Orientation of the N- and C-Terminal Lobes of the Myosin Regulatory Light Chain in Cardiac Muscle

Abstract

The orientations of the N- and C-terminal lobes of the cardiac isoform of the myosin regulatory light chain (cRLC) in the fully dephosphorylated state in ventricular trabeculae from rat heart were determined using polarized fluorescence from bifunctional sulforhodamine probes. cRLC mutants with one of eight pairs of surface-accessible cysteines were expressed, labeled with bifunctional sulforhodamine, and exchanged into demembranated trabeculae to replace some of the native cRLC. Polarized fluorescence data from the probes in each lobe were combined with RLC crystal structures to calculate the lobe orientation distribution with respect to the filament axis. The orientation distribution of the N-lobe had three distinct peaks (N1-N3) at similar angles in relaxation, isometric contraction, and rigor. The orientation distribution of the C-lobe had four peaks (C1-C4) in relaxation and isometric contraction, but only two of these (C2 and C4) remained in rigor. The N3 and C4 orientations are close to those of the corresponding RLC lobes in myosin head fragments bound to isolated actin filaments in the absence of ATP (in rigor), but also close to those of the pair of heads folded back against the filament surface in isolated thick filaments in the so-called J-motif conformation. The N1 and C1 orientations are close to those expected for actin-bound myosin heads with their light chain domains in a pre-powerstroke conformation. The N2 and C3 orientations have not been observed previously. The results show that the average change in orientation of the RLC region of the myosin heads on activation of cardiac muscle is small; the RLC regions of most heads remain in the same conformation as in relaxation. This suggests that the orientation of the dephosphorylated RLC region of myosin heads in cardiac muscle is primarily determined by an interaction with the thick filament surface.

Myosin light chain phosphorylation enhances contraction of heart muscle via structural changes in both thick and thin filaments

“…4B, ○). This insensitivity of BC-cRLC orientation to active force generation is likely to be related to the filament-docking function of the N-terminal lobe of the cRLC and the relatively low fraction of myosin heads bound to actin during active contraction in heart muscle, as discussed previously (34).…”

“…(Fig. 1), provides a sensitive signal for the change in orientation of the RLC region of the myosin heads in cardiac thick filaments induced by cRLC phosphorylation (29,34). In the phosphorylated state of the thick filament, the light chain domains of the myosin heads are more perpendicular to the filament axis, and the dipole of the BC probe (red arrow) becomes more parallel to the filament axis (Fig.…”

“…In the phosphorylated state of the thick filament, the light chain domains of the myosin heads are more perpendicular to the filament axis, and the dipole of the BC probe (red arrow) becomes more parallel to the filament axis (Fig. 1B) (29,34). In the polarized fluorescence technique used here, this is reported as a more positive value of the order parameter <P 2 >, which measures the timeaveraged orientation of the BC probe dipole with respect to the filament axis (35).…”

Conserved Intramolecular Interactions Maintain Myosin Interacting-Heads Motifs Explaining Tarantula Muscle Super-Relaxed State Structural Basis

“…This dual phosphorylation mechanism could be extended to other chelicerate striated muscles, like that of Limulus 6 and scorpion, 7 and possibly other arthropods with thick filaments that exhibit 4-stranded helical tracks of IHMs together with myosin RLCs with a long NTE and two phosphorylatable serines. In contrast, a different activation mechanism is present in vertebrate skeletal 28, 29 and cardiac muscle, 30, 31 which have thick filaments that exhibit a perturbed 3-fold helical array of IHMs, as well as in vertebrate smooth muscle, 32 which also has IHMs but with a proposed ELC activation role, 33 and mollusks, which have 7-fold helical tracks of IHMs with ELC direct Ca 2+ -binding activation control. Below we discuss how the intra- and intermolecular interactions associated with the tarantula model (Figs.…”

“…26, 27 The IHM model has also improved knowledge of the activation mechanism in vertebrate skeletal 28, 29 and cardiac muscle. 30, 31 On the other hand, the smooth muscle IHM (PDB 1I84) structure has improved knowledge of its activation mechanism, 32 including a possible role of myosin ELC. 33 …”