BPS2026 – Conserved patterns vs. rational design: Fold-switching pathways in a phage tail protein

Fold-switching, where a single protein region interconverts between two distinct three-dimensional structures, is increasingly recognized as critical for protein function. A recent striking example is the F-type pyocin, a phage tail-like bactericidal agent. A 160-residue segment of its central tail fiber transitions from a trimeric α-helical coiled-coil to a triangular β-prism upon cell-surface engagement. This structural switch is essential for bactericidal activity, as amino acid substitutions that disrupt it abolish the biological function. Using the CF-random pipeline, we identified similar α-helical coiled-coil to β-prism fold switches in phage protein homologues, suggesting that large-scale fold-switching may be a conserved mechanism for membrane penetration by non-contractile phage tails. Further, sequence analyses reveal conserved hydrophobic and small residues that may stabilize both structures, alongside positions that preferentially support one-fold, indicating evolutionary constraints that encode dual conformations. To test designability, we paralleled two approaches: (1) introducing targeted mutations guided by conservation profiles, and (2) designing sequences using ProteinMPNN. Conservation-guided mutations identified eight substitutions that shifted F7 pyocin predictions toward the helix fold, whereas the wild type predominantly sampled the prism fold. ProteinMPNN-generated sequences produced predictions of both folds, which we will use to screen for residues of potential importance for switching. Overall, we aim to compare the ProteinMPNN designs with those derived from in-depth biological knowledge of the system that often relies on decades of expertise in the biological system of interest. Together, this work provides an outline of complementary approaches that explore how such structural transitions can be rationally engineered.