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.

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