期刊
BIOCHEMICAL JOURNAL
卷 435, 期 -, 页码 55-63出版社
PORTLAND PRESS LTD
DOI: 10.1042/BJ20101593
关键词
avidin; biotin; protein engineering; protein-ligand interaction; streptavidin; traptavidin
资金
- Welcome Trust [085457/Z/OB/Z]
- Biotechnology and Biological Sciences Research Council
- Oxford University
- Worcester College Oxford
The interaction between SA (streptavidin) and biotin is one of the strongest non-covalent interactions in Nature. SA is a widely used tool and a paradigm for protein ligand interactions. We previously developed a SA mutant, termed Tr (traptavidin), possessing a 10-fold lower off-rate for biotin, with increased mechanical and thermal stability. In the present study, we determined the crystal structures of apo-Tr and biotin Tr at 1.5 angstrom resolution. In apo-SA the loop (L3/4), near biotin's valeryl tail, is typically disordered and open, but closes upon biotin binding. In contrast, L3/4 was shut in both apo-Tr and biotin Tr. The reduced flexibility of L3/4 and decreased conformational change on biotin binding provide an explanation for Tr's reduced biotin off- and on-rates. L3/4 includes Ser(45), which forms a hydrogen bond to biotin consistently in Tr, but erratically in SA. Reduced breakage of the biotin-Ser(45) hydrogen bond in Tr is likely to inhibit the initiating event in biotin's dissociation pathway. We generated a Tr with a single biotin-binding site rather than four, which showed a similarly low off-rate, demonstrating that Tr's low off-rate was governed by intrasubunit effects. Understanding the structural features of this tenacious interaction may assist the design of even stronger affinity tags and inhibitors.
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