Improved affinity of engineered streptavidin for the Strep-tag II peptide is due to a fixed open conformation of the lid-like loop at the binding site.
The Strep-tag II is a nine-amino acid peptide that was developed as an affinity tool for the purification of corresponding fusion proteins on streptavidin columns. The peptide recognizes the same pocket of streptavidin where the natural ligand is normally bound so that biotin or its chemical derivatives can be used for competitive elution. We report here the crystal structures of the streptavidin mutants '1' and '2,' which had been engineered for 10-fold higher affinity towards the Strep-tag II. Both streptavidin mutants carry mutations at positions 44, 45, and 47, that is, in a flexible loop region close to the binding site. The crystal structures of the two apo-proteins and their complexes with the Strep-tag II peptide were refined at resolutions below 2 A. Both in the presence and absence of the peptide, the lid-like loop next to the ligand pocket--comprising residues 45 through 52--adopts an 'open' conformation in all four subunits within the asymmetric unit. The same loop was previously described to be disordered in the wild-type apo-streptavidin and to close over the pocket upon complexation of the natural ligand biotin. Our findings suggest that stabilization of the 'open' loop conformation in the absence of a ligand abolishes the need for conformational rearrangement prior to the docking of the voluminous peptide. Because no direct contacts between the flexible part of the loop and the peptide ligand were detected, it seems likely that the higher affinity of the two streptavidin mutants for the Strep-tag II is caused by a predominantly entropic mechanism.
Korndörfer
Ingo P
IP
Lehrstuhl für Biologische Chemie, Technische Universität München, Freising-Weihenstephan, Germany.
Skerra
Arne
A
D001665:Binding Sites; D001667:Binding, Competitive; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D008958:Models, Molecular; D016296:Mutagenesis / Q000235:genetics; D009842:Oligopeptides / Q000378:metabolism*; D011487:Protein Conformation; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification*; D019809:Streptavidin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D009842:Oligopeptides; D011994:Recombinant Proteins; D019809:Streptavidin