title: The structure of the SBP-Tag-streptavidin complex reveals a novel helical scaffold bridging binding pockets on separate subunits.
abstract: The 38-residue SBP-Tag binds to streptavidin more tightly (K(d) -/= 2.5-4.9 nM) than most if not all other known peptide sequences. Crystallographic analysis at 1.75 Å resolution shows that the SBP-Tag binds to streptavidin in an unprecedented manner by simultaneously interacting with biotin-binding pockets from two separate subunits. An N-terminal HVV peptide sequence (residues 12-14) and a C-terminal HPQ sequence (residues 31-33) form the bulk of the direct interactions between the SBP-Tag and the two biotin-binding pockets. Surprisingly, most of the peptide spanning these two sites (residues 17-28) adopts a regular α-helical structure that projects three leucine side chains into a groove formed at the interface between two streptavidin protomers. The crystal structure shows that residues 1-10 and 35-38 of the original SBP-Tag identified through in vitro selection and deletion analysis do not appear to contact streptavidin and thus may not be important for binding. A 25-residue peptide comprising residues 11-34 (SBP-Tag2) was synthesized and shown using surface plasmon resonance to bind streptavidin with very similar affinity and kinetics when compared with the SBP-Tag. The SBP-Tag2 was also added to the C-terminus of β-lactamase and was shown to be just as effective as the full-length SBP-Tag in affinity purification. These results validate the molecular structure of the SBP-Tag-streptavidin complex and establish a minimal bivalent streptavidin-binding tag from which further rational design and optimization can proceed.
authors: Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, Alberta T2N 1N4, Canada.
authors: Isabelle H
authors: IH
authors: Barrette-Ng
authors: Sau Ching
authors: SC
authors: Wu
authors: Wai Mui
authors: WM
authors: Tjia
authors: Sui Lam
authors: SL
authors: Wong
authors: Kenneth K S
authors: KK
authors: Ng
mesh_terms: D000345:Affinity Labels / Q000737:chemistry; D000595:Amino Acid Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D002846:Chromatography, Affinity; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D019809:Streptavidin / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D000345:Affinity Labels; D002352:Carrier Proteins; D011993:Recombinant Fusion Proteins; C452700:streptavidin-binding peptide; D019809:Streptavidin
keywords: molecular recognition; protein engineering; protein–protein recognition
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