Structure of the Flight Muscle Thick Filament from the Bumble Bee, Bombus ignitus, at 6 Å Resolution

Abstract

Four insect orders have flight muscles that are both asynchronous and indirect; they are asynchronous in that the wingbeat frequency is decoupled from the frequency of nervous stimulation and indirect in that the muscles attach to the thoracic exoskeleton instead of directly to the wing. Flight muscle thick filaments from two orders, Hemiptera and Diptera, have been imaged at a subnanometer resolution, both of which revealed a myosin tail arrangement referred to as “curved molecular crystalline layers”. Here, we report a thick filament structure from the indirect flight muscles of a third insect order, Hymenoptera, the Asian bumble bee Bombus ignitus. The myosin tails are in general agreement with previous determinations from Lethocerus indicus and Drosophila melanogaster. The Skip 2 region has the same unusual structure as found in Lethocerus indicus thick filaments, an α-helix discontinuity is also seen at Skip 4, but the orientation of the Skip 1 region on the surface of the backbone is less angled with respect to the filament axis than in the other two species. The heads are disordered as in Drosophila, but we observe no non-myosin proteins on the backbone surface that might prohibit the ordering of myosin heads onto the thick filament backbone. There are strong structural similarities among the three species in their non-myosin proteins within the backbone that suggest how one previously unassigned density in Lethocerus might be assigned. Overall, the structure conforms to the previously observed pattern of high similarity in the myosin tail arrangement, but differences in the non-myosin proteins.

Keywords: asynchronous flight muscle; coiled coil; cryoelectron microscopy; myosin; striated muscle.

Figure 1

Bombus thick filament reconstruction. (A) An electron micrograph of a thick filament. The longitudinal lines in the thick filament backbone are produced by the coiled-coil domains of myosin and paramyosin. The disordered dark transverse densities marked by black arrows are possibly the myosin heads. Occasionally, transverse densities (arrowheads) are observed, which are reminiscent of the Free Heads in Lethocerus thick filaments [8]. (B) The reconstruction shows three full crowns of the Bombus thick filament. The “floating” densities (arrowheads) are the average positions of the myosin heads when helical and 4-fold symmetry are enforced. Non-myosin density, flightin (red) is visible extending from the backbone surface. (C) Transverse view through one crown showing the relative positions of flightin (red), myofilin (yellow), putative paramyosin (purple) and the curved layers (dark gray, light gray and white). Note that both flightin and myofilin have folded domains on the inside surface of the annulus of myosin tails that could contact paramyosin. (D) The 12-crown extended filament, with one curved layer highlighted in blue. The floating densities are not connected to the myosin tail due to the disorder in the proximal S2.

Figure 2

Skip residues and proximal S2. (A) Skip residue accommodation regions of Bombus ignitus (grey map envelope) are well aligned with the Lethocerus indicus atomic model (PDB–7KOG) shown in yellow with skip residue colored in purple for the accommodation region and white elsewhere [21], except for Skip 1 where the Lethocerus atomic model falls outside the Bombus envelope due to the latter’s different azimuthal rotation when compared with Lethocerus (white). (B) Superposition of curved layers from Lethocerus (white) and Bombus (blue). When the backbone is low pass filtered to the same resolution that reveals the average myosin head position additional proximal S2 is revealed up to the arrowhead. The Lethocerus proximal S2 (white) prominently bends away from the trajectory of the coiled coils within the backbone as a consequence of Free Head binding to the filament backbone. In Bombus, the proximal S2 (blue) follows the trajectory of the backbone-embedded tail downwards at least as far as the resolved density shown. The Lethocerus density map has been low pass filtered to 7 Å resolution to make it comparable to the displayed Bombus map.

