Skip residues modulate the structural properties of the myosin rod and guide thick filament assembly

Abstract

The rod of sarcomeric myosins directs thick filament assembly and is characterized by the insertion of four skip residues that introduce discontinuities in the coiled-coil heptad repeats. We report here that the regions surrounding the first three skip residues share high structural similarity despite their low sequence homology. Near each of these skip residues, the coiled-coil transitions to a nonclose-packed structure inducing local relaxation of the superhelical pitch. Moreover, molecular dynamics suggest that these distorted regions can assume different conformationally stable states. In contrast, the last skip residue region constitutes a true molecular hinge, providing C-terminal rod flexibility. Assembly of myosin with mutated skip residues in cardiomyocytes shows that the functional importance of each skip residue is associated with rod position and reveals the unique role of the molecular hinge in promoting myosin antiparallel packing. By defining the biophysical properties of the rod, the structures and molecular dynamic calculations presented here provide insight into thick filament formation, and highlight the structural differences occurring between the coiled-coils of myosin and the stereotypical tropomyosin. In addition to extending our knowledge into the conformational and biological properties of coiled-coil discontinuities, the molecular characterization of the four myosin skip residues also provides a guide to modeling the effects of rod mutations causing cardiac and skeletal myopathies.myosin | cardiac/skeletal myopathies | molecular dynamics | coiled-coils | protein structure M uscle contraction is primarily driven by the interactions between actin and myosin and the associated ATP hydrolysis, but the long-range transmission of force is based on the intrinsic ability of both proteins to self-assemble into organized filaments. The myosin thick filament is a well-characterized bipolar structure. The central area, or bare zone, is

Kinesin-2 KIF3AC and KIF3AB Can Drive Long-Range Transport along Microtubules

“…To examine the microtubule binding properties of the segment of EB1 used for designing the KIF3 constructs reported here, an equivalently designed segment of M. musculus b-cardiac myosin (MYH7 rod; K1783-T1854) was developed (32). The dimerized myosin rod segment contained both an N-terminally fused segment of GP7 scaffolding protein with linker sequence (ASMPLKPEEHEDILNKLLDPELAQSERTEALQQLRVNYGS FVSEYNDLTKS) (33) and a C-terminally fused segment of EB1, matching that used for the KIF3 proteins, including linker residues in-register to the heptad repeat of the rod sequence (EDLEKERDFYFGKLRNIELICQE NEGENDPVLQRIVDILYATDE).…”

A composite approach towards a complete model of the myosin rod

“…The problem of myosin self‐assembly has now been solved by using globular fusion proteins in place of GCN4 . This strategy was developed for investigating the overlap complex for tropomyosin and components of the yeast spindle pole body, and has been recently applied to myosin .…”

“…The problem of myosin self‐assembly has now been solved by using globular fusion proteins in place of GCN4 . This strategy was developed for investigating the overlap complex for tropomyosin and components of the yeast spindle pole body, and has been recently applied to myosin . In those structural studies, the globular domains from human DNA ligase binding protein Xrcc4 and the bacteriophage φ 29 scaffolding protein Gp7 were fused to the N‐terminus of the targeted coiled‐coil domains .…”

Structure and Misfolding of the Flexible Tripartite Coiled-Coil Domain of Glaucoma-Associated Myocilin

“…This crystal grew after in situ proteolysis of mLZ 93-171 , a construct nearly identical to CC 112-185 in sequence and structure by SEC-SAXS (Figures 3C and S4E-F), which failed to crystallize under conditions tested. The susceptibility of mLZ 93-171 to further proteolysis is not immediately clear, but the presence of a so-called skip residue at Thr89/Thr103 (Figure 4A) suggests the possibility of extended unraveling as seen in myosin (Taylor et al, 2015). The structure of mLZ 122-171 , which was solved by combining ab initio modeling with molecular replacement, reveals the C-terminal disulfide bond (Figure 4B and S5A) anticipated from biochemical experiments (Figures 2D, S3D, S3L, and Table 1) capping a parallel dimer with a left-handed supercoiled twist (Figure 4C).…”