Lytle B L, Volkman B F, Westler W M, Wu J H
Department of Chemical Engineering, University of Rochester, Rochester, New York 14627-0166, USA.
Arch Biochem Biophys. 2000 Jul 15;379(2):237-44. doi: 10.1006/abbi.2000.1882.
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 Ca(2+)-binding on the DS structure was first investigated by using 2D (15)N-(1)H HSQC NMR spectroscopy. Changes in the spectra during Ca(2+) titration revealed that Ca(2+) induces folding of DS into its tertiary structure. This Ca(2+)-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 Ca(2+)-binding subdomain deviates from that of the canonical EF-hand motif.
纤维小体是一种大型的细胞外纤维素酶复合物，其组装依赖于众多酶亚基与同源受体或粘着斑结构域的对接，这些受体沿着非催化性支架蛋白串联排列。酶亚基所携带的一个高度保守的结构域——dockerin（DS）介导了与粘着斑结构域的对接。DS由两个22个氨基酸的重复序列组成，每个序列与EF手型钙结合环具有同源性。为了将DS结构与EF手型螺旋-环-螺旋基序的结构进行比较，我们使用多维异核核磁共振波谱分析了嗜热栖热菌纤维小体的纤维二糖水解酶CelS亚基的DS的溶液二级结构。首先通过二维（15）N-（1）H HSQC核磁共振波谱研究了Ca（2+）结合对DS结构的影响。Ca（2+）滴定过程中光谱的变化表明，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手型的蛋白质区分开来。对69个残基中的63个进行了连续的主链归属。对NOE连接性和H（α）化学位移的分析表明，dockerin的每一半仅包含一个α-螺旋，这与EF手型基序的F-螺旋相当。因此，DS的Ca（2+）结合亚结构域的结构不同于典型的EF手型基序的结构。
