Myosin light chain kinase binding to a unique site on F-actin revealed by three-dimensional image reconstruction

Victoria Hatch, Gang Zhi, Lula Smith, James T. Stull, Roger Craig, William Lehman

Research output: Contribution to journalArticle

32 Scopus citations

Abstract

Ca2+-calmodulin-dependent phosphorylation of myosin regulatory light chains by the catalytic COOH-terminal half of myosin light chain kinase (MLCK) activates myosin II in smooth and nonmuscle cells. In addition, MLCK binds to thin filaments in situ and F-actin in vitro via a specific repeat motif in its NH2 terminus at a stoichiometry of one MLCK per three actin monomers. We have investigated the structural basis of MLCK-actin interactions by negative staining and helical reconstruction. F-actin was decorated with a peptide containing the NH2-terminal 147 residues of MLCK (MLCK-147) that binds to F-actin with high affinity. MLCK-147 caused formation of F-actin rafts, and single filaments within rafts were used for structural analysis. Three-dimensional reconstructions showed MLCK density on the extreme periphery of subdomain-1 of each actin monomer forming a bridge to the periphery of subdomain-4 of the azimuthally adjacent actin. Fitting the reconstruction to the atomic model of F-actin revealed interaction of MLCK-147 close to the COOH terminus of the first actin and near residues 228-232 of the second. This unique location enables MLCK to bind to actin without interfering with the binding of any other key actin-binding proteins, including myosin, tropomyosin, caldesmon, and calponin.

Original languageEnglish (US)
Pages (from-to)611-617
Number of pages7
JournalJournal of Cell Biology
Volume154
Issue number3
DOIs
StatePublished - Aug 6 2001

Keywords

  • Actin
  • Electron microscopy
  • Myosin
  • Myosin light chain kinase
  • Phosphorylation

ASJC Scopus subject areas

  • Cell Biology

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