Lessons from a tarantula: new insights into muscle thick filament and myosin interacting-heads motif structure and function

Abstract

The tarantula skeletal muscle X-ray diffraction pattern suggested that the myosin heads were helically arranged on the thick filaments. Electron microscopy (EM) of negatively stained relaxed tarantula thick filaments revealed four helices of heads allowing a helical 3D reconstruction. Due to its low resolution (5.0 nm), the unambiguous interpretation of densities of both heads was not possible. A resolution increase up to 2.5 nm, achieved by cryo-EM of frozen-hydrated relaxed thick filaments and an iterative helical real space reconstruction, allowed the resolving of both heads. The two heads, "free" and "blocked", formed an asymmetric structure named the "interacting-heads motif" (IHM) which explained relaxation by self-inhibition of both heads ATPases. This finding made tarantula an exemplar system for thick filament structure and function studies. Heads were shown to be released and disordered by Ca²⁺-activation through myosin regulatory light chain phosphorylation, leading to EM, small angle X-ray diffraction and scattering, and spectroscopic and biochemical studies of the IHM structure and function. The results from these studies have consequent implications for understanding and explaining myosin super-relaxed state and thick filament activation and regulation. A cooperative phosphorylation mechanism for activation in tarantula skeletal muscle, involving swaying constitutively Ser35 mono-phosphorylated free heads, explains super-relaxation, force potentiation and post-tetanic potentiation through Ser45 mono-phosphorylated blocked heads. Based on this mechanism, we propose a swaying-swinging, tilting crossbridge-sliding filament for tarantula muscle contraction.

Keywords: Muscle; Myosin filaments; Myosin heads; Myosin interacting-heads motif; Tarantula.

Fig. 1

Models of thick filaments proposed by Hugh E. Huxley based on EM of negatively stained thick filaments (a) and low angle X-ray diffraction patterns (b) of skeletal vertebrate muscle. (a) and (b) reproduced with permission from Huxley (1963) and Huxley and Brown (1967) and the Journal of Molecular Biology

Fig. 2

Structural evidence for the thick filament of tarantula striated muscle. a Low-angle X-ray diffraction pattern of demembranated relaxed Brachypelma leg muscle showing layer lines LL1–6 spaced 1/43.5 nm⁻¹ and meridian spots M3 (1/14.5 nm⁻¹) and M6 (1/7.25 nm⁻¹). b Middle-angle pattern showing equatorial reflections E1 (1/4.3 nm⁻¹), E2 (1/2.4 nm^-1) and E3 (1/2.0 nm⁻¹). Electron micrographs of isolated relaxed thick filaments from Brachypelma negatively stained (c), metal shadowed (d) or Aphonophelma frozen-hydrated (e) showing oblique helical tracks spaced at 43.5 nm. These helices are established by heads in the relaxed state (f) that become disrupted under Ca²⁺-activation by Ca²⁺-activated myosin light chain kinase (MLCK) (g). Insets in (e–g) are zoomed 2 times. The 210-nm bare zone in (c–g) is delimited by vertical lines. Bars 200 nm. (a) and (b) provided by Dr. John Wray, (c) and (d) reproduced with permission from Crowther et al. (1985) and the Journal of Molecular Biology, (e) reproduced with permission from Woodhead et al. (2005) and Nature, (f) and (g) reproduced with permission from Craig et al. (1987) and the Journal of Cell Biology

Fig. 3

Structural evidence for the tarantula thick filament backbone. Longitudinal views of negatively stained (a) and frozen-hydrated (b) tarantula muscle thick filaments showing longitudinal striations parallel to the filament axis with ~4.0-nm spacing (red arrows) and (c) transverse ultrathin (~30 nm) section of relaxed tarantula muscle, showing sections of filament backbones (black arrows) with an external ring of 12–16 features ~3.9-nm-thick (inset, between gray circles) surrounding a middle ring of 8–11 features ~3.3 nm thick (inset, between orange circles). d Surface rendition of one 43.5-nm repeat of the 3D–recontruction of frozen-hydrated thick filament (Fig. 4a5) showing 12 density tubes interpreted as ~4-nm subfilaments (red circle). e Density projection (contrast reversed: protein white) of this repeat showing an annulus of 12 white features interpreted as “subfilaments” (red circles). The inset shows a projection of an unsymmetrized reconstruction with features similar to subfilaments at a similar radius, in spite of being much noisier. Twelve subfilaments model (red circle) containing three tails (yellow) (f) based on Wray (1979) and (g) curved molecular crystal model based on Squire (1973) for the tarantula thick filament, both with 36 tails. (b), (d) and (e) reproduced with permission from Woodhead et al. (2005) and Nature, inset in (e) personal communication from Dr. Roger Craig

