Secondary structure and calcium-induced folding of the Clostridium thermocellum dockerin domain determined by NMR spectroscopy
Secondary structure and calcium-induced folding of the Clostridium thermocellum dockerin domain determined by NMR spectroscopy
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DOI：
10.1006/abbi.2000.1882
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发表时间：
2000-07-15
影响因子：
3.9
通讯作者：
Wu, JHD
中科院分区：
文献类型：
--
作者：
Lytle, BL;Volkman, BF;Wu, JHD
多纤维素酶体是一种大的细胞外纤维素酶复合物，它的组装依赖于无数酶亚基与同源受体或粘附素结构域的对接，这些亚基沿着非催化支架蛋白串联排列。与粘附素结构域的对接由每个酶亚基携带的高度保守的结构域dockerin（DS）介导。DS由两个22个氨基酸的重复序列组成，每个序列与EF-手形钙结合环具有同源性。为了比较DS结构与EF-手螺旋-环-螺旋基序的DS结构，我们使用多维杂波NMR光谱分析了来自热纤梭菌纤维素酶体的纤维二糖水解酶CelS亚基的DS的溶液二级结构。通过使用2D N-15-H-1 HSQC MMR光谱首次研究了Ca 2+结合对DS结构的影响。在Ca 2+滴定过程中的光谱变化表明，Ca 2+诱导DS折叠成其三级结构。这种Ca 2+诱导的蛋白质折叠将DS与典型的含EF手的蛋白质区分开来。确定了69个残基中的63个的连续骨架分配。NOE连接性和H-α化学位移的分析表明，每一半的锚定蛋白只包含一个α-螺旋，与EF-手基序的F-螺旋相当。因此，DS Ca 2+结合亚结构域的结构偏离了经典EF-手基序的结构。(C)北京大学出版社.
Assembly of the cellulosome, a large, extracellular cellulase complex, depends upon docking of a myriad of enzymatic subunits to homologous receptors, or cohesin domains, arranged in tandem along a noncatalytic scaffolding protein. Docking to the cohesin domains is mediated by a highly conserved domain, dockerin (DS), borne by each enzymatic subunit. DS consists of two 22-amino-acid duplicated sequences, each bearing homology to the EF-hand calcium-binding loop. To compare the DS structure with that of the EF-hand helix-loop-helix motif, we analyzed the solution secondary structure of the DS from the cellobiohydrolase CelS subunit of the Clostridium thermocellum cellulosome using multidimensional heteronuclear NMR spectroscopy. The effect of Ca2+-binding on the DS structure was first investigated by using 2D N-15-H-1 HSQC MMR spectroscopy. Changes in the spectra during Ca2+ titration revealed that Ca2+ induces folding of DS into its tertiary structure. This Ca2+ induced protein folding distinguishes DS from typical EF-hand-containing proteins. Sequential backbone assignments were determined for 63 of 69 residues. Analysis of the NOE connectivities and H-alpha chemical shifts revealed that each half of the dockerin contains just one alpha-helix, comparable to the F-helix of the EF-hand motif. Thus, the structure of the DS Ca2+-binding sub-domain deviates from that of the canonical EF-hand motif. (C) 2000 Academic Press.