Myosin lever arm orientation in muscle determined with high angular resolution using bifunctional spin labels

Abstract

Despite advances in x-ray crystallography, cryo-electron microscopy (cryo-EM), and fluorescence polarization, none of these techniques provide high-resolution structural information about the myosin light chain domain (LCD; lever arm) under ambient conditions in vertebrate muscle. Here, we measure the orientation of LCD elements in demembranated muscle fibers by electron paramagnetic resonance (EPR) using a bifunctional spin label (BSL) with an angular resolution of 4°. To achieve stereoselective site-directed labeling with BSL, we engineered a pair of cysteines in the myosin regulatory light chain (RLC), either on helix E or helix B, which are roughly parallel or perpendicular to the myosin lever arm, respectively. By exchanging BSL-labeled RLC onto oriented muscle fibers, we obtain EPR spectra from which the angular distributions of BSL, and thus the lever arm, can be determined with high resolution relative to the muscle fiber axis. In the absence of ATP (rigor), each of the two labeled helices exhibits both ordered (σ ∼9-11°) and disordered (σ > 38°) populations. Using these angles to determine the orientation of the lever arm (LCD combined with converter subdomain), we observe that the oriented population corresponds to a lever arm that is perpendicular to the muscle fiber axis and that the addition of ATP in the absence of Ca²⁺ (inducing relaxation) shifts the orientation to a much more disordered orientational distribution. Although the detected orientation of the myosin light chain lever arm is ∼33° different than predicted from a standard "lever arm down" model based on cryo-EM of actin decorated with isolated myosin heads, it is compatible with, and thus augments and clarifies, fluorescence polarization, x-ray interference, and EM data obtained from muscle fibers. These results establish feasibility for high-resolution detection of myosin LCD rotation during muscle contraction.

Figure 1.

The bifunctional spin label (BSL). (A) Chemical structure of BSL reacted with two cysteine residues. (B) BSL bound stereospecifically to an α-helix at positions i and i + 4. (C) Angles that define the orientation of the nitroxide (defined by axes x_N, y_N, and z_N) relative to the applied magnetic field B; these angles directly determine the high-resolution orientation dependence of the EPR spectrum. Coordinates of BSL are from Binder et al., 2018 (Preprint).

Figure 2.

The EPR spectra of oriented BSL-RLC muscle fibers. (A) The effect of changing fiber orientation on EPR spectrum for smRLC helix B (56–60). (B) The parallel field experiment on helix B of skRLC and smRLC homologues labeled at equivalent sites. Field sweep is 120 G.

Figure 3.

EPR spectra of BSL–RLC (blue) and BSL–RLC–HMM (black) in solution (randomly oriented). Structural models show RLC in gray and myosin heavy chain in blue. The position of the label is depicted by red spheres on the B helix (orange) of the N-lobe and the E helix (magenta) of the C-lobe (PDB accession number 5H53; Fujii and Namba, 2017). Vertical bars indicate the positions of outer peaks of the HMM spectra, which indicate a lack of submicrosecond rotational motion. Field sweep is 100 G.

Figure 4.

EPR resolves the angular distributions of BSL-RLC in rigor and relaxation. EPR of BSL-RLC-fiber in (A) rigor (blue) and (B) relaxation (red). Fits are in black, and minced fiber data (randomly oriented control) are green. Field sweep is 100 G. The ${\theta }_{NB}>0^{\circ }$ distribution in C for the nucleotide-free (blue, first component) and ATP-bound (red, single component) state is derived from corresponding helices. Vertical bars represent predicted angles from the PDB accession number 5H53 model.

Figure 5.

EPR spectra of BSL–RLC–HMM–fiber (black) and BSL–RLC–fiber (blue) in parallel orientation. Arrows indicate the most prominent spectral features of the oriented component. Field sweep is 100 G.

Figure 6.

Refinement of the rigor actomyosin model. In blue is the initial model of skeletal myosin attached to actin (yellow) with RLC in red (5H53). Catalytic domains of myosin in other states were aligned with the catalytic domain of 5H53 to find the plane of lever arm rotation (purple), 1DFL and 1DFK (Houdusse et al., 2000). The center of rotation is denoted by a green sphere. In pale blue is the lever arm orientation derived from EPR-derived orientations of helices B and E on the RLC (pale red).

Figure 7.

Comparison of the probe angular distributions measured by EPR and other methods. Spectra of BSL–RLC–fiber in rigor (blue) with a prediction derived from S1 cryo-EM 5H53 model (black), BR fluorescence polarization on skinned fibers (green), and Z-ward head of the double-head model derived from x-ray interference experiment on fibers (magenta).