Fig. 4

For the elucidation of the tarantula thick filament structure three requisites were needed to be fulfilled: a a better resolution of 3D reconstructions from negatively stained isolated filaments (a1–4 resolution 5.0 nm) or frozen-hydrated (a5 2.5 nm, a6 2.0 nm and a7 1.3 nm) with enhanced 3D reconstruction and visualization techniques (HR helical reconstruction, IHRSR iterative helical real space reconstruction), b the myosin head structure (b2) (PDB MYS1) and improved two-heads (b3–7) models and (c) fitting approaches ranging from eye-fitting (c1), 2D fitting (c2), envelope 3D fitting (c3), density rigid 3D fitting (c4, 5) and flexible 3D fitting (c6, 7). The unambiguous interpretation that lead to the myosin interacting-heads motif (IHM) (Woodhead et al. 2005) (a5, red circle) in the relaxed tarantula thick filament, belonging to the Class I proposed by Squire et al. (2005) and Squire (2009), required a resolution higher that 2.5 nm achieved by the IHRSR technique, frozen-hydrated specimens (a5) and an asymmetric head-pair model (b4) (Wendt et al. 2001), but with the S2 properly positioned (b5). In retrospect, the motif (red circles) was present and could be picked out on the 5-nm resolution 3D maps using the right density cutoffs (yellow circles). Bare zone is at the top here as well as in Figs. 6, 8, 9, and 10. Myosin sub-fragment 1 (S1), sub-fragment 2 (S2), free head (FH), blocked head (BH). The following images were reproduced with permission as follows: a1, c1 Crowther et al. (1985) and the Journal of Molecular Biology, a2, c2 Padrón et al. (1995) and the Journal of Structural Biology, c4 Offer et al. (2000) and the Journal of Molecular Biology, a5, b5, c5 Woodhead et al. (2005) and Nature, and c7 Alamo et al. (2016) and the Journal of Molecular Biology

Fig. 5

a Calculated small-angle X-ray solution scattering (SAXS) profile of PDB 3DTP (red line) and 3JBH (blue dashed line) matches the experimental squid heavy meromyosin SAXS profile (green dots) (Gillilan et al. 2013). Integrated scattering intensity (I in arbitrary units) is given as a function of momentum transfer, q = 4π sin(θ)/λ, with a scattering angle of 2θ and a wavelength of λ. b Relative deviation between squid HMM experimental data and PDB 3JBH (green dots) is calculated as (I_model-I_exp)/I_exp. The corresponding difference in scattering between PDB 3DTP and PDB 3JBH models is also shown on the same scale (red dashed line). Comparison shows that the models cannot be distinguished based on currently available scattering data. c Further calculations of PDB 3DTP and PDB 3JBH show that the scattering of the two models is nearly identical at wide angles. SAXS and error calculation described in Alamo et al. (2017b)

Fig. 6

Longitudinal (a) and transverse (b) views of frozen-hydrated tarantula thick 3D reconstruction, filtered to 2-nm resolution (EMD-1950) showing four IHMs helices (blue), packed tails (gray), and PM core (orange). The 3D reconstruction shows four 14.5-nm crowns, each with four IHMs. Densities of two IHMs (top) are shown, with blocked (BH, green) and free (FH, blue) heads and its subfragment 2 (S2, magenta). The model 3JBH (bottom), made from two adjacent IHMs models, shown as spheres, was flexibly fitted to the map. Blocked and free heads MHCIIs in green and blue, ELCs in magenta (FH) and orange (BH), and RLCs in red (FH) and yellow (BH). c Tarantula myosin II molecule based on 3JBH plus tail built with tarantula MHCII sequence (GenBank KT619079). d Tarantula pseudo-filament model built with tarantula myosin II molecules in (c)

Fig. 7

Functional implications of the tarantula asymmetric IHM 3DTP model on the relaxation and activation of thick filaments. a The IHM asymmetric structure 3DTP (inset) produces different environments for the myosin regulatory light chain (RLC) NTEs of the free (blue) and blocked (green) heads containing phosphorylatable Ser35 and Ser45. This enables the MLCK to access only the serines of the free head. b The constitutively mono-phosphorylated Ser35 on the free heads are ready to interact with the Ca²⁺-switched on thin filament through the free head swaying mechanism involving the formation and disruption of intramolecular stabilizing interactions (magenta bars) between the swaying free head and the blocked head and interconnecting intermolecular interactions (Fig. 9b). The un-phosphorylated blocked head is docked onto its own S2 by priming intramolecular interactions (green bars) and with its neighbor myosin tail with anchoring intermolecular interactions (orange bars) (Alamo et al. 2016). c Tarantula thin filament is Ca²⁺- activated according to the steric model of muscle contraction (Huxley 1973) shown by tropomyosin (TM) movements (c4) on thin filament 3D–reconstructions in relaxed (c1, TM strands, red), activating (c2, yellow) and rigor (c3, green). d Model for activation (d1–d2), potentiation (d2–d3), post-tetanic potentiation (d3–d5) and relaxation (d2–d1 and d4–d1) for tarantula striated muscle dual thin and thick filaments regulation. (a) and (d) reproduced with permission from Brito et al. (2011) and the Journal of Molecular Biology, (c) reproduced with permission from Craig and Lehman (2001) and the Journal of Molecular Biology

