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 -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 -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-Tagstreptavidin complex and establish a minimal bivalent streptavidin-binding tag from which further rational design and optimization can proceed.

Structure-Guided Design of an Engineered Streptavidin with Reusability to Purify Streptavidin-Binding Peptide Tagged Proteins or Biotinylated Proteins

“…It binds biotin with high affinity (K d ∼10 −14 M) through extensive hydrogen bonding, hydrophobic interactions and closure of a mobile loop [21], [22]. The mechanism for the tight binding between SBP tag and streptavidin has also been examined recently by determining the structure of the SBP-streptavidin complex via X-ray crystallography [13]. Out of 38 residues in the full-length SBP tag, only 25 amino acids (residues 11–35 colored in Fig.…”

“…1a) form a well-ordered structure that can be visualized in the streptavidin-SBP tag complex. Further truncation of the last residue (residue 35 numbered according to the full-length SBP tag) of the 25-amino-acid SBP sequence leads to the generation of a shorter tag designated SBP-Tag2 which retains comparable binding strength as the full-length SBP tag [13]. This 24-amino-acid sequence can be divided into three functional segments.…”

“…Although the mutein showed a lower binding affinity, its binding strength (K d =  7.67×10 −9 M, Table 1) has the same order of magnitude as that of wild-type streptavidin (1.65×10 −9 M). To examine whether fusing the SBP tag to a protein affects its interaction with streptavidin, a β-lactamase-SBP fusion [13] was used in this study. This fusion has a 19 amino-acid linker as the spacer and SBP is at the C-terminal end.…”

“…The key to the successful development of SAVSBPM18 relies on the ability to create this desirable mutein with minimal changes in the streptavidin sequence. Structural analysis of the streptavidin-SBP complex [13] suggests that both the G48T and S27A mutations should not significantly affect SBP tag binding but should reduce the biotin binding affinity (Fig. 1).…”

“…Wild-type streptavidin (Sigma Canada) was also immobilized to Affi-15 gel in a similar manner. To demonstrate purification of ß-lactamase-SBP [13], Bacillus subtilis WB800[pWB980-BLA-FLSBP] (FLSBP represents the full-length 38-amino-acid streptavidin binding peptide tag) was cultured in a defined medium [12]. After washing the column with PBS, bound ß-lactamase-SBP was eluted using PBS containing 5 mM d-biotin.…”