Hitchcock-DeGregori S E, Varnell T A
Department of Neuroscience and Cell Biology, UMDNJ-Robert Wood Johnson Medical School, Piscataway 08854.
J Mol Biol. 1990 Aug 20;214(4):885-96. doi: 10.1016/0022-2836(90)90343-K.
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.
对原肌球蛋白中氨基酸的周期性分布进行分析后发现存在七个或十四个准等效肌动蛋白结合位点。我们通过使用寡核苷酸定向诱变删除鸡横纹肌α - 原肌球蛋白cDNA来检验周期性肌动蛋白结合位点的假说。基于存在七个位点，这些缺失分别对应于半个（氨基酸残基47至67）、三分之二个（残基47至74）和一个肌动蛋白结合位点（残基47至88）。突变的cDNA在大肠杆菌中表达为融合蛋白和非融合蛋白，并对其肌动蛋白结合和调节功能进行分析。融合原肌球蛋白与肌动蛋白结合的亲和力与肌肉原肌球蛋白相似。在突变的融合原肌球蛋白中，只有删除了完整位点的那种保留了肌动蛋白亲和力和抑制肌动球蛋白S1 ATP酶的能力，不过其效果不如野生型。我们得出结论，原肌球蛋白卷曲螺旋的整数个半圈、随之而来的七重周期性以及两端相对于彼此的正确取向对于肌动蛋白结合很重要。另一方面，非融合原肌球蛋白仅在存在肌钙蛋白的情况下才能很好地与肌动蛋白结合，并且这种结合对钙敏感。对非融合突变原肌球蛋白的检测表明，删除了半个和一个肌动蛋白结合位点的突变体对肌动蛋白都具有高亲和力，等于或略低于野生型。在没有钙的情况下，这两个突变体与肌钙蛋白一起调节肌动球蛋白或肌动蛋白 - S1 ATP酶的能力与野生型没有区别。缺失1/14（去除四分之一圈或半个位点）的突变体的正常调节功能表明，十四重周期性足以进行调节，这与存在两组七个α和七个β准等效肌动蛋白结合位点一致。另一种解释是，α位点最为重要，并且当删除半个位点时，每隔一个原肌球蛋白中α位点的正确排列足以实现正常的调节功能。删除非整数周期（三分之二个位点）会严重损害肌动蛋白结合和调节功能，大概是因为突变体无法在肌动蛋白丝上正确排列。
