Two-Headed Binding of the Unphosphorylated Nonmuscle Heavy Meromyosin·ADP Complex to Actin

M. Kovács, J. Tóth, L. Nyitray, James R. Sellers

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

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 (Kd <10 nM) in the presence or absence of ADP. The heads have high and indistinguishable affinities for ADP (Kd around 1 μM) 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.

Original languageEnglish
Pages (from-to)4219-4226
Number of pages8
JournalBiochemistry
Volume43
Issue number14
DOIs
Publication statusPublished - Apr 13 2004

Fingerprint

Actins
Myosin Subfragments
Adenosine Diphosphate
Head
Actomyosin
Myosins
Phosphorylation
Nucleotides
Nonmuscle Myosin Type IIA
Smooth Muscle Myosins
Myosin Type II
Dimerization
Actin Cytoskeleton
Adenosine Triphosphatases
Fluorescence
Muscle
Light
Adenosine Triphosphate

ASJC Scopus subject areas

  • Biochemistry

Cite this

Two-Headed Binding of the Unphosphorylated Nonmuscle Heavy Meromyosin·ADP Complex to Actin. / Kovács, M.; Tóth, J.; Nyitray, L.; Sellers, James R.

In: Biochemistry, Vol. 43, No. 14, 13.04.2004, p. 4219-4226.

Research output: Contribution to journalArticle

@article{401be97e2a5a44d7a662eeb949bf6f25,
title = "Two-Headed Binding of the Unphosphorylated Nonmuscle Heavy Meromyosin·ADP Complex to Actin",
abstract = "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 (Kd <10 nM) in the presence or absence of ADP. The heads have high and indistinguishable affinities for ADP (Kd around 1 μM) 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 (Kov{\'a}cs 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.",
author = "M. Kov{\'a}cs and J. T{\'o}th and L. Nyitray and Sellers, {James R.}",
year = "2004",
month = "4",
day = "13",
doi = "10.1021/bi036007l",
language = "English",
volume = "43",
pages = "4219--4226",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "14",

}

TY - JOUR

T1 - Two-Headed Binding of the Unphosphorylated Nonmuscle Heavy Meromyosin·ADP Complex to Actin

AU - Kovács, M.

AU - Tóth, J.

AU - Nyitray, L.

AU - Sellers, James R.

PY - 2004/4/13

Y1 - 2004/4/13

N2 - 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 (Kd <10 nM) in the presence or absence of ADP. The heads have high and indistinguishable affinities for ADP (Kd around 1 μM) 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.

AB - 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 (Kd <10 nM) in the presence or absence of ADP. The heads have high and indistinguishable affinities for ADP (Kd around 1 μM) 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.

UR - http://www.scopus.com/inward/record.url?scp=11144355239&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=11144355239&partnerID=8YFLogxK

U2 - 10.1021/bi036007l

DO - 10.1021/bi036007l

M3 - Article

C2 - 15065866

AN - SCOPUS:11144355239

VL - 43

SP - 4219

EP - 4226

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 14

ER -