Abstract
The motor protein nonmuscle myosin II (NMII) must undergo dynamic oligomerization into filaments to perform its cellular functions. A small nonhelical region at the tail of the long coiled-coil region (tailpiece) is a common feature of all dynamically assembling myosin II proteins. This tailpiece is a key regulatory domain affecting NMII filament assembly properties and is subject to phosphorylation in vivo. We previously demonstrated that the positively charged region of the tailpiece binds to assembly-incompetent NMII-C fragments, inducing filament assembly. In the current study, we investigated the molecular mechanisms by which the tailpiece regulates NMII-C self-assembly. Using alanine scan, we found that specific positive and aromatic residues within the positively charged region of the tailpiece are important for inducing NMII-C filament assembly and for filament elongation. Combining peptide arrays with deletion studies allowed us to identify the tailpiece binding sites in the coiled-coil rod. Elucidation of the mechanism by which the tailpiece induces filament assembly permitted us further investigation into the role of tailpiece phosphorylation. Sedimentation and CD spectroscopy identified that phosphorylation of Thr(1957) or Thr(1960) inhibited the ability of the tailpiece to bind the coiled-coil rod and to induce NMII-C filament formation. This study provides molecular insight into the role of specific residues within the NMII-C tailpiece that are responsible for shifting the oligomeric equilibrium of NMII-C toward filament assembly and determining its morphology.
Highlights
nonmuscle myosin II (NMII)-C tailpiece positively charged region binds to the coiled-coil rod, inducing filament assembly
We found that specific positive and aromatic residues within the positively charged region of the tailpiece are important for inducing NMII-C filament assembly and for filament elongation
Sedimentation and CD spectroscopy identified that phosphorylation of Thr1957 or Thr1960 inhibited the ability of the tailpiece to bind the coiled-coil rod and to induce NMII-C filament formation
Summary
NMII-C tailpiece positively charged region binds to the coiled-coil rod, inducing filament assembly. Results: Positive and aromatic residues within the tailpiece are important for filament assembly, and the tailpiece binding sites in the rod were identified. A small nonhelical region at the tail of the long coiled-coil region (tailpiece) is a common feature of all dynamically assembling myosin II proteins. This tailpiece is a key regulatory domain affecting NMII filament assembly properties and is subject to phosphorylation in vivo. This study provides molecular insight into the role of specific residues within the NMII-C tailpiece that are responsible for shifting the oligomeric equilibrium of NMII-C toward filament assembly and determining its morphology
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