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) ac tivates 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 neg ative staining and helical reconstruction. F-actin was decorated with a pep tide 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 o f subdomain-1 of each actin monomer forming a bridge to the periphery of su bdomain-4 of the azimuthally adjacent actin. Fitting the reconstruction to the atomic model of F-actin revealed interaction of MLCK-147 close to the C OOH terminus of the first actin and near residues 228-232 of the second. Th is unique location enables MLCK to bind to actin without interfering with t he binding of any other key actin-binding proteins, including myosin, tropo myosin, caldesmon, and calponin.