PDBsum entry 6qw4

Peptide binding protein

PDB id: 6qw4

Contents

Protein chain

121 a.a.

Ligands

SER-HIS-PRO-GLN-PHE-GLU-LYS-NH2

Waters ×57

PDB id:

6qw4

Name:

Peptide binding protein

Title:

Engineered streptavidin variant (acgr) in complex with the strep-tag ii peptide

Structure:

Streptavidin. Chain: a. Engineered: yes. Strep-tag ii peptide. Chain: p. Engineered: yes. Other_details: the peptide sawshpqfek carries an anthraniloyl/2- aminobenzoyl (be2) group at the n-terminus and an amide group at the c-terminus.

Source:

Streptomyces avidinii. Organism_taxid: 1895. Expressed in: escherichia coli. Expression_system_taxid: 562. Synthetic: yes. Synthetic construct. Organism_taxid: 32630

Resolution:

2.10Å R-factor: 0.202 R-free: 0.240

Authors:

A.Skerra,A.Eichinger

Key ref:

T.G.M.Schmidt et al. (2021). 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. PubMed id: 33639211 DOI: 10.1016/j.jmb.2021.166893

Date:

05-Mar-19 Release date: 25-Mar-20

Protein chain

P22629  (SAV_STRAV) -  Streptavidin from Streptomyces avidinii

Seq: 183 a.a.

Struc: 121 a.a.*

* PDB and UniProt seqs differ at 5 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.?

DOI no: 10.1016/j.jmb.2021.166893

J Mol Biol 433:166893-166893 (2021)

PubMed id: 33639211

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

T.G.M.Schmidt, A.Eichinger, M.Schneider, L.Bonet, U.Carl, D.Karthaus, I.Theobald, A.Skerra.

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.