NMR Solution Structure of SlyD from Escherichia coli: Spatial Separation of Prolyl Isomerase and Chaperone Function
NMR Solution Structure of SlyD from Escherichia coli: Spatial Separation of Prolyl Isomerase and Chaperone Function
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DOI：
10.1016/j.jmb.2009.01.034
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发表时间：
2009-03-27
影响因子：
5.6
通讯作者：
Balbach, Jochen
中科院分区：
文献类型：
--
作者：
Weininger, Ulrich;Haupt, Caroline;Balbach, Jochen
SlyD（对裂解D敏感）是来自细菌胞质溶胶的推定折叠辅助物，并且具有脯氨酰异构酶和伴侣活性。我们确定了来自大肠杆菌（SlyD*）的SlyD（1-165）截短版本的溶液NMR结构，该版本缺乏推测的非结构化C-末端尾部。SlyD* 由两个分离良好的结构域组成：具有脯氨酰异构酶活性的FKBP结构域和具有分子伴侣活性的插入-翻折（IF）结构域。IF结构域插入脯氨酰异构酶活性位点附近的FKBP结构域环中。SlyD* 的NMR结构显示两个结构域相对于彼此没有明显的取向。在FKBP结构域中，Tyr 68指向活性位点，这可能解释了与原型人FKBP 12（具有12 kDa的人FK 506结合蛋白）相比，该蛋白的固有脯氨酰异构酶活性降低以及FK 506结合亲和力低得多。通过荧光共振能量转移定量SlyD* 结合底物的热力学和动力学。NMR滴定实验表明，IF结构域识别并结合未折叠或部分折叠的蛋白质和肽。胰岛素聚集被SlyD* 显著减慢，如通过二维NMR光谱在真实的时间中所证明的，这可能是由于SlyD* 与变性胰岛素结合。IF结构域建立初始碰撞-碰撞复合物的能力，以及两个相互作用结构域的灵活取向，使得SlyD* 成为蛋白质折叠的非常强大的催化剂。(C)2009爱思唯尔有限公司保留所有权利。
SlyD (sensitive to lysis D) is a putative folding helper from the bacterial cytosol and harbors prolyl isomerase and chaperone activities. We determined the solution NMR structure of a truncated version of SlyD (1-165) from Escherichia coli (SlyD*) that lacks the presumably unstructured C-terminal tail. SlyD* consists of two well-separated domains: the FKBP domain, which harbors the prolyl isomerase activity, and the insert-inflap (IF) domain, which harbors the chaperone activity. The IF domain is inserted into a loop of the FKBP domain near the prolyl isomerase active site. The NMR structure of SlyD* showed no distinct orientation of the two domains relative to each other. In the FKBP domain, Tyr68 points into the active site, which might explain the lowered intrinsic prolyl isomerase activity and the much lower FK506 binding affinity of the protein compared with archetype human FKBP12 (human FK506 binding protein with 12 kDa). The thermodynamics and kinetics of substrate binding by SlyD* were quantified by fluorescence resonance energy transfer. NMR titration experiments revealed that the IF domain recognizes and binds unfolded or partially folded proteins and peptides. Insulin aggregation is markedly slowed by SlyD* as evidenced by two-dimensional NMR spectroscopy in real time, probably due to SlyD* binding to denatured insulin. The capacity of the IF domain to establish an initial encounter-collision complex, together with the flexible orientation of the two interacting domains, makes SlyD* a very powerful catalyst of protein folding. (C) 2009 Elsevier Ltd. All rights reserved.