Two-headed binding of the unphosphorylated nonmuscle heavy meromyosin.ADP complex to actin.
The enzymatic and motor function of smooth muscle and nonmuscle myosin II is activated by phosphorylation of the regulatory light chains located in the head portion of myosin. Dimerization of the heads, which is brought about by the coiled-coil tail region, is essential for regulation since single-headed fragments are active regardless of the state of phosphorylation. Utilizing the fluorescence signal on binding of myosin to pyrene-labeled actin filaments, we investigated the interplay of actin and nucleotide binding to thiophosphorylated and unphosphorylated recombinant nonmuscle IIA heavy meromyosin constructs. We show that both heads of either thiophosphorylated or unphosphorylated heavy meromyosin bind very strongly to actin (K(d) < 10 nM) in the presence or absence of ADP. The heads have high and indistinguishable affinities for ADP (K(d) around 1 microM) when bound to actin. These findings are in line with the previously observed unusually loose coupling between nucleotide and actin binding to nonmuscle myosin IIA subfragment-1 (Kovács et al. (2003) J. Biol. Chem. 278, 38132.). Furthermore, they imply that the structure of the two heads in the ternary actomyosin-ADP complex is symmetrical and that the asymmetrical structure observed in the presence of ATP and the absence of actin in previous investigations (Wendt et al. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 4361) is likely to represent an ATPase intermediate that precedes the actomyosin-ADP state.
Kovács
Mihály
M
Laboratory of Molecular Cardiology, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892-1762, USA.
Tóth
Judit
J
Nyitray
László
L
Sellers
James R
JR
D000199:Actins / Q000737:chemistry*; D000205:Actomyosin / Q000737:chemistry; D000244:Adenosine Diphosphate / Q000737:chemistry*; D000251:Adenosine Triphosphatases / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites; D002417:Cattle; D002645:Chickens; D004789:Enzyme Activation; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008956:Models, Chemical; D015879:Myosin Subfragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011721:Pyrenes / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D018411:Spodoptera / Q000235:genetics
D000199:Actins; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D015879:Myosin Subfragments; D011721:Pyrenes; C022571:adenosine 5'-O-(3-thiotriphosphate); D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000205:Actomyosin; C030984:pyrene; D000251:Adenosine Triphosphatases