Requirement of left-handed glycine residue for high stability of the Tk-subtilisin propeptide as revealed by mutational and crystallographic analyses
Requirement of left-handed glycine residue for high stability of the Tk-subtilisin propeptide as revealed by mutational and crystallographic analyses
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DOI：
10.1016/j.jmb.2007.10.030
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发表时间：
2007-12-14
影响因子：
5.6
通讯作者：
Kanaya, Shigenori
中科院分区：
文献类型：
--
作者：
Pulido, Marian A.;Tanaka, Shun-ichi;Kanaya, Shigenori
Tk-subtilisin[活性形式的pro -Tk-subtilisin的成熟结构域（Gly70-Gly398）]来自超嗜热古细菌Thermococcus kodakaraensis，由pro -Tk-subtilisin[来自T. kodakaraensis的前形式的枯草素同源物（Gly1-Gly398）]经过前肽的自动加工和降解而成熟。Pro-Tk-subtilisin的特点是在温和的温度下成熟非常缓慢，但由于前肽区域的单个Gly56 -> Ser突变，这一成熟速度大大提高。为了分析Gly56的作用，构建了Gly56上氨基酸完全取代的Pro-Tk-subtilisin变体。对它们的光圈形成活性进行比较表明，除了Pro-G56W [Pro-G56X， Pro-Tk-subtilisin与Gly56 -> X突变（X =任何氨基酸）]外，所有变体的成熟速度都快于WT。在19个变体中，Pro-G56W和Pro-G56E分别具有最低和最高的成熟率，以及WT和Pro-G56S的过量生产，纯化和表征。SDS-PAGE分析和Tk-subtilisin活性测定表明，它们的成熟率依次为WT = g56w -前肽> g56s -前肽> g56e -前肽，表明它们与Pro7-Tk-subtilisin及其衍生物的成熟率呈负相关。与S324A-subtilisin复合物测定的这些前肽的晶体结构表明，突变改变了前肽的构象，例如56位的非甘氨酸残基呈现右手构象，并且核心区域的疏水相互作用减少。这些结果表明，Gly56是稳定前肽折叠所必需的。这一褶皱的稳定导致tk -前肽与Tk-subtilisin的强结合，tk -前肽对蛋白水解降解具有高抗性，并且Pro-Tk-subtilisin成熟缓慢。(c) 2007 Elsevier Ltd.版权所有。
Tk-subtilisin [the mature domain of Pro-Tk-subtilisin in active form (Gly70-Gly398)] from the hyperthermophilic archaeon Thermococcus kodakaraensis is matured from Pro-Tk-subtilisin [a subtilisin homologue from T. kodakaraensis in pro form (Gly1-Gly398)] upon autoprocessing and degradation of propeptide. Pro-Tk-subtilisin is characterized by extremely slow maturation at mild temperatures, but this maturation rate is greatly increased by a single Gly56 -> Ser mutation in the propeptide region. To analyze the role of Gly56, which assumes a left-handed conformation, Pro-Tk-subtilisin variants with complete amino acid substitutions at Gly56 were constructed. A comparison of their halo-forming activities suggests that all variants, except for Pro-G56W [Pro-G56X, Pro-Tk-subtilisin with Gly56 -> X mutation (X = any amino acid)], mature faster than WT. Pro-G56W and Pro-G56E with the lowest and highest maturation rates, respectively, among 19 variants, as well as WT and Pro-G56S, were overproduced, purified, and characterized. SDS-PAGE analyses and Tk-subtilisin activity assay indicated that their maturation rates increased in the order WT = G56W-propeptide >G56S-propeptide>G56E-propeptide, indicating that they are inversely correlated with the maturation rates of Pro7-Tk-subtilisin and its derivatives. The crystal structures of these propeptides determined in complex with S324A-subtilisin indicate that the conformation of the propeptide is altered by the mutation, such that non-glycine residues at position 56 assume a right-handed conformation and hydrophobic interactions at the core region decrease. These results indicate that Gly56 is required in stabilizing the propeptide fold. Stabilization of this fold leads to strong binding of Tk-propeptide to Tk-subtilisin, high resistance of Tk-propeptide to proteolytic degradation, and slow maturation of Pro-Tk-subtilisin. (c) 2007 Elsevier Ltd. All rights reserved.