Nagy Tibor, Tunnicliffe Richard B, Higgins Lee D, Walters Chris, Gilbert Harry J, Williamson Mike P
Institute for Cell and Molecular Biosciences, The University of Newcastle upon Tyne, The Medical School, Framlington Place, Newcastle upon Tyne NE2 4HH, UK.
J Mol Biol. 2007 Oct 26;373(3):612-22. doi: 10.1016/j.jmb.2007.08.007. Epub 2007 Aug 19.
The assembly into supramolecular complexes of proteins having complementary activities is central to cellular function. One such complex of considerable biological and industrial significance is the plant cell wall-degrading apparatus of anaerobic microorganisms, termed the cellulosome. A central feature of bacterial cellulosomes is a large non-catalytic protein, the scaffoldin, which contains multiple cohesin domains. An array of digestive enzymes is incorporated into the cellulosome through the interaction of the dockerin domains, present in the catalytic subunits, with the cohesin domains that are present in the scaffoldin. By contrast, in anaerobic fungi, such as Piromyces equi, the dockerins of cellulosomal enzymes are often present in tandem copies; however, the identity of the cognate cohesin domains in these organisms is unclear, hindering further biotechnological development of the fungal cellulosome. Here, we characterise the solution structure and function of a double-dockerin construct from the P. equi endoglucanase Cel45A. We show that the two domains are connected by a flexible linker that is short enough to keep the binding sites of the two domains on adjacent surfaces, and allows the double-dockerin construct to bind more tightly to cellulosomes than a single domain and with greater coverage. The double dockerin binds to the GH3 beta-glucosidase component of the fungal cellulosome, which is thereby identified as a potential scaffoldin.
具有互补活性的蛋白质组装成超分子复合物是细胞功能的核心。具有重要生物学和工业意义的一种这样的复合物是厌氧微生物的植物细胞壁降解装置，称为纤维小体。细菌纤维小体的一个主要特征是一种大型非催化蛋白，即支架蛋白，它含有多个粘着蛋白结构域。一系列消化酶通过催化亚基中存在的dockerin结构域与支架蛋白中存在的粘着蛋白结构域的相互作用而被整合到纤维小体中。相比之下，在厌氧真菌中，如马氏梨形孢，纤维小体酶的dockerin结构域通常以串联形式存在；然而，这些生物体中同源粘着蛋白结构域的身份尚不清楚，这阻碍了真菌纤维小体的进一步生物技术开发。在这里，我们表征了来自马氏梨形孢内切葡聚糖酶Cel45A的双dockerin构建体的溶液结构和功能。我们表明，这两个结构域由一个柔性接头连接，该接头足够短，以保持两个结构域的结合位点在相邻表面上，并使双dockerin构建体比单个结构域更紧密地结合到纤维小体上，且覆盖范围更大。双dockerin与真菌纤维小体的GH3β-葡萄糖苷酶成分结合，因此该成分被确定为潜在的支架蛋白。
