Abstract
The motor protein myosin uses energy derived from ATP hydrolysis to produce force and motion. Important conserved components (P-loop, switch I, and switch II) help propagate small conformational changes at the active site into large scale conformational changes in distal regions of the protein. Structural and biochemical studies have indicated that switch I may be directly responsible for the reciprocal opening and closing of the actin and nucleotide-binding pockets during the ATPase cycle, thereby aiding in the coordination of these important substrate-binding sites. Smooth muscle myosin has displayed the ability to simultaneously bind tightly to both actin and ADP, although it is unclear how both substrate-binding clefts could be closed if they are rigidly coupled to switch I. Here we use single tryptophan mutants of smooth muscle myosin to determine how conformational changes in switch I are correlated with structural changes in the nucleotide and actin-binding clefts in the presence of actin and ADP. Our results suggest that a closed switch I conformation in the strongly bound actomyosin-ADP complex is responsible for maintaining tight nucleotide binding despite an open nucleotide-binding pocket. This unique state is likely to be crucial for prolonged tension maintenance in smooth muscle.
Highlights
Myosin is a molecular motor that uses energy derived from ATP hydrolysis to produce force and motion along an actin filament during muscle contraction and other forms of cell motility
In this study we use site-specific probes of the upper 50-kDa domain and switch I to examine the coordination of these two important regions of the smooth muscle myosin motor domain in the actomyosin-ADP state
We show for the first time an actin-dependent closure of switch I in the ADP state that has only been observed in smooth muscle myosin to date, because actin has been shown to open switch I in skeletal and Dictyostelium discoideum myosin where it has been studied previously [19, 40]
Summary
Myosin is a molecular motor that uses energy derived from ATP hydrolysis to produce force and motion along an actin filament during muscle contraction and other forms of cell motility. The available crystal structures suggest that opening of switch I within the active site of myosin is associated with a rotation of the upper 50-kDa domain causing closing of the actin-binding cleft and subsequent strong binding to actin [12]. Strong binding to both actin and ADP has been proposed to be necessary for the formation of the latch state, where a decrease in regulatory light chain phosphorylation leads to maintained tension for long periods of time with little hydrolysis of ATP [21] and which may be related to the closed active site state previously observed at low temperature by EPR and transient kinetics (16 –19). The structural basis of such a state is currently unknown but is likely
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