Interacting-heads motif has been conserved as a mechanism of myosin II inhibition since before the origin of animals

Abstract

Electron microscope studies have shown that the switched-off state of myosin II in muscle involves intramolecular interaction between the two heads of myosin and between one head and the tail. The interaction, seen in both myosin filaments and isolated molecules, inhibits activity by blocking actin-binding and ATPase sites on myosin. This interacting-heads motif is highly conserved, occurring in invertebrates and vertebrates, in striated, smooth, and nonmuscle myosin IIs, and in myosins regulated by both Ca binding and regulatory light-chain phosphorylation. Our goal was to determine how early this motif arose by studying the structure of inhibited myosin II molecules from primitive animals and from earlier, unicellular species that predate animals. Myosin II from (sea anemones, jellyfish), the most primitive animals with muscles, and (sponges), the most primitive of all animals (lacking muscle tissue) showed the same interacting-heads structure as myosins from higher animals, confirming the early origin of the motif. The social amoeba showed a similar, but modified, version of the motif, while the amoeba and fission yeast () showed no head-head interaction, consistent with the different sequences and regulatory mechanisms of these myosins compared with animal myosin IIs. Our results suggest that head-head/head-tail interactions have been conserved, with slight modifications, as a mechanism for regulating myosin II activity from the emergence of the first animals and before. The early origins of these interactions highlight their importance in generating the inhibited (relaxed) state of myosin in muscle and nonmuscle cells.

Mavacamten stabilizes the auto-inhibited state of two-headed cardiac myosin

“…It also indicates that actin interaction with the auto-inhibited HMM accelerates the structural disruption of the inhibited state. This provides an explanation for why the IHM has been difficult to observe in cardiac and skeletal myosin by EM-interactions with the UV-treated carbon surface or charged mica surface could disrupt it in the absence of cross-linking [13] or stabilizing small-molecules [11].…”

“…Regulation of the cardiac myosin thick filament-independent of the actomyosin interaction-is hypothesized to be a critical determinant of contraction and contribute to the Frank-Starling relationship, a fundamental regulator of cardiac performance [26], and to cardiac dysfunction in inherited cardiomyopathies [18,27]. The structural state of cardiac thick-filaments is hypothesized to be controlled by an evolutionarily-conserved mechanism where the heads of each myosin dimer fold back and interact with their S2 coiled-coil domain and with the thick-filament backbone [13]. This structural state, with myosin heads arranged on the thick filament, has been observed in relaxed muscle with X-ray diffraction [23] and with fluorescence polarization in a specific, head-head conformation termed the interacting heads motif (IHM) [22,28].…”

“…As a result of these interactions, the folded heads are hypothesized to turn over ATP much more slowly than the unfolded heads or than S1 heads isolated in solution. This is likely because the structural changes in the ATPase site that are required for dissociation of the ATP hydrolysis products require movement of the myosin light-chain binding domain and actin-binding interface [12,13]. Movement of these elements would be prevented in the folded state due to direct interaction of the heads and the head-S2 domain, and thus, ATP-turnover would be slowed in this state.The interactions between myosin heads and the S2 coiled-coil domain and thick-filament backbone described above are collectively termed the interacting-heads motif (IHM, Fig.…”

“…1). The IHM is hypothesized to be an evolutionarily conserved mechanism for controlling myosin II driven motility [13][14][15][16]. Though electron microscopy (EM) studies suggest that the individual myosin heads in the cardiac thick-filament can form the IHM state [10,17] it has not been observed in cardiac myosin in solution without chemical stabilization, nor have its associated transient biochemical and structural kinetics been fully explored.We tested the hypothesis that cardiac myosin is auto-inhibited by direct head-head interaction and that mavacamten selectively targets this intrinsic inhibition by comparing the actin-activated and nonactivated (basal) single ATP turnover kinetics of single and two-headed bovine ventricular cardiac myosin fragments.…”

“…This dimerization allows the two heads to directly and allosterically interact with each other. Head-head interactions play critical roles in regulating actin-activation in other myosin II family members [13]. The selective effect of mavacamten on the amplitudes in HMM but not S1 at saturating concentrations of actin indicates that mavacamten stabilizes a unique state in HMM, which occurs infrequently in S1 when the myosin heavy chain fragment lacks a regulatory light chain (RLC) and is not tethered to a second myosin by the S2 coiled-coil domain.…”