Structural basis of the relaxed state of a Ca ²⁺ -regulated myosin filament and its evolutionary implications

Abstract

Myosin filaments of muscle are regulated either by phosphorylation of their regulatory light chains or Ca 2+ binding to the essential light chains, contributing to on-off switching or modulation of contraction. Phosphorylation-regulated filaments in the relaxed state are characterized by an asymmetric interaction between the two myosin heads, inhibiting their actin binding or ATPase activity. Here, we have tested whether a similar interaction switches off activity in myosin filaments regulated by Ca 2+ binding. Cryo-electron microscopy and single-particle image reconstruction of Ca 2+ -regulated (scallop) filaments reveals a helical array of myosin headpair motifs above the filament surface. Docking of atomic models of scallop myosin head domains into the motifs reveals that the heads interact in a similar way to those in phosphorylation-regulated filaments. The results imply that the two major evolutionary branches of myosin regulation-involving phosphorylation or Ca 2+ binding-share a common structural mechanism for switching off thick-filament activity in relaxed muscle. We suggest that the Ca 2+ -binding mechanism evolved from the more ancient phosphorylation-based system to enable rapid response of myosin-regulated muscles to activation. Although the motifs are similar in both systems, the scallop structure is more tilted and higher above the filament backbone, leading to different intermolecular interactions. The reconstruction reveals how the myosin tail emerges from the motif, connecting the heads to the filament backbone, and shows that the backbone is built from supramolecular assemblies of myosin tails. The reconstruction provides a native structural context for understanding past biochemical and biophysical studies of this model Ca 2+ -regulated myosin.scallop muscle | molluscan muscle | thick-filament structure | 3D reconstruction | muscle regulation C ontractile activity in muscle is switched on and off by protein subunits on the thick (myosin-containing) and thin (actincontaining) filaments (1). Regulation via myosin is based on either Ca 2+ -dependent phosphorylation of the myosin regulatory light chains (RLCs) (this mode of regulation occurs in vertebrate smooth muscle and some invertebrate striated muscles) or Ca 2+ binding to the essential light chains (ELCs) (occurring in some invertebrate striated muscles) (2-6). In some muscles (vertebrate striated and some invertebrate striated), phosphorylation modulates activity but is not required for muscle activation (2,7,8). Electron-microscopic studies of vertebrate smooth muscle myosin molecules have revealed that, in the relaxed (dephosphorylated) state, the two myosin heads in a molecule interact with each other asymmetrically so that the actin-binding region of one (the "blocked" head) is blocked by interaction with the converter domain and ELC of the other (the "free" head). It is thought that this switches off actin-binding and ATPase activity of the blocked and free heads, respectively (9, 10), contributing to muscle relaxation. Phosphorylati...

An invertebrate smooth muscle with striated muscle myosin filaments

“…Table 2. Sliding of native tarantula thin filaments over tarantula or S. mansoni thick filaments This motif has now been demonstrated in striated muscle thick filaments of chelicerates (22)(23)(24), molluscs (53), vertebrates (54,55), and here in the smooth muscle thick filaments of a Platyhelminth. Observation of the motif in single myosin molecules (in cases where it has not yet been demonstrated in thick filaments) extends this list to vertebrate smooth muscle (25,56) and nonmuscle myosin (57), and to the most primitive animals with muscles, Cnidaria (sea anemones, jellyfish) (58).…”

The myosin mesa and the basis of hypercontractility caused by hypertrophic cardiomyopathy mutations

“…1,2,25,40,43,44 ). The IHM has also been observed in other classes of muscle myosin, including striated muscle myosins 41 , and in images of intact thick filaments isolated from a variety of muscle types 1–6 . Thick-filament structures of 2.0- to 4.0-nm resolution have been obtained with cryo-EM for tarantula skeletal muscle 1,2 , Limulus telson muscle 3 , and vertebrate cardiac muscle 7,8 .…”

“…The myosin thick filament alone is a complex structure containing myosin, MyBP-C, titin, and other proteins. Important low-resolution structures have been determined by a number of investigators using both skeletal 1–6 and cardiac muscle 7–9 isolated thick filaments. However, the complexity of this multiprotein assembly makes the locations of its various components difficult to determine.…”

Coupling between myosin head conformation and the thick filament backbone structure

“…1C) (Woodhead et al, 2005). Since then, it has been found in all relaxed thick filament structures investigated (Al-Khayat et al, 2013; Pinto et al, 2012; Sulbaran et al, 2015; Woodhead et al, 2013; Zhao et al, 2009), usually in an orientation in which the IHM is approximately tangential to the surface of the filament backbone with the blocked head contacting the S2 fragment of the myosin rod. More recently and surprisingly, the IHM was found to characterize the relaxed thick filament structure from Lethocerus asynchronous flight muscle (Hu et al, 2016) but in what is so far a unique orientation; it is almost perpendicular to the filament backbone (Fig.…”