4BX5

cis-divalent streptavidin

4BX5 の概要

• エントリーDOI: 10.2210/pdb4bx5/pdb
• 関連するPDBエントリー: 4BX6 4BX7
• 分子名称: STREPTAVIDIN, 1,2-ETHANEDIOL, TETRAETHYLENE GLYCOL, ... (5 entities in total)
• 機能のキーワード: biotin-binding protein, biotin, avidin
• 由来する生物種: STREPTOMYCES AVIDINII; 詳細
• 細胞内の位置: Secreted: P22629 P22629
• タンパク質・核酸の鎖数: 4
• 化学式量合計: 55885.73

構造登録者

Fairhead, M.,Krndija, D.,Lowe, E.D.,Howarth, M. (登録日: 2013-07-08, 公開日: 2013-09-25, 最終更新日: 2023-12-20)

主引用文献

Fairhead, M.,Krndija, D.,Lowe, E.D.,Howarth, M.
Plug-and-Play Pairing Via Defined Divalent Streptavidins.
J.Mol.Biol., 426:199-, 2014

PubMed Abstract: Streptavidin is one of the most important hubs for molecular biology, either multimerizing biomolecules, bridging one molecule to another, or anchoring to a biotinylated surface/nanoparticle. Streptavidin has the advantage of rapid ultra-stable binding to biotin. However, the ability of streptavidin to bind four biotinylated molecules in a heterogeneous manner is often limiting. Here, we present an efficient approach to isolate streptavidin tetramers with two biotin-binding sites in a precise arrangement, cis or trans. We genetically modified specific subunits with negatively charged tags, refolded a mixture of monomers, and used ion-exchange chromatography to resolve tetramers according to the number and orientation of tags. We solved the crystal structures of cis-divalent streptavidin to 1.4Å resolution and trans-divalent streptavidin to 1.6Å resolution, validating the isolation strategy and explaining the behavior of the Dead streptavidin variant. cis- and trans-divalent streptavidins retained tetravalent streptavidin's high thermostability and low off-rate. These defined divalent streptavidins enabled us to uncover how streptavidin binding depends on the nature of the biotin ligand. Biotinylated DNA showed strong negative cooperativity of binding to cis-divalent but not trans-divalent streptavidin. A small biotinylated protein bound readily to cis and trans binding sites. We also solved the structure of trans-divalent streptavidin bound to biotin-4-fluorescein, showing how one ligand obstructs binding to an adjacent biotin-binding site. Using a hexaglutamate tag proved a more powerful way to isolate monovalent streptavidin, for ultra-stable labeling without undesired clustering. These forms of streptavidin allow this key hub to be used with a new level of precision, for homogeneous molecular assembly.

PubMed: 24056174
DOI: 10.1016/J.JMB.2013.09.016

実験手法

X-RAY DIFFRACTION (1.431 Å)