High resolution structure of streptavidin in complex with a novel high affinity peptide tag mimicking the biotin binding motif.
A novel peptide was designed which possesses nanomolar affinity of less than 20 nM for streptavidin. Therefore it was termed Nano-tag and has been used as an affinity tag for recombinant proteins. The minimized version of the wild type Nano-tag is a seven-amino acid peptide with the sequence fMDVEAWL. The three-dimensional structure of wild type streptavidin in complex with the minimized Nano-tag was analyzed at atomic resolution of 1.15 A and the details of the binding motif were investigated. The peptide recognizes the same pocket of streptavidin where the natural ligand biotin is bound, but the peptide requires significantly more space than biotin. Therefore the binding loop adopts an "open" conformation in order to release additional space for the peptide. The conformation of the bound Nano-tag corresponds to a 3(10) helix. However, the analysis of the intermolecular interactions of the Nano-tag with residues of the binding pocket of streptavidin reveals astonishing similarities to the biotin binding motif. In principle the three-dimensional conformation of the Nano-tag mimics the binding mode of biotin. Our results explain why the use of the Nano-tag in fusion with recombinant proteins is restricted to their N-terminus and we describe the special significance of the fMet residue for the high affinity binding mode.
Perbandt
M
M
Department of Biochemistry and Molecular Biology, University of Hamburg, 20146 Hamburg, Germany.
Bruns
O
O
Vallazza
M
M
Lamla
T
T
Betzel
Ch
Ch
Erdmann
V A
VA
D020816:Amino Acid Motifs; D001665:Binding Sites; D001710:Biotin / Q000737:chemistry* / Q000378:metabolism*; D018360:Crystallography, X-Ray; D008024:Ligands; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D019809:Streptavidin / Q000737:chemistry* / Q000378:metabolism*
D008024:Ligands; D010455:Peptides; D001710:Biotin; D019809:Streptavidin