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 C9^CARD 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 C9^CARD, 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-1^CARD, indicating reuse of a common interaction hotspot across distinct assembly states. Complementary biophysical analyses show that C9^CARD remains predominantly monomeric under physiological conditions, while sulfate induces changes in its hydrodynamic properties. Compared to WT C9^CARD, a triple-arginine mutant (R10E/R52E/R56E) shows different sulfate-dependent behavior and abolishes formation of higher-order assemblies. Additionally, WT C9^CARD 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 C9^CARD. This work highlights conserved electrostatic interaction modes in CARD-CARD association and suggests that environmental conditions can modulate their assembly behavior.