Dimension, Shape, and Conformational Flexibility of a Two Domain Fungal Cellulase in Solution Probed by Small Angle X-ray Scattering*
Dimension, Shape, and Conformational Flexibility of a Two Domain Fungal Cellulase in Solution Probed by Small Angle X-ray Scattering*
复制标题
DOI：
10.1074/jbc.m205404200
复制
发表时间：
2002-10
期刊：
影响因子：
--
通讯作者：
V. Receveur;M. Czjzek;M. Schülein;P. Panine;B. Henrissat
中科院分区：
文献类型：
--
作者：
V. Receveur;M. Czjzek;M. Schülein;P. Panine;B. Henrissat
来自Humicola insolens的纤维素酶Cel45具有一个由催化模块和纤维素结合模块(CBM)组成的模块化结构，该模块由一个36个氨基酸的糖基化连接肽隔开。通过小角X射线散射结合已知的模块三维结构，研究了整个两个结构域Cel45蛋白的溶液构象，以及连接子的长度和柔性对组成模块空间排列的影响。酶的测量尺寸表明，连接体表现出延伸的构象，导致每个模块的两个质量中心之间的最大延伸，对应于纤维素链上的大约四个纤维二糖单元。连接子的糖基化是决定其延伸构象的关键因素，连接子上的五个脯氨酸拉伸突变被发现赋予了酶更高的刚性。我们的研究表明，催化模块和煤层气在不溶性底物上的定位很可能受到连接体结构和灵活性的影响。我们的结果与纤维素酶可以在纤维素酶表面以毛毛虫一样的位移在自由能限制下移动的模型一致。
Cellulase Cel45 from Humicola insolens has a modular structure with a catalytic module and a cellulose-binding module (CBM) separated by a 36 amino acid, glycosylated, linker peptide. The solution conformation of the entire two domain Cel45 protein as well as the effect of the length and flexibility of the linker on the spatial arrangement of the constitutive modules were studied by small angle x-ray scattering combined with the known three-dimensional structure of the individual modules. The measured dimensions of the enzyme show that the linker exhibits an extended conformation leading to a maximum extension between the two centers of mass of each module corresponding to about four cellobiose units on a cellulose chain. The glycosylation of the linker is the key factor defining its extended conformation, and a five proline stretch mutation on the linker was found to confer a higher rigidity to the enzyme. Our study shows that the respective positioning of the catalytic module and the CBM onto the insoluble substrate is most likely influenced by the linker structure and flexibility. Our results are consistent with a model where cellulases can move on the surface of cellulose with a caterpillar-like displacement with free energy restrictions.