Computational Methods

For a scaffold database, we curated 1718 high-resolution, monomeric crystal structures from the PDB and relaxed them into the Rosetta energy function. Using Epigraft²², segments of varying lengths extracted from the heavy chain CDR3 of the C05 antibody (PDB ID 4FP8) ranging from Ser97 to Leu100K (or Ala100I) were aligned with each scaffold. If the rms distance between the joining backbone endpoints was less than 0.25 Å and diverged less than 1.0 Å when aligning the replaced scaffold fragment with the CDR fragment motif, grafting was attempted. All predicted interface residues of suitable scaffolds were substituted with alanine and, using small fragment insertion and backbone minimization, we attempted to close broken bonds. For the top 10 closed solutions, the remainder was briefly redesigned by Epigraft²². Both intra- and intermolecular interfaces were then extensively redesigned using 3 rounds of the most current RosettaDesign²⁸ while also allowing rigid-body minimization, but maintaining key contacts (100A, 100C-F) of the grafted piece. The best scoring design for each successful graft solution was kept if hydrogen bonds to the HA 130 loop and Tyr98 were maintained and had a computed binding energy below -12 units. All residues that were changed and did not contribute to the computed ΔΔG of binding were reverted to their native identities. Source code is freely available to academic users through the Rosetta Commons agreement (http://www.rosettacommons.org/).

To identify possible locations for the incorporation of two cysteine residues to form a disulfide bridge in HSB variants without disturbing the backbone, we utilized RosettaRemodel²⁹. Briefly, Cβ distances between all residue pairs were calculated and disulfide bridges were introduced and the model was relaxed and minimized when the appropriate distances were identified.

To prevent domain swapping and enable crystallization with HA, loop 2 of HSB was redesigned. Ideal phi-psi angles and loop truncations were sampled using blueprint files³⁰ and sequence design was biased towards amino acid identities commonly seen within beta-sheet turns. Out of 10 tested designs, a 3-residue truncated version, HSB.2A, expressed well and ran as a mono-disperse peak on SEC.

The query sequence was threaded onto the HA structure with the closest sequence similarity as identified by BLAST (ncbi.nlm.nih.gov). The model was then subjected to several rounds of relaxation while constraining the backbone atoms to their original coordinates. We used following PDBs as templates for homology models: chain A of 3sm5 for H1 SI and H1 NewCal, and chain A of 4hfu for H2 Ada.

Non-redundant human influenza A H3 sequences for HA were downloaded from the Influenza Research Database (fludb.org) and only sequences from complete genomes were extracted and aligned using Muscle³¹. Shannon Entropy was calculated using method calls from the Python-based ProDy package³².

Translations and rotations of 300 homo-trimeric structures were sampled along the symmetry axis of the HA trimers and scored based on their proximity to either termini while avoiding clashes. Out of the successful solutions, we focused on small trimers of thermophilic organisms with compatible geometry for loop closure between the identified trimer and HSB. Residues of the trimer that could interfere with binding to HA were “shaved off”, e.g. charges were designed to become neutral residues, and potentially clashing bulky residues were changed to smaller amino acids in FoldIt³³. The original trimeric scaffolds were selected to have a minimal distance towards either terminus of HSB, which allows loop closure with a simple glycine-serine linker. Loops were closed via RosettaRemodel²⁹. Blueprint files were generated so that 2, 3, 5 or 8 residue insertion between the trimerizing unit and HSB were modeled. Trimierization is based on the 1nza PDB coordinates. For initial experimental characterization, we fused the trimer adapter to the HSB.1C variant. Different loop lengths for most successful design were cloned, and expressed.