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A 29 residue region of the sarcomeric myosin rod is necessary for filament formation
R L Sohn1, K L Vikstrom, M Strauss
1Department of Molecular, Cellular and Developmental Biology, Universityof Colorado, Boulder 80309-0347, USA.
Abstract:
Myosin is a motor protein whose functional unit in the sarcomere is the thick filament. The myosin molecule is capable of self-assembly into thick filaments through its alpha-helical coiled-coil rod domain. To define more precisely the sequence requirements for this assembly, segments of the human fast IId skeletal myosin rod were expressed in Escherichia coli and examined differential solubility and the formation of ordered paracrystals. We show that both properties appear to require a 29 residue sequence (residues 1874 to 1902) near the C terminus of the rod region. To test further the role of this region in assembly, a protein was constructed which consisted of this assembly competence domain (ACD) fused to the carboxy terminus of an assembly-incompetent myosin rod fragment. This chimeric fragment exhibited myosin's characteristic solubility properties and formed ordered paracrystals. To complement these in vitro experiments, both a full-length myosin heavy chain (MYH) and one from which the 29 residues were deleted were transfected into cultured mammalian cells. While the full-length construct formed the spindle-shaped structures characteristic of arrays of thick filaments, the deleted MYH showed only diffuse staining throughout the cytoplasm by light microscopy. Thus, there appears to be a specific sequence in the C-terminal region of the myosin heavy chain rod which is necessary for ordered paracrystal formation and is sufficient to confer assembly properties to an assembly-incompetent rod fragment.
Insights
A specific 29-residue sequence in myosin heavy chain (MYH) rods is crucial for thick filament assembly. This sequence is sufficient to restore assembly to incompetent fragments and is necessary for proper thick filament formation in cells.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Myosin is a motor protein essential for muscle contraction.
- Myosin self-assembles into thick filaments via its rod domain.
- Understanding myosin assembly is key to muscle function.
Purpose of the Study:
- To identify specific sequences in the myosin rod required for thick filament assembly.
- To investigate the role of the C-terminal rod region in myosin assembly.
Main Methods:
- Expression of human fast IId skeletal myosin rod segments in E. coli.
- Analysis of protein solubility and paracrystal formation in vitro.
- Construction of chimeric myosin fragments and transfection into mammalian cells.
Main Results:
- A 29-residue sequence (residues 1874-1902) near the C terminus is critical for solubility and paracrystal formation.
- This sequence, termed the assembly competence domain (ACD), conferred assembly properties to an incompetent fragment.
- Full-length MYH transfected into cells formed characteristic thick filament structures, while MYH lacking the ACD showed diffuse cytoplasmic staining.
Conclusions:
- A specific C-terminal sequence in the myosin heavy chain rod is necessary and sufficient for ordered thick filament assembly.
- This discovery provides insights into the molecular mechanisms of sarcomere formation and muscle structure.