Myosin light chain kinase binding to a unique site on F-actin revealed by three-dimensional image reconstruction.
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.
Hatch
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Department of Physiology and Biophysics, Boston University School of Medicine, Boston, MA 02118-2526, USA.
Zhi
G
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Smith
L
L
Stull
J T
JT
Craig
R
R
Lehman
W
W
D000199:Actins / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D000818:Animals; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D008854:Microscopy, Electron; D008958:Models, Molecular; D018482:Muscle, Skeletal / Q000737:chemistry / Q000378:metabolism; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011817:Rabbits
D000199:Actins; D010455:Peptides; C065738:MLCK peptide; D009219:Myosin-Light-Chain Kinase