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 Ca²⁺ 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 Cnidaria (sea anemones, jellyfish), the most primitive animals with muscles, and Porifera (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 Dictyostelium discoideum showed a similar, but modified, version of the motif, while the amoeba Acanthamoeba castellanii and fission yeast (Schizosaccharomyces pombe) 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.

Keywords: evolution; interacting-heads motif; muscle; myosin II; myosin regulation.

Fig. 1.

The myosin II IHM in smooth muscle myosin. In the inhibited state, the tail folds into three segments, and the heads bend back onto the tail; the blocked head motor domain (green) interacts with the free head motor domain (blue) and its ELC (pink) and with all three segments of the tail, including S2. Blocked-head ELC and blocked and free RLCs are orange, yellow, and red respectively.

Fig. 2.

Vertebrate smooth muscle myosin molecules. Molecules were imaged by rotary shadowing (A and C) or negative staining (B and D) under high-salt (A and B; extended molecules) or low-salt/MgATP (C and D; folded molecules with interacting heads) conditions. (Scale bar: 100 nm.)

Fig. 3.

Conformations of different myosin II molecules. Cartoons (approximately to scale) show folded or extended tail accompanied respectively by folding back and interaction of heads with each other and with the tail or the absence of these interactions. Below the cartoons are class averages of folded molecules and single images of unfolded molecules (averaging was not possible due to the varied conformation of these molecules). (A) In high salt, all the myosins had an extended tail and noninteracting heads; sponge myosin is shown as an example. (B–I) In low salt/MgATP, most myosins show an IHM (B–G), but Acanthamoeba and fission yeast do not (H and I). Folding of the lower half of the tail in Dictyostelium is variable and difficult to define, due to the close apposition of the segments. The cartoon shows one likely arrangement based on an analysis of the lengths of the tail regions projecting on the two sides of the heads. BH, blocked head; FH, free head; arrow shows second fold in tail. Scale bars: A, H, and I, 50 nm; B–G, 15 nm.

Fig. 4.

Insect muscle myosin II molecules. IFM and EMB myosin under high-salt (A–D; extended molecules) or low-salt/MgATP (E–H; folded molecules) conditions were imaged by rotary shadowing or negative staining. (Scale bar: 100 nm.)

Fig. 5.

Sea anemone and sponge myosin II molecules. Myosin under high-salt (A, C, and E; extended molecules) or low-salt/MgATP (B, D, and F; folded molecules) conditions was imaged by rotary shadowing or negative staining. (Scale bar: 100 nm.)

Fig. 6.

Acanthamoeba and yeast (S. pombe) myosin II molecules. Myosin under high-salt (A–D) or low-salt/MgATP (E–H) conditions was imaged by rotary shadowing or negative staining. In all cases molecules were extended (but with a shorter tail than in the animal and Dictyostelium myosin), and there was no sign of head–head interaction. (Scale bar: 100 nm.)

Fig. 7.

D. discoideum myosin II molecules. Myosin under high-salt (A and B; extended molecules) or low-salt/MgATP (C and D; folded molecules) conditions was imaged by rotary shadowing or negative staining. Molecules had a longer tail than the animal, amoeba, and yeast myosins, and part of the tail extended in the opposite direction to the folded heads (see Fig. 3G). (Scale bar: 100 nm.)

Fig. 8.

Average distance tree for MHCs from sequence alignment. The MHC sequences segregate into two major groups according to their MHC type [a similar segregation is obtained by comparing ELC and RLC sequences (72)]: (i) striated-like MHCs in vertebrates (cyan) and invertebrates (blue); (ii) smooth/nonmuscle-like MHCs in vertebrates (brown), invertebrates (orange), and amoebozoa and fungi (purple). Within the smooth/nonmuscle-like group, amoebozoa and fungi were distinct from animals. The bottom group in green (myosin V isoforms) is shown for comparison, as myosin V does not establish IHMs. The horizontal axis reflects sequence similarity (horizontal lines are shorter for more similar sequences), not evolutionary divergence times. The latter have been estimated for Amoebozoa, fungi, and Metazoa as 1,425–1,675, 975–1,225, and 725–850 Mya, based on the time-calibrated tree of ref. .