Proteomic QSAR analysis of calpain substrate specificity

Calpains are intracellular Ca2+-regulated cysteine proteases that are essential for various cellular functions. Mammalian conventional calpains (calpain-1 and calpain-2) modulate the structure and function of their substrates by limited proteolysis; however, their substrate specificity remains unclear because the amino acid (aa) sequences around their cleavage sites are very diverse. To clarify calpains’ substrate specificities, 84 20-mer oligopeptides, corresponding to P10-P10’ of reported cleavage site sequences, were proteolyzed by calpains, and the catalytic efficiencies (kcat/Km) were globally determined by LC/MS. This analysis revealed 483 cleavage site sequences, including 360 novel ones. The kcat/Kms for 119 sites ranged from 12.5~1,710 M-1s-1. Most sites were cleaved by both calpain-1 and -2 with a similar kcat/Km. The aa compositions of the novel sites were not significantly different from the 420 previously reported sites, suggesting calpains have a strict implicit rule for sequence specificity, and that the limited proteolysis of intact substrates is due to the substrates’ higher-order structures. Cleavage position frequencies indicated that longer sequences N-terminal to the cleavage site (P-sites) than C-terminal (P’-sites) were preferred for proteolysis. Quantitative structure-activity relationship (QSAR) analyses using partial least-squares regression and >1,300 aa descriptors achieved kcat/Km prediction with r=0.834, and binary-QSAR modeling attained 64.8% prediction accuracy for 132 reported calpain cleavage sites independent of our model construction. These results outperformed previous calpain cleavage predictors, and revealed the importance of the P2, P3’, P4’, and P1-P2 contexts. This study increases our understanding of calpain substrate specificities, and opens calpains to “next-generation,” i.e., activity-related quantitative and context-dependent analyses.