Prodomain mutations at the subtilisin interface: correlation of binding energy and the rate of catalyzed folding.
Prodomain mutations at the subtilisin interface: correlation of binding energy and the rate of catalyzed folding.
复制标题
枯草杆菌蛋白酶界面的前结构域突变：结合能与催化折叠速率的相关性。
DOI：
10.1021/bi00047a004
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发表时间：
1995
期刊：
影响因子：
2.9
通讯作者：
Bryan,PN
中科院分区：
文献类型：
--
作者：
Wang,L;Ruvinov,S;Strausberg,S;Gallagher,DT;Gilliland,G;Bryan,PN
摘要：枯草菌素的体内折叠依赖于一个77个氨基酸的序列，该序列最终从枯草菌素的n端切割出来，形成275个氨基酸的成熟酶。最近对原结构体和无钙枯草菌素突变体复合物结构的测定表明，原结构体可能催化枯草菌素折叠[Bryan， P., Wang， L., Hoskins， J., Ruvinov， S., Strausberg， S., Alexander， P., Almog， O., Gilliland， G., & Gallagher， T.（1995）生物化学34,10310-10318]。在该复合体中，原结构域围绕枯草菌素的两个平行表面螺旋（残基104-116和残基133-144），并为这两个螺旋的n端提供帽状物。结合位点几乎全部包含在枯草杆菌素的线性序列100-144中。原域的c端（残基72-77）从其中心部分向外延伸，在枯草菌素的活性位点间隙中与底物结合。催化折叠最简单的模型是，观察到的复合物中的结合相互作用通过稳定含有枯草菌素中45个氨基酸亚结构的中间体来加速折叠。根据我们的假设，氨基酸100-144在中间会有一个原结构域稳定的天然折叠。根据枯草菌素及其原结构域的双分子复合物的结构，我们在原结构域的c端区域构建了突变。对五个突变体的分析揭示了原结构域与天然枯草菌素结合的能力与其加速枯草菌素折叠的能力之间的普遍相关性。SI底物结合袋（Y77）中原域侧链的取代对枯草菌素折叠和与折叠枯草菌素结合（3倍或更少）的催化作用相对较小。删除两个或五个c端氨基酸，会去除复合物中的氢键，产生更大的影响（25 - 500倍）。与天然枯草菌素的结合与折叠酶活性之间的相关性表明，原结构域在折叠反应中结合并稳定的枯草菌素中间体具有类似天然的结构特征。275个氨基酸的丝氨酸蛋白酶枯草菌素和其他几种细胞外微生物蛋白酶是不寻常的，但不是唯一的例子，蛋白质具有稳定的天然状态，难以从未折叠状态获得（Baker & Agard, 1994）。枯草菌素的生物合成依赖于一个77个氨基酸的序列，该序列最终从枯草菌素的n端切割出来，产生275个氨基酸的成熟酶（Ikemura等人，1987；Power等人，1986；Vasantha等人，1984；Wells等人，1983）。加工过的枯草菌素的再折叠是有问题的。在没有原结构域的情况下，枯草菌素的再折叠非常缓慢（在0.1 M KPi, pH 7.0条件下，大约需要100周）。即使在双分子反应中被分离的原域催化，枯草杆菌素的再折叠速率也只有0.2 M~！s-1 15℃（Eder等人，1993年）。为了简化枯草菌素折叠的研究，我们使用了去除高亲和力钙结合位点的枯草菌素突变体（Bryan et al., 1992）。2 .与野生型枯草菌素不同，“无钙”的再折叠
Revised Manuscript Received September 18, 1995® abstract: The in vivo folding of subtilisin is dependent on a 77 amino acid prosequence, which is eventually cleaved from the N-terminus of subtilisin to create the 275 amino acid mature form of the enzyme. The recent determination of the structure of a complex of the prodomain and a calcium-free subtilisin mutant has suggested how the prodomain may catalyze subtilisin folding [Bryan, P., Wang, L., Hoskins, J., Ruvinov, S., Strausberg, S., Alexander, P., Almog, O., Gilliland, G., & Gallagher, T.(1995) Biochemistry 34, 10310—10318]. In the complex, the prodomain packs against the two parallel surface helices of subtilisin (residues 104—116 and residues 133—144) and supplies caps to the N-termini of the two helices. The binding site is contained almost entirelyin the linear sequence 100—144 of subtilisin. The C-terminus of the prodomain (residues 72—77) extends out from its central part to bindlike a substrate in subtilisin’s activesite cleft. The simplest model of catalyzed folding is one in which the observed binding interaction in the complex accelerates folding by stabilizing an intermediate which includes the 45 amino acid afia substructure in subtilisin. According to our hypothesis, amino acids 100—144 would have a native-like fold in the intermediatewhich the prodomain stabilizes. Guided bythe structure of the bimolecular complex of subtilisin and its prodomain, we have constructed mutations in the C-terminal region of the prodomain. Analysis of five mutants reveals a general correlation between the ability of the prodomain to bind to native subtilisin and its ability to accelerate subtilisin folding. Substitutions of the prodomain side chain in the SI subtrate binding pocket (Y77) have relatively small effects on catalysis of subtilisin folding and binding to folded subtilisin (3-fold or less). Deletion of two or five C-terminal amino acids, which removes hydrogen bonds in the complex, has much larger effects (25—500-fold). The correlation between binding to native subtilisin and foldase activity suggests that the subtilisin intermediate which the prodomain binds and stabilizes in the folding reaction has native-like structural features.The 275 amino acid serine protease subtilisin and several other extracellular microbial proteases are unusual but not unique examples of proteins with a stable native state which is difficult to access from the unfolded state (Baker & Agard, 1994). The biosynthesis of subtilisin is dependent on a 77 amino acid prosequence, which is eventually cleaved from the N-terminus of subtilisin to create the 275 amino acid mature form of the enzyme (Ikemura et al., 1987; Power et al., 1986; Vasantha et al., 1984; Wells et al., 1983). Refolding of processed subtilisin is problematic. Without the prodomain, subtilisin refolds very slowly (r> weeks in 0.1 M KPi,'pH 7.0). Even when catalyzed by the isolated prodomain in a bimolecular reaction, refolding of subtilisin occurs at a rate of only 0.2 M~! s-1 at 15 C (Eder et al., 1993). In order to simplify the study of subtilisin folding, we have employedsubtilisin mutants from which the high-affinity calcium binding site is removed (Bryan et al., 1992). 2 Unlike wild-type subtilisin, refolding of “calcium-free”