Despite a large body of work characterizing the interaction between streptavidin and biotin, the structural basis of high affinity biotin binding remains poorly understood. Studies have shown that W120 plays a critical role in biotin binding, since mutating the residue reduces the biotin affinity by many orders of magnitude. Yet, the mutation disrupts the native tetramer conformation and therefore, the observed change in affinity represents combined effects from multiple sources, including changes in residue contacts as well as intersubunit contacts. To evaluate the energetic contribution from direct W120 contacts toward biotin binding, we constructed and analyzed a domain swapped dimer formed across the tetramer interface, in which the W120 contacts and tetramerization are decoupled. The dimer is stabilized through hydrophobic and hydrophilic contacts and can be detected using disulfide crosslinking both on the yeast surface and in vitro. Although the binding pockets of the domain swapped dimer are similar to those of wt, the biotin affinity of the mutant is only marginally higher than that of a monomer and significantly lower than wt tetramer. Therefore, the W120 contacts alone do not significantly increase the binding affinity and other factors besides W120 are important to ensure high affinity biotin interaction.
This preliminary program is subject to change.
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Construction and Analysis of Novel Domain Swapped Streptavidin Dimer
Despite a large body of work characterizing the interaction between streptavidin and biotin, the structural basis of high affinity biotin binding remains poorly understood. Studies have shown that W120 plays a critical role in biotin binding, since mutating the residue reduces the biotin affinity by many orders of magnitude. Yet, the mutation disrupts the native tetramer conformation and therefore, the observed change in affinity represents combined effects from multiple sources, including changes in residue contacts as well as intersubunit contacts. To evaluate the energetic contribution from direct W120 contacts toward biotin binding, we constructed and analyzed a domain swapped dimer formed across the tetramer interface, in which the W120 contacts and tetramerization are decoupled. The dimer is stabilized through hydrophobic and hydrophilic contacts and can be detected using disulfide crosslinking both on the yeast surface and in vitro. Although the binding pockets of the domain swapped dimer are similar to those of wt, the biotin affinity of the mutant is only marginally higher than that of a monomer and significantly lower than wt tetramer. Therefore, the W120 contacts alone do not significantly increase the binding affinity and other factors besides W120 are important to ensure high affinity biotin interaction.