light chains. Cations, when in the presence of L2, protected the head region of myosin from attack by chymotrypsin (5,43), but were unable to protect this region from attack by CAF. In this regard, CAF was similar to trypsin (42) and to papain (44).…”

Effect of calcium on synthetic myosin minifilaments

“…Weeds & Pope (1977) and Bagshaw (1977) have shown that binding of divalent cations to LC-2 subunits in filamentous myosin results in protection of the S-l/S-2 junction from chymotryptic attack and promotes cleavage at the HMM/LMM junction. Since both Ca2+ and Mg2+ and even high concentrations of monovalent salts (Mrakovcic et al, 1979;Oda et al, 1980;Kardami & Gratzer, 1982) produce this striking effect, it is most probably unrelated to the function of the LC-2 chains. In fact, it appears now that the Ca2+-and Mg2+-induced transitions in the proteolysis of myosin can be explained without evoking structural changes in the HMM/LMM hinge (Oda et al, 1980;Borejdo & Werber, 1982).…”

Probing Conformational Changes within LC2 Domains of Cardiac Myosin

“…Calcium and phosphorylation induce conformational changes in LC2s, as demonstrated by fluorescence studies and circular dichroism of isolated skeletal muscle light chains (Alexis & Gratzer, 1978). Calcium and EDTA, also by inducing conformational changes in LC2, influence chymotryptic cleavage of skeletal myosin LC2 (Kardami & Gratzer, 1982;Roulet et al, 1993). Using an anti-LC2 monoclonal antibody, Shimizu et al (1985) showed that Ca2+ or Mg2+ cause a conformational change around the first helical domain of the calcium binding site of LC2 from chicken skeletal myosin.…”