A histidine switch controls the pH-responsive self-assembly of a helical protein filament.

Self-assembling helical protein filaments underlie diverse biological processes, from signaling pathways to cell motility. Encoding tunable self-assembly into the sequences of filamentous proteins remains a major challenge. Here, we discovered that the caspase-9 CARD can natively self-assemble into helical filaments in a pH-regulated manner. We defined the determinants of filament assembly using an integrative structural, biophysical, and computational approach. Using NMR spectroscopy, we found that the protonation of a single histidine residue near an N-terminal helix dipole, H38, regulates the pH-dependent self-assembly process. Charge-altering mutations at this site tune thermodynamic stability and filament self-assembly across solution and pH conditions. We solved 3.3- and 3.5-Å cryo-EM structures of the wild-type and H38R filaments, respectively, which show H38 positioned directly at a filament interface. Molecular dynamics simulations show that H38 functions as a molecular switch, whereby protonation rotates its positively charged side-chain toward solvent and away from the partial-positive charge at the N-terminal helix dipole. This reflects a fine balance between stabilizing intermolecular association and a destabilizing intramolecular electrostatic clash at the helix dipole. More broadly, across 350 helix-containing protein domains we identified electrostatic contributions to protein stability near helix dipoles by integrating AlphaFold2 predictions with deep mutational scanning data. Together, our results identify a native, pH-sensitive histidine switch that regulates a self-assembling helical protein filament. Our results establish a mechanism by which charge-altering mutations near helical N-termini can be engineered to control side-chain rotamers, protein stability, and self-assembly.