Figure 2. Complementing HA structural diversity with designed HSB variants.

(a) Sequence and structural diversity around the receptor binding site of HA (4wea) presents a challenge for binding of molecules larger than sialic acid (PDB entries 4fqr, 3qqi, 4o5n and homology models, see Supp. Info). (b) Design of HSB library: residues of HSB (purple spheres) that come in contact with HA (4fp8) were allowed to mutate to match the diversity of nearby segments of HA. HA is colored by Shannon Entropy over a set of ∼3700 H3 sequences (highly conserved in blue, less in red, Suppl. Info). (c) (left) Sequence alignment of HA-contacting residues for isolates identified in independent sorting trajectories against different flu strains (HSB.1A and B were selected for H1 A/Solomon Islands/3/2006 (SI), HSB.1C for H1 A/New Caledonia/20/1999 (NewCal), HSB.1D and E alternately against H3 HK68/Vic11 and H2 A/Adachi/2/1957 (Ada)). (right) mean of the apparent K_d (nM) from yeast surface titrations of two independent yeast cultures measured on different days. (d) Model of HSB.1A (grey cartoon) with H3 HK68 (colored by electrostatic potential, see Fig. S3) indicates electrostatic repulsion between K79 and the positively charged RBS of H3 HK68; H1 SI is the only strain tested with a net negative charge within Loop 140 (Supplementary Fig. 5) explaining the high preference of HSB.1A for this strain (e) Substitution E79V of HSB.1E (grey cartoon) promotes binding to H2 strains (green) which commonly have a proline at position 145 (Fig.2a, panel IV). (f) Between loop 150 and helix 190 of HA (Fig. 2a, panel II), HSB.1B projects a positive charge (K47) which interacts with the negatively charged E156 of H2 A/Japan/305/1957 (H2 Jap). In contrast, HSB.1D has a negative charge within this loop (D45), which diminishes binding to H2 Jap.