Structure Guided Engineering of Monomeric Streptavidin With Improved Ligand Binding

We recently reported the engineering of monomeric streptavidin, mSA, corresponding to a single subunit of wild type (wt) streptavidin tetramer. The monomer was designed by homology modeling, in which the streptavidin and rhizavidin sequences were combined to engineer a high affinity binding pocket containing residues from a single subunit only. Although mSA is stable and binds biotin with nanomolar affinity, its fast off rate (koff) creates practical challenges during applications. We obtained a 1.9 Å crystal structure of mSA bound to biotin to understand their interaction in detail, and used the structure to introduce targeted mutations to improve its binding kinetics. To this end, we analyzed the structure of a streptavidin analog, shwanavidin, which binds biotin with high affinity as a dimer. The binding pocket of shwanavidin differs from that of mSA or rhizavidin in that it contains an aromatic residue that appears to form a lid over bound biotin. This led us to hypothesize that a comparable lid would likewise enhance the binding affinity of mSA. However, the T48F mutation in mSA only resulted in a modest 20 – 40% improvement in the measured koff. On the other hand, introducing the S25H mutation near the bicyclic ring of bound biotin increases the dissociation half life (t½) by an order of magnitude. Molecular dynamics (MD) simulations suggest that the mutation stabilizes the binding loop L3,4 by interacting with a residue at the base of the binding loop, and protects key intermolecular hydrogen bonds by limiting solvent entry into the binding pocket. The latest results from a directed evolution study will also be presented. The engineered binding kinetics of the mSA mutant significantly improves the usability of the streptavidin monomer in applications that require stable ligand binding.