Structural basis of the activation of MARTX cysteine protease domain from Vibrio vulnificus

The multifunctional autoprocessing repeat-in-toxin (MARTX) toxin is the primary virulence factor of Vibrio vulnificus displaying cytotoxic and hemolytic properties. The cysteine protease domain (CPD) is responsible for activating the MARTX toxin by cleaving the toxin precursor and releasing the mature toxin fragments. To investigate the structural determinants for inositol hexakisphosphate (InsP(6))-mediated activation of the CPD, we determined the crystal structures of unprocessed and beta-flap truncated MARTX CPDs of Vibrio vulnificus strain MO6-24/O in complex with InsP(6) at 1.3 and 2.2 & Aring; resolution, respectively. The CPD displays a conserved domain with a central seven-stranded beta-sheet flanked by three alpha-helices. The scissile bond Leu3587-Ala3588 is bound in the catalytic site of the InsP(6)-loaded form of the Cys3727Ala mutant. InsP(6) interacts with the conserved basic cleft and the beta-flap inducing the active conformation of catalytic residues. The beta-flap of the post-CPD is flexible in the InsP(6)-unbound state. The structure of the CPD Delta beta-flap showed an inactive conformation of the catalytic residues due to the absence of interaction between the active site and the beta-flap. This study confirms the InsP(6)-mediated activation of the MARTX CPDs in which InsP(6)-binding induces conformational changes of the catalytic residues and the beta-flap that holds the N terminus of the CPD in the active site, facilitating hydrolysis of the scissile bond.