Acta Crystallographica Section D

Biological Crystallography

Volume 69, Part 5 (May 2013)


research papers



Acta Cryst. (2013). D69, 879-887    [ doi:10.1107/S0907444913002576 ]

The structure of the SBP-Tag-streptavidin complex reveals a novel helical scaffold bridging binding pockets on separate subunits

I. H. Barrette-Ng, S.-C. Wu, W.-M. Tjia, S.-L. Wong and K. K. S. Ng

Abstract: The 38-residue SBP-Tag binds to streptavidin more tightly (Kd [asymptotically equal to] 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 [alpha]-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 [beta]-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.

PDB reference: 4jo6

Keywords: protein-protein recognition; protein engineering; molecular recognition.


pdfdisplay filedownload file

Portable Document Format (PDF) file
[ doi:10.1107/S0907444913002576/mv5083sup1.pdf ]
Supplementary material


Notes:

To open or display or play some files, you may need to set your browser up to use the appropriate software. See the full list of file types for an explanation of the different file types and their related mime types and, where available links to sites from where the appropriate software may be obtained.

The download button will force most browsers to prompt for a file name to store the data on your hard disk.

Where possible, images are represented by thumbnails.

 bibliographic record in  format

  Find reference:   Volume   Page   
  Search:     From   to      Advanced search

Copyright © International Union of Crystallography
IUCr Webmaster