6QBB

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

Summary for 6QBB

• Entry DOI: 10.2210/pdb6qbb/pdb
• Related: 1RST
• Descriptor: Streptavidin, Strep-tag II peptide (3 entities in total)
• Functional Keywords: loop engineering, peptide binding protein, protein engineering, strep-tag, streptavidin
• Biological source: Streptomyces avidinii; More
• Total number of polymer chains: 4
• Total formula weight: 29841.11

Authors

Skerra, A.,Eichinger, A. (deposition date: 2018-12-20, release date: 2020-01-29, Last modification date: 2024-01-24)

Primary citation

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

Experimental method

X-RAY DIFFRACTION (1.52 Å)