Structural insights into human caspase-9 CARD assembly modes driven by conserved arginine hotspots.

Caspase-9 (C9) is a key initiator of the intrinsic apoptotic pathway and contains an N-terminal caspase activation and recruitment domain (CARD) that mediates homotypic and heterotypic interactions. While previous studies have shown that C9CARD can form filamentous assemblies under specific conditions, its intrinsic structural properties and interaction modes below saturation concentration remain incompletely understood. Here, we present a 2.4 Å resolution crystal structure of the isolated human C9CARD, revealing a sulfate-mediated dimer-of-dimers assembly involving a conserved arginine-rich surface. Notably, these residues overlap with those previously shown in filament formation and in interactions with APAF-1CARD, indicating reuse of a common interaction hotspot across distinct assembly states. Complementary biophysical analyses show that C9CARD remains predominantly monomeric under physiological conditions, while sulfate induces changes in its hydrodynamic properties. Compared to WT C9CARD, a triple-arginine mutant (R10E/R52E/R56E) shows different sulfate-dependent behavior and abolishes formation of higher-order assemblies. Additionally, WT C9CARD exhibits reversible temperature-dependent transitions between filamentous and droplet-like assemblies. Finally, AlphaFold3 predictions suggest that filament-forming propensity and electrostatically regulated higher-order assemblies may extend to other human CARDs. Together, our findings suggest a model in which a conserved electrostatic interaction motif enables multiple structurally distinct assembly modes of C9CARD. This work highlights conserved electrostatic interaction modes in CARD-CARD association and suggests that environmental conditions can modulate their assembly behavior.