CAS Key Laboratory of Biofuels, Shandong Provincial Key Laboratory of Synthetic Biology, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, China.
Shandong Energy Institute, Qingdao, China.
Protein Sci. 2024 Apr;33(4):e4937. doi: 10.1002/pro.4937.
Cellulosomes are intricate cellulose-degrading multi-enzymatic complexes produced by anaerobic bacteria, which are valuable for bioenergy development and biotechnology. Cellulosome assembly relies on the selective interaction between cohesin modules in structural scaffolding proteins (scaffoldins) and dockerin modules in enzymes. Although the number of tandem cohesins in the scaffoldins is believed to determine the complexity of the cellulosomes, tandem dockerins also exist, albeit very rare, in some cellulosomal components whose assembly and functional roles are currently unclear. In this study, we characterized the structure and mode of assembly of a tandem bimodular double-dockerin, which is connected to a putative S8 protease in the cellulosome-producing bacterium, Clostridium thermocellum. Crystal and NMR structures of the double-dockerin revealed two typical type I dockerin folds with significant interactions between them. Interaction analysis by isothermal titration calorimetry and NMR titration experiments revealed that the double-dockerin displays a preference for binding to the cell-wall anchoring scaffoldin ScaD through the first dockerin with a canonical dual-binding mode, while the second dockerin module was unable to bind to any of the tested cohesins. Surprisingly, the double-dockerin showed a much higher affinity to a cohesin from the CipC scaffoldin of Clostridium cellulolyticum than to the resident cohesins from C. thermocellum. These results contribute valuable insights into the structure and assembly of the double-dockerin module, and provide the basis for further functional studies on multiple-dockerin modules and cellulosomal proteases, thus highlighting the complexity and diversity of cellulosomal components.
纤维小体是由厌氧细菌产生的复杂纤维素降解多酶复合物，对于生物能源开发和生物技术具有重要价值。纤维小体的组装依赖于结构支架蛋白（支架蛋白）中的黏合模块和酶中的 dockerin 模块之间的选择性相互作用。尽管支架蛋白中的串联黏合模块数量被认为决定了纤维小体的复杂性，但在一些纤维小体组件中也存在串联 dockerin，尽管非常罕见，但其组装和功能作用目前尚不清楚。在这项研究中，我们对来自产纤维小体的细菌 Clostridium thermocellum 中的串联双模块双 dockerin 的结构和组装方式进行了表征。双 dockerin 的晶体和 NMR 结构揭示了两个具有显著相互作用的典型 I 型 dockerin 折叠。通过等温滴定量热法和 NMR 滴定实验的相互作用分析表明，双 dockerin 显示出通过第一个 dockerin 与典型的双重结合模式优先与细胞壁锚定支架蛋白 ScaD 结合的倾向，而第二个 dockerin 模块无法与任何测试的黏合模块结合。令人惊讶的是，与来自 Clostridium cellulolyticum 的 CipC 支架蛋白的黏合模块相比，双 dockerin 对来自 C. thermocellum 的常驻黏合模块表现出更高的亲和力。这些结果为双 dockerin 模块的结构和组装提供了有价值的见解，并为进一步研究多 dockerin 模块和纤维小体蛋白酶的功能提供了基础，从而突出了纤维小体组件的复杂性和多样性。
