Assembly of Acanthamoeba myosin-II minifilaments. Definition of C-terminal residues required to form coiled-coils, dimers, and octamers.
Acanthamoeba myosin-II forms bipolar octamers by three successive steps of dimerization of the C-terminal, coiled-coil tail. In this study, we generated N-terminal and C-terminal truncation constructs and point mutants of the Acanthamoeba myosin-II tail to delineate the structural requirements for assembly of bipolar mini-filaments. By the use of light-scattering, CD spectroscopy, analytical ultracentrifugation, and tryptophan fluorescence experiments, we determined that: (1) the C-terminal 14 heptad repeats plus most of the tailpiece (residues 1381-1509) are required to form antiparallel dimers of coiled-coils; (2) amino acid residues within heptads 23-32 (residues 1254-1325) are required to form tetramers; (3) the C-terminal 32 heptad repeats suffice to assemble octameric minifilaments; (4) A1378 is outside of the interaction interface; (5) the mutation L1475W inhibits dimerization; and (6) F1443 is involved in the dimerization interface but is exposed to the solvent. We propose that the tailpiece (residues 1483-1509) interacts with two heptads (13 and 14, residues 1381-1393), which are important for dimerization and coiled-coil formation. These results support a model in which hydrophobic as well as electrostatic interactions control the register between myosin-II coiled-coils and guide sequential steps of dimerization that generate stable, octameric mini-filaments.
Turbedsky
Kirsi
K
Structural Biology Laboratory, Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA. turbedsky@access4less.net
Pollard
Thomas D
TD
D000048:Acanthamoeba / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D003001:Cloning, Molecular; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D007525:Isoelectric Focusing; D007700:Kinetics; D008027:Light; D008969:Molecular Sequence Data; D009154:Mutation; D024462:Myosin Type II / Q000737:chemistry*; D009218:Myosins / Q000737:chemistry; D017354:Point Mutation; D011189:Potassium Chloride / Q000737:chemistry; D011392:Proline / Q000737:chemistry; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012542:Scattering, Radiation; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D014461:Ultracentrifugation
D011189:Potassium Chloride; D011392:Proline; D024462:Myosin Type II; D009218:Myosins