Functions of tropomyosin's periodic repeats.
Tropomyosin binds along actin filaments and regulates actin-myosin interaction in muscle and nonmuscle cells. Seven periodic amino acid repeats are proposed to correspond to actin binding sites, and the middle periods are important for cooperative activation of actin by myosin. The functional contributions of individual periods were studied in mutants in which periods 2-6 were individually deleted from rat striated muscle alphaalpha-tropomyosin or replaced with a leucine zipper sequence. Unacetylated recombinant tropomyosins were assayed for actin binding, regulation of the actomyosin ATPase with troponin, cooperative myosin S1-induced binding to actin, and thermal stability. Tropomyosin function is relatively insensitive to deletion of period 2, but loss increases as the deletion is shifted toward the C-terminus. Retention of function upon deletion of the periodic repeats is in the order of 2 > 3 approximately 4 approximately 6 >> 5. Internal periods are important for specific functions and are not quasiequivalent. Deletion of period 5 (residues 166-207), and especially deletion or replacement of residues 166-188, a constitutively expressed region encoded by exon 5, had severe consequences on actin affinity and cooperative myosin S1-induced binding to actin. Period 6, residues 208-242, part of the troponin binding site, is required for full inhibition of the actomyosin ATPase in the absence of calcium. The effect of the deletion can depend on its context, suggesting that sequence alone is not the only factor important for function. We propose that the local structure and stability, and consequent flexibility, of the coiled coil are major determinants of actin affinity.
Hitchcock-DeGregori
Sarah E
SE
Department of Neuroscience and Cell Biology, UMDNJ-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA. hitchcoc@umdnj.edu
Song
Yuhua
Y
Greenfield
Norma J
NJ
D000199:Actins / Q000737:chemistry / Q000378:metabolism; D000205:Actomyosin / Q000737:chemistry / Q000378:metabolism; D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000737:chemistry; D002942:Circular Dichroism; D016350:Leucine Zippers / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015879:Myosin Subfragments / Q000737:chemistry / Q000378:metabolism; D009218:Myosins / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D051381:Rats; D020449:Repetitive Sequences, Amino Acid / Q000235:genetics; D017384:Sequence Deletion; D014335:Tropomyosin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D000199:Actins; D015879:Myosin Subfragments; D014335:Tropomyosin; D000205:Actomyosin; D009218:Myosins; D002118:Calcium