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Published online 30 July 2001. doi:10.1083/jcb.200105079
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© The Rockefeller University Press, 0021-9525/2001/8/611 $5.00
The Journal of Cell Biology, Volume 154, Number 3, August 6, 2001 611-618

Article

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



Victoria Hatch1, Gang Zhi2, Lula Smith2, James T. Stull2, Roger Craig3 and William Lehman1

1 Department of Physiology and Biophysics, Boston University School of Medicine, Boston, MA
2 Department of Physiology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX
3 Department of Cell Biology, University of Massachusetts Medical School, Worcester, MA

Address correspondence to William Lehman, Department of Physiology and Biophysics, Boston University School of Medicine, 715 Albany St., Boston, MA 02118-2526. Tel.: (617) 638-4397. Fax: (617) 638-4273. E-mail: wlehman{at}bu.edu

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.

Key Words: actin; electron microscopy; myosin; myosin light chain kinase; phosphorylation


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