Head–Head and Head–Tail Interaction: A General Mechanism for Switching Off Myosin II Activity in Cells

Abstract

Intramolecular interaction between myosin heads, blocking key sites involved in actin-binding and ATPase activity, appears to be a critical mechanism for switching off vertebrate smooth-muscle myosin molecules, leading to relaxation. We have tested the hypothesis that this interaction is a general mechanism for switching off myosin II-based motile activity in both muscle and nonmuscle cells. Electron microscopic images of negatively stained myosin II molecules were analyzed by single particle image processing. Molecules from invertebrate striated muscles with phosphorylation-dependent regulation showed head-head interactions in the off-state similar to those in vertebrate smooth muscle. A similar structure was observed in nonmuscle myosin II (also phosphorylation-regulated). Surprisingly, myosins from vertebrate skeletal and cardiac muscle, which are not intrinsically regulated, undergo similar head-head interactions in relaxing conditions. In all of these myosins, we also observe conserved interactions between the 'blocked' myosin head and the myosin tail, which may contribute to the switched-off state. These results suggest that intramolecular head-head and head-tail interactions are a general mechanism both for inducing muscle relaxation and for switching off myosin II-based motile activity in nonmuscle cells. These interactions are broken when myosin is activated.

Pseudophosphorylation of cardiac myosin regulatory light chain: a promising new tool for treatment of cardiomyopathy

“…Interestingly, the authors reported that cardiac RLC phosphorylation does not lead to a disordered structure and an ordered RLC-thick filam e n t ba c kb o n e i n t e r a c t i o n i s m a i n t a i n e d po s tphosphorylation even in the calcium-activated systolic state (Kampourakis and Irving 2015). This evidence challenges the previous studies that (1) implicated Bweakening/breakingm echanism by which RLC phosphorylation controls myosin head conformation by disrupting a compact OFF conformation of myosin (Jung et al 2008) or (2) maintained that RLC phosphorylation produces an order-to-disorder transition in skeletal muscle thick filaments (Levine et al 1995;Stewart et al 2010). The study also suggested a rather global change in RLC environment associated with changes in tertiary structure/intermolecular interactions, which argues against a previously reported unchanged conformation postphosphorylation in scallop catch muscle myosin (Kampourakis and Irving 2015;Kumar et al 2011).…”

“…The predicted structures were then modeled using the PyMOL (www.pymol.org) molecular visualization system to allow determination of the C-α distances (Å) between neighboring amino acid residues. From Yuan et al (2015) of isolated myosins and thick filaments suggested a conserved molecular mechanism in which RLC phosphorylation activates or potentiates contractility by disrupting a compact 'OFF' conformation of myosin in which the myosin heads are folded back on the myosin tail (Jung et al 2008;Wendt et al 2001;Woodhead et al 2005). Using bifunctional rhodamine probes on the cardiac RLC, Kampourakis et al have reported that phosphorylation enhances active force and its Ca 2+ -sensitivity and alters thick filament structure, with the myosin head domains becoming more perpendicular to the filament axis (Fig.…”

Myosin-10 produces its power-stroke in two phases and moves processively along a single actin filament under low load

“…However, it is worth noting that earlier work using a 93 amino acid peptide from the coiled-coil forming region of scallop muscle myosin-2 also gave rise to an APCC structure (53), but this was known to form a parallel coiled-coil in the context of the full-length molecule (54). At present, the resolution of our negatively stained EMs, obtained using recombinant M10HMM with the leucine zipper sequence after amino acid 936, does not allow us to discern the tail structure directly.…”

Structural insights into the regulation of sialic acid catabolism by the Vibrio vulnificus transcriptional repressor NanR

“…Purified NanR, the NanR/ ligand complex, and the NanR/DNA complex were diluted to a final concentration of 300 nM with 50 mM Tris·HCl (pH 7.0) and 300 mM NaCl. Five microliters of the sample solution were applied to a carbon-coated grid, which had been glow-discharged (Harrick Plasma) for 3 min in air, and the grid was negatively stained immediately by using 1% uranyl acetate (44). The grids were examined by using a Technai G2 Spirit Twin transmission electron microscope (FEI) operated at 120 kV, and the images were recorded on a 4K × 4K Ultrascan 895 CCD (Gatan) at a magnification of 0.36 nm/pixel.…”