Fig. 8

a Low-angle synchrotron radiation X-ray diffraction pattern (60 s exposure) of skinned relaxed tarantula Brachypelma sp. leg muscle showing layer-lines LL1–6 and meridian spots M3 and M6. b Intensity profiles of LL1–6 (black lines) from this pattern. The LL1–6 intensities (orange) calculated from the tarantula thick pseudo-filament model (Fig. 6d) using the 3JBH-based tarantula myosin molecule model (Fig. 6c) matches well the experimental intensities up to ~1/0.125 nm⁻¹. c Effect on the LL1 profile of a small shift (green: +0.5 nm, cyan: +1.0 nm) in the radial position of the IHM. This suggests that in intact relaxed tarantula muscle heads are organized as in the pseudo-filament model (Fig. 6d) with IHMs protruding ~2 nm above the backbone. Fourier transforms F₁ on the l-th layer line of the heads were calculated according to the formula F₁ (R,ψ) = Σ_n G _nl (R) exp (−inψ) (Vainstein 1963) where the Fourier–Bessel structural factor G _nl is G _nl (R) = Σ_j f _jJ _n(2πRr _j) exp(i[n(π/2 – φ_j) + 2π z _jl/c]), f _j is the structural factor of j-th amino acid (number of electrons); r _j, ψ_j, z _j are polar coordinates of C_α atom of j-th amino acid (cylindrical coordinates r, ψ, z where the z-axis coincides with the axis of the thick filament); J _n is the n^th order Bessel function of the first kind; R, ψ are the radial and azimuthal coordinates in the reciprocal space; and c = 3 × 145 Å = 435 Å is the axial size of the unit cell. Layer line intensities were calculated as the azimuthally averaged square of the Fourier transform (Vainstein 1963) as I_l = Σ_n | F_nl|² with –NB < n < NB, NB = 20. X-ray diffraction patterns in (a) kindly provided by Dr. John Wray

Fig. 9

The tarantula IHM 3JBH quasi-atomic model shown flexibly fitted to 3D reconstruction (in gray) of frozen-hydrated relaxed tarantula thick filament (EMD-1950) (Alamo et al. 2008). The model is made by a free (FH, blue) and blocked (BH, green) heads, seen from its back in (b) and is established by five intramolecular interactions: two priming interactions “f” and “g” between the blocked head and the S2 (BH–S2 precursor) and three stabilizing interactions “a”, “d” and “e” between the free head and the blocked head and S2, that keep the IHM stable. The four helices of IHMs above the backbone surface (Figs. 6a, 7a, 9d) are established by five inter-molecular interactions: the heads along each helix are connected by two inter-chaining intermolecular interactions “b” and “c” established between the free head motor domain (MD) and the RLC and ELC of the neighboring blocked head regulatory domain shown in (c) and maintained above the backbone surface by three anchoring intermolecular interactions: “h” with the neighboring S2 shown in (b–d), and “i” and “j” with a neighbor “subfilament” shown on the 90° rotated view in (d). For clarity, in (c), the surface of the 3D map corresponding to the IHM in the center is highlighted in yellow, and the S2 of the model of the two IHMs on the left has been extended as coiled-coil α-helices (pink). Since the backbone structure of tarantula thick filament is not known, the two neighboring “subfilaments” tails are depicted in (d) as cylinders with ~2.2 nm diameters. In (d), it is shown that the model of the IHMs is present only in the slice between the two blue dotted lines in (a), causing the neighboring “h” interaction to be far and the “i” and “j” to be closer to the reader. In relaxed state, the S2 of the IHM emerges from the bare zone (top) with a slight angle of 6° (d), causing the helix of IHMs to “float”, separated from the backbone surface by ~2 nm. The blocked head is the only part of the IHM that is in contact with the backbone and is covalently connected to it via the S2 and electrostatically connected by the three “anchoring” intermolecular interactions: “h” (blocked head SH3 domain) with the extended S2 of an adjacent tail and “i” (blocked head relay/converter) and “j” (blocked head ELC) with the neighboring S2. The MHCII in 3JBH shows six surface loops (2, H, CM, 3, C, and I) that are involved in the interactions. Also, the ELC in the 3JBH model shows the extra two amino acids that are missing in the chicken ELC sequence. Bar 5 nm. (a–d) Reproduced with permission from Alamo et al. (2016) and the Journal of Molecular Biology

