X-ray diffraction analysis of the effects of myosin regulatory light chain phosphorylation and butanedione monoxime on skinned skeletal muscle fibers

Abstract

The phosphorylation of the myosin regulatory light chain (RLC) is an important modulator of skeletal muscle performance and plays a key role in posttetanic potentiation and staircase potentiation of twitch contractions. The structural basis for these phenomena within the filament lattice has not been thoroughly investigated. Using a synchrotron radiation source at SPring8, we obtained X-ray diffraction patterns from skinned rabbit psoas muscle fibers before and after phosphorylation of myosin RLC in the presence of myosin light chain kinase, calmodulin, and calcium at a concentration below the threshold for tension development ([Ca(2+)] = 10(-6.8)M). After phosphorylation, the first myosin layer line slightly decreased in intensity at ∼0.05 nm(-1)along the equatorial axis, indicating a partial loss of the helical order of myosin heads along the thick filament. Concomitantly, the (1,1/1,0) intensity ratio of the equatorial reflections increased. These results provide a firm structural basis for the hypothesis that phosphorylation of myosin RLC caused the myosin heads to move away from the thick filaments towards the thin filaments, thereby enhancing the probability of interaction with actin. In contrast, 2,3-butanedione monoxime (BDM), known to inhibit contraction by impeding phosphate release from myosin, had exactly the opposite effects on meridional and equatorial reflections to those of phosphorylation. We hypothesize that these antagonistic effects are due to the acceleration of phosphate release from myosin by phosphorylation and its inhibition by BDM, the consequent shifts in crossbridge equilibria leading to opposite changes in abundance of the myosin-ADP-inorganic phosphate complex state associated with helical order of thick filaments.

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

“…7d1, 2, 10d, 11b) (Linari et al 2015). Seventh, X-ray diffraction of rabbit skeletal muscle suggests that RLC phosphorylation move heads away from thick towards thin filaments (Yamaguchi et al 2016) confirming tarantula equatorial X-ray diffraction results . Eighth, spectroscopic studies of the super-relaxed state of skeletal muscle supports the putative RLC-RLC interface identified by cryo-EM showing that both the divalent cation bound to RLC and the RLC NTE play a role in the stability of the superrelaxed state (Nogara et al 2016).…”

“…7d) (Brito et al 2011) suggested a cooperative phosphorylation mechanism for activation of tarantula thick filaments (Sulbarán et al 2013) extended by further work (Espinoza-Fonseca et al 2007, 2008Kast et al 2010;Alamo et al 2015Alamo et al , 2016Yamaguchi et al 2016). The CPA mechanism is shown in Fig.…”

“…Bar 5 nm. (a-d) Reproduced with permission from Alamo et al (2016) and the Journal of Molecular Biology (Alamo et al 2008(Alamo et al , 2016Brito et al 2011;Sulbarán et al 2013;Espinoza-Fonseca et al 2015;Yamaguchi et al 2016;reviewed by (Vandenboom 2017).…”

Effects of myosin inhibitors on the X-ray diffraction patterns of relaxed and calcium-activated rabbit skeletal muscle fibers

“…The M·ADP·Pi state is one of the states that favor the ‘closed’ conformation, but this conformation is also favored by the presence of so-called phosphate analogs, i.e., aluminofluoride and beryllium fluoride [ 18 ], and a myosin inhibitor blebbistatin [ 19 ], resulting in an increased helical order. An increased helical order is also observed in the presence of BDM [ 20 ]. Here the ‘closed’ conformation refers to the one consisting of “the switch-2 element of myosin head being in a position that interacts with the γ-phosphate-binding pocket; open conformation, the switch-2 loop being rotated away ~4 Å from the γ-phosphate-binding pocket, facilitating phosphate release” (quoted from ref.…”

X-ray Diffraction Studies on the Structural Origin of Dynamic Tension Recovery Following Ramp-Shaped Releases in High-Ca Rigor Muscle Fibers

“…The wavelength and the size of X-ray beam was 0.09 nm and size, 250 × 150 µm, respectively, and the specimen-to-detector distance was 1.8 m. The fiber was kept at its slack length Lo (~3 mm) at a sarcomere length of 2.0-2.4 µm, which was measured by optical diffraction by He-Ne laser light [9]. Dithiothreitol (5 mM) and catalase (1000 U/mL) were added to the experimental solution to prevent damage of the fibers from free radicals produced by X-rays [25].…”