A potential yeast actin allosteric conduit dependent on hydrophobic core residues val-76 and trp-79.
A potential yeast actin allosteric conduit dependent on hydrophobic core residues val-76 and trp-79.
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潜在的酵母肌动蛋白变构管道依赖于疏水性核心残基 val-76 和 trp-79。
DOI：
10.1074/jbc.m110.121426
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发表时间：
2010
期刊：
影响因子：
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通讯作者：
Rubenstein,PeterA
中科院分区：
文献类型：
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作者：
Wen,Kuo-Kuang;McKane,Melissa;Stokasimov,Ema;Fields,Jonathon;Rubenstein,PeterA
负责肌动蛋白构象调节的分子内变构相互作用在很大程度上是未知的。先前的研究表明，用肌动蛋白Ile代替酵母肌动蛋白Val-76会导致核苷酸交换减少。在6个残基线性阵列中，残基76与Trp-79相邻，表面以Lys-118开始，核苷酸间隙以His-73结束。为了测试改变这两个残基的包装程度是否会影响肌动蛋白的动力学，我们构建了V76I、W79F和W79Y单突变体以及Ile-76/ ph -79和Ile-76/Tyr-79双突变体。Tyr或Phe应该减少拥挤并增加蛋白质的灵活性。随后引入Ile应恢复包装和抑制变化。所有突变体在液体培养基中生长均下降。单独的W79Y具有严重的渗透敏感性，并表现出液泡异常。这两处房产都被伊尔-76救起。ph -79或Tyr降低了肌动蛋白的热稳定性，提高了其核苷酸交换速率。这些作用，通常Tyr比Phe更大，被引入Ile-76逆转。HD交换表明突变引起所有四个亚结构域的传播构象变化。根据磷酸盐释放和光散射试验的结果，单个突变对聚合的影响从最小到最大依次为Ile、Phe和Tyr。Ile-76的引入部分地弥补了Tyr-79或phee -79引起的聚合缺陷。因此，76-79残基对的拥挤变化可以强烈影响肌动蛋白的构象和行为，这些结果支持了它们所在的氨基酸阵列可能在肌动蛋白调控中发挥核心作用的理论。
Intramolecular allosteric interactions responsible for actin conformational regulation are largely unknown. Previous work demonstrated that replacing yeast actin Val-76 with muscle actin Ile caused decreased nucleotide exchange. Residue 76 abuts Trp-79 in a six-residue linear array beginning with Lys-118 on the surface and ending with His-73 in the nucleotide cleft. To test if altering the degree of packing of these two residues would affect actin dynamics, we constructed V76I, W79F, and W79Y single mutants as well as the Ile-76/Phe-79 and Ile-76/Tyr-79 double mutants. Tyr or Phe should decrease crowding and increase protein flexibility. Subsequent introduction of Ile should restore packing and dampen changes. All mutants showed decreased growth in liquid medium. W79Y alone was severely osmosensitive and exhibited vacuole abnormalities. Both properties were rescued by Ile-76. Phe-79 or Tyr decreased the thermostability of actin and increased its nucleotide exchange rate. These effects, generally greater for Tyr than for Phe, were reversed by introduction of Ile-76. HD exchange showed that the mutations caused propagated conformational changes to all four subdomains. Based on results from phosphate release and light-scattering assays, single mutations affected polymerization in the order of Ile, Phe, and Tyr from least to most. Introduction of Ile-76 partially rescued the polymerization defects caused by either Tyr-79 or Phe-79. Thus, alterations in crowding of the 76–79 residue pair can strongly affect actin conformation and behavior, and these results support the theory that the amino acid array in which they are located may play a central role in actin regulation.