title: Tropomyosin has discrete actin-binding sites with sevenfold and fourteenfold periodicities.
abstract: Analysis of the periodic distribution of amino acids in tropomyosin has revealed the presence of seven or 14 quasi-equivalent actin-binding sites. We tested the hypothesis of periodic actin-binding sites by making deletions of chicken striated alpha-tropomyosin cDNA using oligonucleotide-directed mutagenesis. The deletions corresponded to one-half (amino acid residues 47 to 67), two-thirds (residues 47 to 74) and one actin-binding site (residues 47 to 88), on the basis of there being seven sites. The mutant cDNAs were expressed as fusion and non-fusion proteins in Escherichia coli and analyzed for actin binding and regulatory function. Fusion tropomyosin binds to actin with an affinity similar to that of muscle tropomyosin. Of the mutant fusion tropomyosins, only that with a full site deleted retained actin affinity and the ability to inhibit the actomyosin S1 ATPase, though it was less effective than wild-type. We conclude that an integral number of half-turns of the tropomyosin coiled-coil, and the consequential sevenfold periodicity, as well as the correct orientation of the ends with respect to each other, are important for actin binding. On the other hand, non-fusion tropomyosin binds well to actin only in the presence of troponin, and the binding is calcium-sensitive. Assay of non-fusion mutant tropomyosins showed that mutants with deletion of one-half and one actin binding site both had high affinity for actin, equal to or slightly less than wild-type. The ability of these two mutants to regulate the actomyosin or acto-S1 ATPase with troponin in the absence of calcium was indistinguishable from that of the wild-type. The normal regulatory function of the mutant with a 1/14 deletion (removal of a quarter turn or half a site) indicates that a 14-fold periodicity is adequate for regulation, consistent with the presence of two sets of seven alpha and seven beta quasi-equivalent actin-binding sites. An alternative explanation is that the alpha-sites are of primary importance and that proper alignment of the alpha-sites in every second tropomyosin, as when half a site is deleted, is sufficient for normal regulatory function. Deletion of a non-integral period (2/3 of a site) severely compromised actin-binding and regulatory function, presumably due to the inability of the mutant to align properly on the actin filament.
authors: Department of Neuroscience and Cell Biology, UMDNJ-Robert Wood Johnson Medical School, Piscataway 08854.
authors: S E
authors: SE
authors: Hitchcock-DeGregori
authors: T A
authors: TA
authors: Varnell
mesh_terms: D000199:Actins / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002645:Chickens; D002872:Chromosome Deletion; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D007460:Iodoacetamide / Q000378:metabolism; D007700:Kinetics; D008961:Models, Structural; D008969:Molecular Sequence Data; D009154:Mutation; D009218:Myosins / Q000378:metabolism; D015345:Oligonucleotide Probes; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D014335:Tropomyosin / Q000235:genetics / Q000378:metabolism*; D014336:Troponin / Q000378:metabolism
chemical_list: D000199:Actins; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D014335:Tropomyosin; D014336:Troponin; D004247:DNA; D009218:Myosins; D007460:Iodoacetamide