IBA GmbH, Rudolf-Wissell-Str. 28, 37079 Göttingen, Germany.
Lehrstuhl für Biologische Chemie, Technische Universität München, 85354 Freising, Germany.
J Mol Biol. 2021 Apr 30;433(9):166893. doi: 10.1016/j.jmb.2021.166893. Epub 2021 Feb 24.
The affinity system based on the artificial peptide ligand Strep-tag® II and engineered tetrameric streptavidin, known as Strep-Tactin®, offers attractive applications for the study of recombinant proteins, from detection and purification to functional immobilization. To further improve binding of the Strep-tag II to streptavidin we have subjected two protruding loops that shape its ligand pocket for the peptide - instead of D-biotin recognized by the natural protein - to iterative random mutagenesis. Sequence analyses of hits from functional screening assays revealed several unexpected structural motifs, such as a disulfide bridge at the base of one loop, replacement of the crucial residue Trp120 by Gly and a two-residue deletion in the second loop. The mutant m1-9 (dubbed Strep-Tactin XT) showed strongly enhanced affinity towards the Strep-tag II, which was further boosted in case of the bivalent Twin-Strep-tag®. Four representative streptavidin mutants were crystallized in complex with the Strep-tag II peptide and their X-ray structures were solved at high resolutions. In addition, the crystal structure of the complex between Strep-Tactin XT and the Twin-Strep-tag was elucidated, indicating a bivalent mode of binding and explaining the experimentally observed avidity effect. Our study illustrates the structural plasticity of streptavidin as a scaffold for ligand binding and reveals interaction modes that would have been difficult to predict. As result, Strep-Tactin XT offers a convenient reagent for the kinetically stable immobilization of recombinant proteins fused with the Twin-Strep-tag. The possibility of reversibly dissociating such complexes simply with D-biotin as a competing ligand enables functional studies in protein science as well as cell biology.
基于人工肽配体 Strep-tag® II 和工程化四聚体链霉亲和素（称为 Strep-Tactin®）的亲和系统，为研究重组蛋白提供了有吸引力的应用，从检测和纯化到功能固定化。为了进一步提高 Strep-tag II 与链霉亲和素的结合，我们对形成其配体口袋的两个突出环进行了迭代随机诱变，这些环用于肽 - 而不是天然蛋白识别的 D-生物素。功能筛选分析的命中序列分析揭示了几个意想不到的结构基序，例如一个环底部的二硫键，关键残基 Trp120 被 Gly 取代以及第二个环中的两个残基缺失。突变体 m1-9（称为 Strep-Tactin XT）显示出对 Strep-tag II 的强烈增强亲和力，而在双价 Twin-Strep-tag®的情况下亲和力进一步增强。四个代表性的链霉亲和素突变体与 Strep-tag II 肽复合物进行了结晶，并以高分辨率解决了它们的 X 射线结构。此外，还阐明了 Strep-Tactin XT 与 Twin-Strep-tag 复合物的晶体结构，表明了二价结合模式，并解释了实验观察到的亲和力效应。我们的研究说明了链霉亲和素作为配体结合支架的结构可塑性，并揭示了难以预测的相互作用模式。结果，Strep-Tactin XT 为融合了 Twin-Strep-tag 的重组蛋白的动力学稳定固定化提供了方便的试剂。简单地用 D-生物素作为竞争配体可逆解离这种复合物的可能性，使得在蛋白质科学和细胞生物学中进行功能研究成为可能。
