Structural Insights into the Unique Activation Mechanisms of a Non-Classical Calpain and its Disease-Causing Variants

Abstract

Increased calpain activity is linked to neuroinflammation including a heritable retinal disease caused by hyper-activating mutations in the calcium-activated calpain-5 (CAPN5) protease. Although structures for classical calpains have been described, the structure of CAPN5, a non-classical calpain, remains undetermined. Herein, we report the 2.8 A crystal structure of the human CAPN5 protease core (CAPN5-PC). Compared to classical calpains, CAPN5-PC requires high Ca2+ concentrations for maximal activity. Structure-based phylogenetic analysis revealed that CAPN5-PC contains three elongated flexible loops compared to its classical counterparts. The presence of a disease-causing mutation (c.799G>A, p.Gly267Ser) on the unique C2 loop revealed a novel function for this region in regulating enzymatic activity. This mechanism could be transferred to distant calpains, using synthetic calpain hybrids, suggesting an evolutionary mechanism for fine-tuning calpain function by modifying flexible loops. Further, the open (inactive) conformation of the CAPN5-PC structure provides an opportunity for identifying inhibitors that bind to CAPN5-specific residues and lock it in the inactivated form.