Actin Filament Dynamics in the Actomyosin VI Complex Is Regulated Allosterically by Calcium–Calmodulin Light Chain

• Published in: Journal of molecular biology Vol. 413; no. 3; pp. 584 - 592
• Main Authors: Prochniewicz, Ewa, Pierre, Anaëlle, McCullough, Brannon R., Chin, Harvey F., Cao, Wenxiang, Saunders, Lauren P., Thomas, David D., De La Cruz, Enrique M.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 28.10.2011
• Subjects: Actin; Actin Cytoskeleton - metabolism; Actins - metabolism; actomyosin; Actomyosin - metabolism; adenosine triphosphate; Allosteric properties; Allosteric Regulation; Animals; Anisotropy; ATP; binding sites; Calcium; Calcium - metabolism; Calcium-binding protein; Calmodulin; Calmodulin - metabolism; cooperativity; Cytoskeleton - metabolism; Data processing; dissociation; Enzymatic activity; enzyme activity; Erythrosine - metabolism; Filaments; fluorescence microscopy; Fluorescence Polarization; Light chains; Luminescent Measurements; mechanics; microfilaments; Motility; Muscle contraction; Muscle, Skeletal - metabolism; Muscles; Myosin; Myosin Heavy Chains - metabolism; Myosin Light Chains - metabolism; myosin VI; Phosphorescence; phosphorescence spectroscopy; Rabbits; Swine; 2D; actin; myosin VI; TPA; ErIA; cooperativity; phosphorescence spectroscopy; calmodulin; CaM; EGTA
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2011.08.058

The contractile and enzymatic activities of myosin VI are regulated by calcium binding to associated calmodulin (CaM) light chains. We have used transient phosphorescence anisotropy to monitor the microsecond rotational dynamics of erythrosin-iodoacetamide-labeled actin with strongly bound myosin VI (MVI) and to evaluate the effect of MVI-bound CaM light chain on actin filament dynamics. MVI binding lowers the amplitude but accelerates actin filament microsecond dynamics in a Ca2+- and CaM-dependent manner, as indicated from an increase in the final anisotropy and a decrease in the correlation time of transient phosphorescence anisotropy decays. MVI with bound apo-CaM or Ca2+–CaM weakly affects actin filament microsecond dynamics, relative to other myosins (e.g., muscle myosin II and myosin Va). CaM dissociation from bound MVI damps filament rotational dynamics (i.e., increases the torsional rigidity), such that the perturbation is comparable to that induced by other characterized myosins. Analysis of individual actin filament shape fluctuations imaged by fluorescence microscopy reveals a correlated effect on filament bending mechanics. These data support a model in which Ca2+-dependent CaM binding to the IQ domain of MVI is linked to an allosteric reorganization of the actin binding site(s), which alters the structural dynamics and the mechanical rigidity of actin filaments. Such modulation of filament dynamics may contribute to the Ca2+- and CaM-dependent regulation of myosin VI motility and ATP utilization. ► Myosin VI (MVI) with bound calmodulin weakly affects actin filament stiffness. ► CaM dissociation from bound MVI stiffens filaments to an extent comparable to other myosins. ► CaM binding to MVI IQ domain is linked to allosteric reorganization of the actin-binding site. ► Three MVI states with distinct actin interactions exist in a Ca2+– and CaM– linked equilibrium. ► The actomyosin interface is allosterically regulated by the myosin light chain-binding domain.