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Structural and Biochemical Insights into the Activation and Substrate Selectivity of Clostripain‐like Proteases Secreted From Commensal Gut Bacteria

Commensal gut bacteria secrete a broad variety of biological and chemical signals to influence intestinal homeostasis and modulate immune responses, including active proteases. Enteric cysteine proteases are among the most abundant hydrolytic enzymes produced by bacteria and they play a crucial role in intestinal inflammation as they cleave host proteins, activate cell surface receptors, and disrupt epithelial barrier integrity. Recently, members of the clostripain‐like (C11) family of cysteine proteases were shown to mediate immune responses by inducing neutrophil phagocytosis and activating bacterial pathogenic toxins. We have structurally and functionally characterized two additional members of this family from the predominant gut symbiont Bacteroides thetaiotaomicron: BT1308 and BT0727. These secreted proteases are predicted to be packaged into outer membrane vesicles capable of interacting with host epithelial cells and are known to be activated through internal cleavage events. We verified the necessity of self‐cleavage for maturation, showed this cleavage event occurs in trans, and identified the sites of cleavage following designated arginine residues. Using Multiplex Substrate Profiling by Mass Spectrometry we identified a unique cleavage motif of X‐X‐T/S‐K/R common among C11 proteases and determined protease‐specific differences in P3–P4 recognition. To supplement this biochemical data, we solved the structures of both active enzymes in complex with a tetrapeptide inhibitor at under 2.4Å resolution, and these structures highlighted subtle variations in active site architecture that confer selectivity in substrate recognition. Furthermore, comparing our structures to an inactive mutant of a C11 protease from Parabacteroides distasonis revealed that proteolytic activity could be dependent on a conformational change that reorients the active site histidine upon self‐cleavage. Our findings provide insight into the mechanism and specificity of C11 protease activity and will aid in the rational design of protease‐specific probes that can be used to interrogate the role of C11 proteases in inflammation and bacterial fitness.