4BX6

trans-divalent streptavidin

Summary for 4BX6

• Entry DOI: 10.2210/pdb4bx6/pdb
• Related: 4BX5 4BX7
• Descriptor: STREPTAVIDIN, (4S)-2-METHYL-2,4-PENTANEDIOL, ... (4 entities in total)
• Functional Keywords: biotin-binding protein, avidin
• Biological source: STREPTOMYCES AVIDINII; More
• Total number of polymer chains: 4
• Total formula weight: 54967.20

Authors

Fairhead, M.,Krndija, D.,Lowe, E.D.,Howarth, M. (deposition date: 2013-07-08, release date: 2013-09-25, Last modification date: 2023-12-20)

Primary citation

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

Experimental method

X-RAY DIFFRACTION (1.595 Å)