Critical regions for assembly of vertebrate nonmuscle myosin II.
Myosin II molecules assemble and form filaments through their C-terminal rod region, and the dynamic filament assembly-disassembly process of nonmuscle myosin II molecules is important for cellular activities. To estimate the critical region for filament formation of vertebrate nonmuscle myosin II, we assessed the solubility of a series of truncated recombinant rod fragments of nonmuscle myosin IIB at various concentrations of NaCl. A C-terminal 248-residue rod fragment (Asp 1729-Glu 1976) was shown by its solubility behavior to retain native assembly features, and two regions within it were found to be necessary for assembly: 35 amino acid residues from Asp 1729 to Thr 1763 and 39 amino acid residues from Ala 1875 to Ala 1913, the latter containing a sequence similar to the assembly competence domain (ACD) of skeletal muscle myosin. Fragments lacking either of the two regions were soluble at any NaCl concentration. We referred to these two regions as nonmuscle myosin ACD1 (nACD1) and nACD2, respectively. In addition, we constructed an alpha-helical coiled-coil model of the rod fragment, and found that a remarkable negative charge cluster (termed N1) and a positive charge cluster (termed P2) were present within nACD1 and nACD2, respectively, besides another positive charge cluster (termed P1) in the amino-terminal vicinity of nACD2. From these results, we propose two major electrostatic interactions that are essential for filament formation of nonmuscle myosin II: the antiparallel interaction between P2 and N1 which is essential for the nucleation step and the parallel interaction between P1 and N1 which is important for the elongation step.
Nakasawa
Takashi
T
Division of Chemistry, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan.
Takahashi
Masayuki
M
Matsuzawa
Fumiko
F
Aikawa
Seiichi
S
Togashi
Yuki
Y
Saitoh
Takayuki
T
Yamagishi
Akihiko
A
Yazawa
Michio
M
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D024462:Myosin Type II / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D010446:Peptide Fragments / Q000737:chemistry; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D020449:Repetitive Sequences, Amino Acid; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012965:Sodium Chloride; D012995:Solubility; D014714:Vertebrates
D017931:DNA Primers; D018076:DNA, Complementary; D010446:Peptide Fragments; D012333:RNA, Messenger; D011994:Recombinant Proteins; D012965:Sodium Chloride; D024462:Myosin Type II