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bioRxiv · 10.1101/2025.06.02.657148

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

Rawal, S.·Kolaric, Đesika·Bohn, S.·Kolb, D.·Pavkov-Keller, T.·Pritisanac, I.·Madl, T.·Desfosses, A.·Alderson, T. R.
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Abstract

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 (C9CARD) self assembles into filaments in vitro at physiological pH and salt concentrations. The C9CARD 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 pKa 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-[A] structures of the wild-type and H38R C9CARD filaments, respectively, which provide new insights into the molecular basis of C9CARD polymerization and its pH dependence via H38.

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Rawal, S., Kolaric, Đesika, Bohn, S., Kolb, D., Pavkov-Keller, T., Pritisanac, I., Madl, T., Desfosses, A., Alderson, T. R.. 2025-06-03. A histidine switch regulates pH-dependent filament formation by the caspase-9 CARD. https://doi.org/10.1101/2025.06.02.657148

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