Molecular basis for the effect of the L31F mutation on CARD function in ARC

The apoptosis repressor with caspase-recruiting domain (ARC) is aberrantly overexpressed in various cancers. ARC contains a caspase recruitment domain (CARD) that is the main mediator of protein-protein interactions. Mutation of Leu31 within the CARD of ARC to Phe (ARC_L31F) is widely used as a functionally defective mutant of ARC despite a lack of clear experimental evidence regarding how its functionality is lost. In this study, we show that L31 in helix 2 (H2) is critical for stabilization of the helix bundle fold in the CARD domain. In addition, the L31F mutation disrupts homodimer formation that is critical to ARC functions. Our current study reveals the molecular basis for the L31F mutation disrupting the ARC CARD functions.