Structural insights into the inactivation of Ras and Rap1 small GTPases by Ras/Rap1-specific endopeptidase from the sepsis-causing pathogen Vibrio vulnificus

Abstract

Pathogenic bacteria have various survival strategies to incapacitate or evade the host’s immune responses. One of the potent strategies is to secrete diverse effectors. Vibrio vulnificus, causing gastroenteritis, primary septicemia, and necrotizing fasciitis in humans, secretes a broad range of toxins as well. Among them, multifunctional autoprocessing repeats-in-toxin (MARTX) toxins are known to be the most potent virulence factor. They consist of several effector domains and a cysteine protease domain (CPD) responsible for releasing effector domains into the cytosol of the host cells. Among the released effector domains, Ras/Rap1-specific endopeptidase (RRSP) of MARTX toxins secreted from sepsis causing the pathogen, V. vulnificus strain CMCP6 catalyzes the sequence-specific cleavage of the switch I region of Ras and Rap1, which is crucial for activation of the innate immune response during infections. To understand the recognition and cleavage mechanism of Vibrio vulnificus RRSP ($V_V RRSP$) against the Ras and Rap1, first we determined the structure of VvRRSP. The $V_V RRSP$ structure consists of an N-terminal domain (N lobe) involved in membrane localization, a C-terminal domain (C lobe) belonging to TIKI superfamily proteins, and a long loop connecting the two lobes. Through structure-based biochemical assays and cellular analysis we determined that 2His/2Glu catalytic residues are conserved across the TIKI superfamily enzyme. In addition, through a protease inhibitor screening assay, we identified that $V_V RRSP$ is a metal-independent TIKI family endopeptidase. Moreover, based on the results of in vitro KRas cleavage assays, we found that the N lobe of $V_V RRSP$ is important for enzymatic activity. Additionally, through endogenous Ras cleavage assays and confocal microscopy analysis, we showed that the N lobe not only functions in membrane localization by the $\alpha1$ -helix but also supports cleavage of the cellular substrates by inducing functional conformation of the C lobe. $V_V RRSP$ is thus a potent virulence factor that disrupts the signaling network by cleaving the Ras and Rap1 as a metal-independent TIKI family protease.