Structure of the shutdown state of myosin-2

Abstract

Myosin-2 is essential for processes as diverse as cell division and muscle contraction. Dephosphorylation of its regulatory light chain (RLC) promotes an inactive, 'shutdown' state with the filament-forming tail folded onto the two heads 1 , preventing filament formation and inactivating the motors 2 . The mechanism by which this happens is obscure.Here we report a cryo-electron microscopy structure of shutdown smooth muscle myosin, with a resolution of 6 Å in the head region. 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. The N-terminal extension of one RLC interacts with the tail and the other with the partner head, revealing how the RLCs stabilise the shutdown state in different ways and how their phosphorylation would allow myosin activation. Additional interactions between the three segments of the coiled coil, the motor domains and LCs stabilise the shutdown molecule. The structure of the lever in each head is competent to generate force upon activation. This shutdown structure is relevant to all myosin-2 isoforms and provides a framework for understanding their disease-causing mutations.Myosin-2 is a molecular motor that interacts with actin using the energy from ATP hydrolysis to generate force and movement. It comprises two heavy chains, two essential light chains (ELC) and two regulatory light chains (RLC). The first ~800 residues of the heavy chain form the globular motor, which contains binding sites for actin and nucleotide. The remaining heavy chain forms an α-helix, in which the proximal region complexes with one ELC and one RLC to form a stiff light chain domain (LCD) that transmits motor force via a lever mechanism. The motor and the LCD comprise the myosin head. C-terminal to the LCD, the two heavy chains dimerise to form a 160-nm coiled-coil tail. The coiled coil contains the typical seven residue heptad repeat 3 , interrupted by three 'skip' residues in smooth muscle and non-muscle myosin-2 isoforms.In the active state of smooth muscle myosin (SmM), the two RLCs are phosphorylated 4 and the tails are polymerised into thick filaments. Dephosphorylation of S20 and/or T19 within the N-terminal extension of the RLCs 4,5 triggers an extraordinary transformation into a monomeric, shutdown state, in which both motors are trapped in a primed state, phosphate release is inhibited and ATPase activity is very low 6 . The two motor domains are organised into an asymmetric, interacting heads motif (IHM) (Fig. 1a), in which part of the actin-binding interface of the motor in the 'blocked' head is positioned onto the converter domain of the motor in the 'free' head, preventing interaction with actin 7 . The coiled-coil tail folds into three segments and wraps around the heads 8,9 .

Cardiac myosin super relaxation (SRX): a perspective on fundamental biology, human disease and therapeutics

“…The majority of actin-attached and force producing myosin heads are located in the C-zone whereas proximal (P) and distal (D) zones primarily comprises disordered and folded myosin heads. myosin by cryo-EM (Scarff et al, 2020;Yang et al, 2020). The IHM has been conserved and diversified evolutionarily, yet the IHMs importance in regulating cardiac muscle has largely been overlooked, until recently, when it became clear that residues within the IHM defined a significant proportion of cases of the genetic cardiomyopathy HCM (Alamo et al, 2017).…”

“…IHM interactions must be adjustable to regulate and maintain sarcomeric contractility an especially important characteristic to adapt cardiac output in the heart. The IHM regulatory complex includes the ELC, which possess a Ca 2+ binding site, and RLC (Rayment et al, 1993;Scarff et al, 2020), which has a single serine in human myocardium that is phosphorylatable by the cardiac myosin light chain kinase (Chan et al, 2008;Chang et al, 2015;Davis et al, 2001;Ding et al, 2010). In addition to Ca 2+ binding and RLC phosphorylation, stretch activation and mechanosensing have likewise been suggested to trigger the disruption of IHM interactions and thus initiate activation of the thick filament (Padrón et al, 2020).…”

Cardiac myosin contraction and mechanotransduction in health and disease

“…Although the regulation of molecular motors by autoinhibition has been recognized for a while (51)(52)(53)(54)(55)(56), even in the context of myosin II (57)(58)(59)(60)(61), recognition of the functional importance of autoinhibition in striated muscle myosin has emerged more recently. Key evidence of thick filament-based regulation of striated muscle was provided by measurements in Roger Cooke's lab of the single turnover rate of ATP in relaxed skeletal muscle (i.e., at low calcium) using a fluorescent ATP analog, Mant-ATP (62).…”

Filament evanescence of myosin II and smooth muscle function

“…The AA sequence of myosin II is of mechanistic significance in determining how the filaments are formed. As shown by cryoelectron microscopy ( Scarff et al, 2020 ; Yang et al, 2020 ), each myosin molecule consists of a pair of intertwined MHC as illustrated schematically in Fig. 1 .…”