A histidine switch regulates pH-dependent filament formation by the caspase-9 CARD.

The caspase activation and recruitment domain (CARD) mediates protein-protein interactions in apoptotic and inflammatory signaling pathways. In humans, more than 30 proteins contain a CARD, several of which have been reported to polymerize into helical filaments. Here we found that the CARD from the apoptotic protease caspase-9 (C9) self assembles into filaments at physiological pH and salt concentrations. The C9 more readily polymerizes under low-salt or mildly acidic conditions, suggesting a significant role for electrostatic interactions in mediating filament formation. Using NMR spectroscopy, we determined the p of the lone histidine residue, H38, which supports a role for histidine protonation in enhancing filament formation. Indeed, mutation of H38 to introduce a positive (H38R) or negative (H38D) charge, or to remove the pH-dependence of the side chain at this site altogether (H38N), dramatically alters the filament propensity of the domain. Using cryo-election microscopy, we determined 3.4- and 3.2-Å structures of the wild-type and H38R C9 filaments, respectively, which provide new insights into the molecular basis of C9 polymerization and its pH dependence via H38.