Slutzki Michal, Reshef Dan, Barak Yoav, Haimovitz Rachel, Rotem-Bamberger Shahar, Lamed Raphael, Bayer Edward A, Schueler-Furman Ora
From the Department of Biological Chemistry, The Weizmann Institute of Science, 7610001 Rehovot, Israel.
Department of Microbiology and Molecular Genetics, Institute for Medical Research Israel-Canada, Hadassah Medical School, The Hebrew University, 9112102 Jerusalem, Israel, and.
J Biol Chem. 2015 May 29;290(22):13654-66. doi: 10.1074/jbc.M115.651208. Epub 2015 Apr 1.
Interactions between cohesin and dockerin modules play a crucial role in the assembly of multienzyme cellulosome complexes. Although intraspecies cohesin and dockerin modules bind in general with high affinity but indiscriminately, cross-species binding is rare. Here, we combined ELISA-based experiments with Rosetta-based computational design to evaluate the contribution of distinct residues at the Clostridium thermocellum cohesin-dockerin interface to binding affinity, specificity, and promiscuity. We found that single mutations can show distinct and significant effects on binding affinity and specificity. In particular, mutations at cohesin position Asn(37) show dramatic variability in their effect on dockerin binding affinity and specificity: the N37A mutant binds promiscuously both to cognate (C. thermocellum) as well as to non-cognate Clostridium cellulolyticum dockerin. N37L in turn switches binding specificity: compared with the wild-type C. thermocellum cohesin, this mutant shows significantly increased preference for C. cellulolyticum dockerin combined with strongly reduced binding to its cognate C. thermocellum dockerin. The observation that a single mutation can overcome the naturally observed specificity barrier provides insights into the evolutionary dynamics of this system that allows rapid modulation of binding specificity within a high affinity background.
黏连蛋白和锚定蛋白模块之间的相互作用在多酶纤维小体复合物的组装中起着关键作用。尽管种内黏连蛋白和锚定蛋白模块通常以高亲和力结合，但缺乏特异性，跨物种结合则很罕见。在这里，我们将基于酶联免疫吸附测定（ELISA）的实验与基于罗塞塔（Rosetta）的计算设计相结合，以评估嗜热栖热菌黏连蛋白-锚定蛋白界面上不同残基对结合亲和力、特异性和混杂性的贡献。我们发现，单个突变可对结合亲和力和特异性产生明显且显著的影响。特别是，黏连蛋白第37位天冬酰胺（Asn）处的突变对锚定蛋白结合亲和力和特异性的影响表现出极大的变异性：N37A突变体既能与同源（嗜热栖热菌）的锚定蛋白混杂结合，也能与非同源的解纤维梭菌锚定蛋白结合。而N37L则改变了结合特异性：与野生型嗜热栖热菌黏连蛋白相比，该突变体对解纤维梭菌锚定蛋白的偏好性显著增加，同时与其同源的嗜热栖热菌锚定蛋白的结合力大幅降低。单个突变能够克服自然存在的特异性障碍这一观察结果，为该系统的进化动力学提供了见解，该系统能够在高亲和力背景下快速调节结合特异性。
