The second half of the fourth period of tropomyosin is a key region for Ca(2+)-dependent regulation of striated muscle thin filaments.
Rabbit skeletal muscle alpha-tropomyosin (Tm), a 284-residue dimeric coiled-coil protein, spans seven actin monomers and contains seven quasiequivalent periods. X-ray analysis of cocrystals of Tm and troponin (Tn) placed the Tn core domain near residues 150-180 of Tm. To identify the Ca(2+)-sensitive Tn interaction site on Tm, we generated three Tm mutants to compare the consequences of sequence substitution inside and outside of the Tn core domain-binding region. Residues 152-165 and 156-162 in the second half of period 4 were replaced by corresponding residues 33-46 and 37-43 in the second half of period 1, respectively (termed mTm152-165 and mTm156-162, respectively), and residues 134-147 in the first half of period 4 were replaced with residues 15-28 in the first half of period 1 (mTm134-147). Recombinant Tms designed with an additional tripeptide, Ala-Ala-Ser, at the N-terminus were expressed in Escherichia coli. Both mTm152-165 and mTm156-162 suppressed the actin-activated myosin subfragment-1 Mg(2+)-ATPase rate regardless of whether Ca(2+) and Tn were present. On the other hand, mTm134-147 retained the normal Ca(2+)-sensitive regulation, although the actin binding of mTm alone was significantly impaired. Differential scanning calorimetry showed that the sequence substitution in the second half of period 4 affected the thermal stability of the complete Tm molecule and also the actin-induced stabilization. These results suggest that the second half of period 4 of Tm is a key region for inducing conformational changes of the regulated thin filament required for its fully activated state.
Sakuma
Akiko
A
Department of Applied Chemistry and Biotechnology, Fukui University, 3-9-1 Bunkyo, Fukui 910-8507, Japan.
Kimura-Sakiyama
Chieko
C
Onoue
Atsuhiro
A
Shitaka
Yuji
Y
Kusakabe
Takahisa
T
Miki
Masao
M
D008841:Actin Cytoskeleton / Q000737:chemistry / Q000378:metabolism*; D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D018360:Crystallography, X-Ray; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000378:metabolism*; D009154:Mutation; D015879:Myosin Subfragments / Q000737:chemistry / Q000378:metabolism; D010590:Phalloidine / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D014335:Tropomyosin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014336:Troponin / Q000737:chemistry
D000199:Actins; D015879:Myosin Subfragments; D011994:Recombinant Proteins; D014335:Tropomyosin; D014336:Troponin; D010590:Phalloidine; D000251:Adenosine Triphosphatases; C035123:magnesium sodium ATPase; D002118:Calcium