Identification of the Interactions Critical for Propeptide-Catalyzed Folding of Tk-Subtilisin
Identification of the Interactions Critical for Propeptide-Catalyzed Folding of Tk-Subtilisin
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DOI：
10.1016/j.jmb.2009.09.028
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发表时间：
2009-11-27
影响因子：
5.6
通讯作者：
Kanaya, Shigenori
中科院分区：
文献类型：
--
作者：
Tanaka, Shun-ichi;Matsumura, Hiroyoshi;Kanaya, Shigenori
TK-枯草杆菌蛋白酶需要Ca 2+进行折叠。这种折叠通过其前肽（Tkpro）的伴侣功能加速。构建了几种Tkpro和Tk-枯草杆菌蛋白酶衍生物，以检测Tkpro和Tk-枯草杆菌蛋白酶的C-末端延伸区与Glu 61/Asp 63-和Glu 201-介导的氢之间的相互作用。结构域界面的键对于Tkpro的伴侣功能是重要的。具有一系列C-末端截短和Glu 61和Asp 63双突变的Tkpro衍生物对SA-枯草杆菌蛋白酶（Tk-subtilisin的活性位点突变体）的伴侣功能比Tkpro弱。良好的相关性，观察到他们的伴侣功能和折叠SA-枯草杆菌蛋白酶蛋白的结合能力之间。这些结果表明，C-末端延伸区，Glu 61，和Asp 63的Tkpro的Tk-枯草杆菌蛋白酶的折叠不是关键的，但加速它通过结合到一个折叠中间的Tk-枯草杆菌蛋白酶与天然样结构在其结合位点。相比之下，Tkpro对E201 A/SA-枯草杆菌蛋白酶几乎没有伴侣功能。它可以与折叠的E201 A/SA-枯草杆菌蛋白酶蛋白结合，结合常数低于SA-枯草杆菌蛋白酶。这些结果表明Tkpro的环，其通过氢键与Tk-枯草杆菌蛋白酶的Glu 201相互作用，并且通过结合到具有规范结构的Tk-枯草杆菌蛋白酶的折叠中间体而为Tk-枯草杆菌蛋白酶的折叠所需。因为该环是相当疏水的，并且紧密地包裹在TK-枯草杆菌蛋白酶的中心α β α亚结构的表面平行螺旋上，所以该环与Glu 201的结合可以诱导这两个螺旋的缔合，从而形成α β α亚结构。我们建议，Glu 201介导的相互作用是至关重要的Tkpro催化折叠的TK-枯草杆菌蛋白酶的启动。(C)2009爱思唯尔有限公司保留所有权利。
Tk-subtilisin requires Ca2+, for folding. This folding is accelerated by the chaperone function of its propeptide (Tkpro). Several Tkpro and Tk-subtilisin derivatives were constructed to examine whether the interactions between the C-terminal extended region of Tkpro and Tk-subtilisin and Glu61/Asp63- and Glu201-mediated hydrogen. bonds at the domain interface are important for the chaperone function of Tkpro. The Tkpro derivatives with a series of C-terminal truncations and double mutations at Glu61 and Asp63 exhibited weaker chaperone functions than Tkpro for SA-subtilisin (active-site mutant of Tk-subtilisin). Good correlation was observed between their chaperone functions and binding abilities to the folded SA-subtilisin protein. These results suggest that the C-terminal extended region, Glu61, and Asp63 of Tkpro are not critical for folding of Tk-subtilisin but accelerate it by binding to a folding intermediate of Tk-subtilisin with a native-like structure at their binding sites. In contrast, Tkpro exhibited little chaperone function for E201A/SA-subtilisin. It could bind to the folded E201A/SA-subtilisin protein with a lower association constant than that for SA-subtilisin. These results suggest a loop of Tkpro, which interacts with Glu201 of Tk-subtilisin through hydrogen bonds and is required for folding of Tk-subtilisin by binding to a folding intermediate of Tk-subtilisin with a normative structure. Because this loop is fairly hydrophobic and tightly packs to the surface parallel helices of the central alpha beta alpha substructure of Tk-subtilisin, binding of this loop to Glu201 may induce association of these two helices and thereby formation of the alpha beta alpha substructure. We propose that Glu201-mediated interactions are critical for initiation of Tkpro-catalyzed folding of Tk-subtilisin. (C) 2009 Elsevier Ltd. All rights reserved.