Figure 3

Disordered myosin heads of Bombus compared with those of Lethocerus. The Bombus density map (blue) is superimposed on the Lethocerus density map (grey) in both side view (A) and top view (B). (C) An atomic model of the IHM (Blocked Head heavy chain, red; Essential Lght Chain (ELC), blue; Regulatory Light Chain (RLC), yellow; Free Head heavy chain, purple; ELC, green; RLC, orange) along with the myosin head density of the Lethocerus reconstruction is shown. The floating densities from Bombus (blue) represent the average position of otherwise highly disordered myosin heads. The crystal structure of cardiac S2 (red) is aligned to the Bombus S2 density. The S2 atomic model points directly at the floating myosin head density. Note that the Lethocerus features (map and atomic model) are aligned to the cardiac S2 atomic model and not to their position in the relaxed Lethocerus thick filaments. (D) Axial view with atomic models of the Free Head (left) and IHM (right) from relaxed Lethocerus thick filament superimposed on the Bombus density map with the N-terminal domain of the RLC under the proximal S2. The position of the proximal S2 (black arrowheads) is juxtaposed with the inner edge of the floating density. The atomic models serve to illustrate how a floating density might be explained by mostly azimuthal movements of either individual myosin heads or interacting heads motifs. The floating density in Bombus would appear to mostly represent the average position of the disordered RLC.

Figure 4

Multiple sequence alignment of flightin and myofilin of Drosophila, Lethocerus and Bombus flight muscle. (A) The alignment of flightin shows the highly conserved “WYR” motif, which probably corresponds to the common globular flightin density seen in 3-D image reconstructions of thick filaments from the three species. Comparatively, the N- and C- termini are less conserved, although some improvement in conservation is seen at the C-terminus. Among all 112 flightin sequences available in Uniprot, both predicted and observed experimentally, 6 out of 32 C-terminal residues have over 60% similarity (indicated as blue dots below), and if the entries are narrowed down to endopteryogota taxonomy, a total of 80 flightin sequences, 11 out of 32 have high similarity of over 60% (indicated as purple dots below). (B) For myofilin, the N-terminal sequence is well conserved, as is the shape and position of the folded domain seen among the three species, which suggests that they represent the same entity. Conservation decreases after the N-terminal domain, improves somewhat after about 30 residues in Bombus, and becomes very poor toward the C-terminus suggesting that the highly variable parts of the putative myofilin density represent these less well-conserved other segments of the sequence.

Figure 5

Three non-myosin densities in Bombus thick filament. (A) Bombus flightin molecules (red, solid) are similar among all three species with a small folded domain (labeled 1) in the middle corresponding to the WYR domain [28], a partially ordered extension outside of the filament backbone (labelled 2) on one side of the WYR domain and an extension on the other side (labelled 3). At the N-terminus (labelled 2) Lethocerus (red, mesh) shows the largest extension outside the filament backbone, stabilized by contacts to its proximal S2 [8]. Drosophila has the same feature but with a shorter extension [13] corresponding to only the overlap between Lethocerus and Bombus. The flightin N-terminal extension seen in Bombus leaves the backbone density at a different angle (solid red surface). The flightin C-terminus (labelled 3) overlaps a density (blue, mesh) from a Lethocerus thick filament reconstruction. (B) Bombus myofilin (yellow, solid) shows a novel structure, with two densities, that overlap and contact the Lethocerus myofilin density (yellow, mesh). (C) The Bombus paramyosin (purple, solid) in the central core. (D) The distribution of all three non-myosin proteins in the context of the thick filament. (E) Flightin and myofilin decorating a single curved layer. (F) The top view of a full crown slab containing all three non-myosin proteins. (G) Side view showing the relationship of the three non-myosin proteins to the five curved layers colored white, light gray and dark gray. (H) Top view and (I) side view showing the relationship of flightin and myofilin with the paramyosin core. Both myofilin and flightin appear to contact paramyosin.

Figure 6

AlphaFold2 modeling of Bombus myofilin and flightin. (A) The AlphaFold2 atomic model of the flightin WYR motif fits well into the globular domain in a zoomed-in view (right) that roughly extends from residue 65 to residue 110, which is also the region of conserved sequence and similar reconstruction density in Drosophila and Lethocerus. The full atomic model starts from residue 41 to the residue 151 at the C-terminus (left). (B) The AlphaFold2 atomic model of myofilin only fits well at the N-terminal LKG domain up to residue 36 (right), which corresponds to the region of high sequence and density conservation in Drosophila and Lethocerus, while the rest of the AlphaFold2 model has poor reliability (left).