The Role of Changing Loop Conformations in Streptavidin Versions Engineered for High-affinity Binding of the Strep-tag II Peptide

• Published in: Journal of molecular biology 2021-04, Vol.433 (9), p.166893, Article 166893
• Main Authors: Schmidt, Thomas G.M., Eichinger, Andreas, Schneider, Markus, Bonet, Lidia, Carl, Uwe, Karthaus, Dennis, Theobald, Ina, Skerra, Arne
• Format: Article
• Language: English
• Subjects: affinity system; avidity effect; peptide ligand; protein-peptide complex; streptavidin
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2021.166893

[Display omitted] •Engineering of streptavidin for improved/tighter binding of the Strep-tag II peptide.•Discovery of several unexpected structural motifs in two of the binding site loops.•Elucidation of four representative crystal structures in complex with the Strep-tag II.•Structural analysis of the complex between Strep-Tactin XT and the Twin-Strep-tag.•The resulting avidity effect allows ultra-strong, yet reversible complexation of proteins. 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.