Chapon V, Czjzek M, El Hassouni M, Py B, Juy M, Barras F
Laboratoire de Chimie Bactérienne , Institut de Biologie Structurale et Microbiologie CNRS-Marseille, France.
J Mol Biol. 2001 Jul 27;310(5):1055-66. doi: 10.1006/jmbi.2001.4787.
Erwinia chrysanthemi, a Gram-negative plant pathogen, secretes the cellulase Cel5 (formerly EGZ) via the type II secretion pathway (referred to as Out). Cel5 is composed of two domains, a large N-terminal catalytic domain (390 amino acid residues) and a small C-terminal cellulose-binding domain (62 amino acid residues) separated by a linker region. A combination of mutagenesis and structural analysis permitted us to investigate the structure/secretion relationships with respect to the catalytic domain of Cel5. The 3D structure of the catalytic domain was solved by molecular replacement at 2.3 A resolution. Cel5 exhibits the (beta/alpha)8 structural fold and two extra-barrel features. Our previous genetic study based upon tRNA-mediated suppression allowed us to predict positions of importance in the molecule in relation to structure and catalysis. Remarkably, all of the predictions proved to be correct when compared with the present structural information. Mutations of Arg57, which is located at the heart of the catalytic domain, allowed us to test the consequences of structural modifications on the secretion efficiency. The results revealed that secretability imposes remarkably strong constraints upon folding. In particular, an Arg-to-His mutation yielded a species that folded to a stable conformation close to, but distinct from the wild-type, which however was not secretable. We discuss the relationships between folding of a protein in the periplasm, en route to the cell exterior, and presentation of secretion information. We propose that different solutions have been selected for type II secreted exoproteins in order to meet the constraints imposed by their interaction with their respective secretion machineries. We propose that evolutionary pressure has led to the adaptation of different secretion motifs for different type II exoproteins.
菊欧文氏菌是一种革兰氏阴性植物病原体，它通过II型分泌途径（称为Out）分泌纤维素酶Cel5（以前称为EGZ）。Cel5由两个结构域组成，一个大的N端催化结构域（390个氨基酸残基）和一个小的C端纤维素结合结构域（62个氨基酸残基），中间由一个连接区隔开。通过诱变和结构分析相结合的方法，我们得以研究Cel5催化结构域的结构/分泌关系。催化结构域的三维结构通过分子置换法在2.3埃分辨率下解析出来。Cel5呈现出(β/α)8结构折叠和两个额外的桶状特征。我们之前基于tRNA介导的抑制作用进行的遗传学研究，使我们能够预测该分子中与结构和催化相关的重要位置。值得注意的是，与目前的结构信息相比，所有这些预测都被证明是正确的。位于催化结构域核心位置的Arg57的突变，使我们能够测试结构修饰对分泌效率的影响。结果表明，可分泌性对折叠施加了非常强的限制。特别是，Arg-to-His突变产生了一种折叠成接近但不同于野生型的稳定构象的物种，然而它是不可分泌的。我们讨论了周质中蛋白质折叠、通向细胞外的过程以及分泌信息呈现之间的关系。我们提出，为了满足与各自分泌机制相互作用所施加的限制，II型分泌外蛋白选择了不同的解决方案。我们提出，进化压力导致了不同的II型外蛋白适应不同的分泌基序。
