Abstract
The strong interaction between streptavidin (SA) and biotin is widely utilized in biotechnological applications. A SA variant, monovalent SA, was developed with a single and high affinity biotin-binding site within the intact tetramer. However, its structural characterization remains undetermined. Here, we seek to determine the crystal structure of monovalent SA at 1.7-Å resolution. We show that, in contrast to its ‘close-state’ in the only wild-type subunit, the L3,4 loops of three Dead SA subunits are free from crystal packing and remain in an ‘open state’, stabilized by a consistent H-bonding network involving S52. This H-bonding network also applies to the previously reported open state of the wild-type apo-SA. These results suggest that specific substitutions (N23A/S27D/S45A) at biotin-binding sites stabilize the open state of SA L3,4 loop, thereby further reducing biotin-binding affinity. The general features of the ‘open state’ SA among different SA variants may facilitate its rational design. The structural information of monovalent SA will be valuable for its applications across a wide range of biotechnological areas.
Highlights
The strong interaction between streptavidin (SA) and biotin is widely utilized in biotechnological applications
This H-bonding network applies to the previously reported open state of the wildtype apo-SA. These results suggest that specific substitutions (N23A/S27D/S45A) at biotin-binding sites stabilize the open state of SA L3,4 loop, thereby further reducing biotin-binding affinity
By solving the structure of monovalent streptavidin, we found substantial conformational changes between the single wild-type subunit and three Dead subunits, localized to their L3,4 loops
Summary
The strong interaction between streptavidin (SA) and biotin is widely utilized in biotechnological applications. In contrast to its ‘close-state’ in the only wild-type subunit, the L3,4 loops of three Dead SA subunits are free from crystal packing and remain in an ‘open state’, stabilized by a consistent H-bonding network involving S52 This H-bonding network applies to the previously reported open state of the wildtype apo-SA. Howarth et al.[12] reported the production of monovalent streptavidin with N23A/S27D/S45A triple mutants in three Dead subunits This chimeric SA variant has a single biotin-binding site with high (femtomolar) affinity and is still in tetramer form. These results suggest that the substitutions at biotin-binding sites (N23A/S27D/ S45A) may stabilize the open conformation of the L3,4 loop, and further reduce biotin-binding These insights from monovalent SA structure promote our understanding of this widely used biotechnological system and can facilitate its rational modification and application
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