6QW4

Engineered streptavidin variant (ACGR) in complex with the Strep-tag II peptide

6QW4 の概要

• エントリーDOI: 10.2210/pdb6qw4/pdb
• 関連するPDBエントリー: 1RST 6QBB 6QSY
• 分子名称: Streptavidin, Strep-tag II peptide (3 entities in total)
• 機能のキーワード: loop engineering, peptide binding protein, protein engineering, strep-tag, streptavidin
• 由来する生物種: Streptomyces avidinii; 詳細
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 14695.03

構造登録者

Skerra, A.,Eichinger, A. (登録日: 2019-03-05, 公開日: 2020-03-25, 最終更新日: 2024-01-24)

主引用文献

Schmidt, T.G.M.,Eichinger, A.,Schneider, M.,Bonet, L.,Carl, U.,Karthaus, D.,Theobald, I.,Skerra, A.
The Role of Changing Loop Conformations in Streptavidin Versions Engineered for High-affinity Binding of the Strep-tag II Peptide.
J.Mol.Biol., 433:166893-166893, 2021

PubMed Abstract: 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.

PubMed: 33639211
DOI: 10.1016/j.jmb.2021.166893

実験手法

X-RAY DIFFRACTION (2.1 Å)