The structure of the SBP-Tag-streptavidin complex reveals a novel helical scaffold bridging binding pockets on separate subunits
The 38‐residue SBP‐Tag binds to streptavidin more tightly (Kd ≃ 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 poc...
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Published in | Acta crystallographica. Section D, Biological crystallography. Vol. 69; no. 5; pp. 879 - 887 |
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Main Authors | , , , , |
Format | Journal Article |
Language | English |
Published |
5 Abbey Square, Chester, Cheshire CH1 2HU, England
International Union of Crystallography
01.05.2013
Wiley Subscription Services, Inc |
Subjects | |
Online Access | Get full text |
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Summary: | The 38‐residue SBP‐Tag binds to streptavidin more tightly (Kd ≃ 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. |
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Bibliography: | ark:/67375/WNG-ZPMBBN1S-5 ArticleID:AYDMV5083 istex:5C8C310442FC00A116C1934125E75D9B23A71F82 ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 ObjectType-Article-2 ObjectType-Feature-1 |
ISSN: | 1399-0047 0907-4449 1399-0047 |
DOI: | 10.1107/S0907444913002576 |