Structure of the Shutdown State of Myosin-2

Abstract

Myosin-2 is a molecular motor that is essential for cell movement, cell division and muscle contraction. In its active state, it forms filaments, interacts with actin, and uses the energy from ATP hydrolysis to generate force. It also forms an inactive compact shutdown state, conserved across myosin-2 isoforms, in which phosphate release is inhibited and ATPase activity is very low. The compact nature of the shutdown state likely enables it to easily diffuse or be transported through the cell. Despite its important, structural studies of the shutdown state have been limited to 20 Å, restricting our understanding of its formation, maintenance and release. Here we report a sub-nanometre cryo-electron microscopy structure of shutdown smooth muscle myosin (6 Å in the heads region), which allows the structure of a two-headed myosin to be seen in unprecedented detail. A pseudo-atomic model, obtained by flexible fitting of crystal structures into the density and molecular dynamics simulations, describes interaction interfaces at the atomic level. Our structure reveals how the shutdown state is stabilised through multiple interaction interfaces between the three segments of the myosin molecule coiled-coil, the motor domains and the essential and regulatory light chains (RLCs). Crucially, our structure reveals the paths of the N-terminal extensions of the RLCs, and explains how phosphorylation would disrupt key stabilising interactions of the shutdown state allowing myosin activation. Unlike in many previous models of the shutdown state, the structure of the lever in each head is competent to generate force upon activation. Our shutdown structure is relevant to all myosin-2 isoforms, and provides a foundation for understanding their disease-causing mutations.