Fig. 10

Cooperative phosphorylation activation (CPA) mechanism for recruiting active heads in tarantula thick filament activation. a Thick filament model with heads disordered around S2 with RLC NTEs un-phosphorylated. Heads do not make any inter- or intramolecular interactions (see interaction table at the bottom). b On myogenesis only one head of each myosin, in pre-powerstroke state (Zoghbi et al. ; Llinas et al. 2015) can establish the two required priming intramolecular interactions docking them onto their own S2 as “blocked” heads (“BH–S2 precursor”, light green) and to a neighbor myosin tail (pink cylinder) on the backbone by anchoring intermolecular interactions (Alamo et al. 2016). The other partner (“free”) head, also in pre-powerstroke state, remains disordered without making interactions with the docked blocked head as needed for establishing an IHM. c Precursor IHMs become fully functional in relaxed state (yellow box) after Ser35 of disordered free head (blue) becomes mono-phosphorylated (yellow circle) by a temporarily activated PKC which can only phosphorylate the fully exposed Ser35 of free heads, since blocked heads Ser35 (green) are not accessible (Alamo et al. ; Brito et al. ; Sulbarán et al. 2013). To fully assemble a functional IHM, each partner free head should establish three intramolecular stabilizing interactions and one regulating RLC-RLC intramolecular interaction with its partner docked blocked head (Alamo et al. 2016). The constitutive Ser35 mono-phosphorylation (Brito et al. ; Sulbarán et al. 2013) cannot be dephosphorylated (Brito et al. ; Sulbarán et al. 2013). Free heads Ser35 mono-phosphorylation promotes their swaying away and back by Brownian motion (“swaying” heads; Fig. 7b) by breaking and reforming the intramolecular stabilizing interactions (dotted curved arrows, “±” in table) (Brito et al. 2011). d–g The CPA mechanism supports the recruiting of heads for controlled force production during brief (red box) and long Ca²⁺ exposures on unfused or fused tetani (Brito et al. ; Sulbarán et al. ; Espinoza-Fonseca et al. ; Alamo et al. 2015, 2016) as explained in the text (cf. Fig. 11c, d). The very slow, slow, fast and very fast tarantula ATP turnover life times are labeled with VS, S, F and VF on each head. The mechanism explains the structural origin of these life times according to each head type. The table shows how priming intramolecular and stabilizing intermolecular interactions are sequentially established in relaxation (c), being progressively removed (crossed out) on activation (c–f), ending in a disordered array (f) with fewer interactions. Conversely, it also shows how interactions are reformed from (f) to a newly achieved ordered array (c) after lowering [Ca²⁺] and MLCP dephophorylate Ser45 of bi-phosphorylated and mono-phosphorylated RLCs. Modified from (Alamo et al. 2016)

Fig. 11

Swaying-swinging, tilting crossbridge-sliding filament mechanism (cf. (Huxley 1969, 2004b)). The number of recruited heads producing force in the tarantula striated muscle half-sarcomere under contraction according to the CPA mechanism (Fig. 10) depends on the time [Ca²⁺] is below the threshold, as in the relaxed state (a), or above it, as in activated state (b–d). In relaxed state (a) about half of the total number of heads (free heads) could be released swaying heads while about the other half are docked swaying heads and docked blocked heads (Fig. 7d and 10). The released swaying heads move out with a swaying duty cycle D_s = 0.6 (i.e. they are released 60% of cycle time, see text) (Fig. 7b). As thin filaments are not Ca²⁺-activated (Fig. 7c1) these heads cannot bind actin neither produce force. When [Ca²⁺] is high thin filaments become activated (b–d, Fig. 7 c2). For brief Ca²⁺ exposures (blue box), only the detached swaying heads can bind to the thin filament producing force in twitches and twitch summation (b), while for longer Ca²⁺ exposures CaM.MLCK (red box) become activated (Ca²⁺ ₄.CaM.MLCK). Thus, blocked heads are recruited by phosphorylation swaying away binding to actin, potentiating force in unfused tetani (c). With a much longer Ca²⁺ exposure, many more of the remaining blocked heads become recruited, producing near maximal force in fused tetani (d). Tarantula thick filaments are ~5 μm long with a ~210-nm central bare zone, so, since there are four free (blue) and blocked (green) heads per 14.5 nm crown, each filament halve has ~1320 free and blocked heads. To simplify, only heads from 20 crowns (1 crown per repeat) are shown on each filament segment halve. From the four IHMs on each crown, only the top and bottom ones are shown. M M-band, Z Z-disk. Not to scale (Padrón 2007)