Structural and Molecular Mechanism for Autoprocessing of MARTX Toxin of Vibrio cholerae at Multiple Sites

Abstract

The multifunctional autoprocessing repeats-in-toxin (MARTX) toxin of Vibrio cholerae causes destruction of the actin cytoskeleton by covalent cross-linking of actin and inactivation of Rho GTPases. The effector domains responsible for these activities are here shown to be independent proteins released from the large toxin by autoproteolysis catalyzed by an embedded cysteine protease domain (CPD). The CPD is activated upon binding inositol hexakisphosphate (InsP 6 ). In this study, we demonstrated that InsP 6 is not simply an allosteric cofactor, but rather binding of InsP 6 stabilized the CPD structure, facilitating formation of the enzyme-substrate complex. The 1.95-Å crystal structure of this InsP 6 -bound unprocessed form of CPD was determined and revealed the scissile bond Leu 3428 -Ala 3429 captured in the catalytic site. Upon processing at this site, CPD was converted to a form with 500-fold reduced affinity for InsP 6 , but was reactivated for high affinity binding of InsP 6 by cooperative binding of both a new substrate and InsP 6 . Reactivation of CPD allowed cleavage of the MARTX toxin at other sites, specifically at leucine residues between the effector domains. Processed CPD also cleaved other proteins in trans, including the leucine-rich protein YopM, demonstrating that it is a promiscuous leucine-specific protease.Multifunctional-autoprocessing repeats-in-toxin (MARTX) 3 toxins are a family of large bacterial protein toxins with conserved repeat regions at the N and C termini that are predicted to transfer effector domains located between the repeats across the eukaryotic cell plasma membrane (1). The best characterized MARTX is the Ͼ450-kDa secreted virulence-associated MARTX of Vibrio cholerae. This toxin causes disassembly of the actin cytoskeleton and enhances V. cholerae colonization of the small intestine, possibly by facilitating evasion of phagocytic cells (2, 3). The central region of the V. cholerae MARTX toxin contains four discrete domains: the actin cross-linking domain (ACD) that introduces lysine-glutamate cross-links between actin protomers (4, 5), the Rho-inactivating domain (RID) that disables small Rho GTPases (6), an ␣␤ hydrolase of unknown function (1), and an autoprocessing cysteine protease domain (CPD) (7,8).The CPD is a 25-kDa domain found in all MARTX toxins located just before the start of the C-terminal repeats (7,8). This domain is activated for autoproteolysis upon binding inositol hexakisphosphate (InsP 6 ) (7), a molecule ubiquitously present in eukaryotic cell cytosol (9 -11), but absent in extracellular spaces and bacteria. Thus, autocatalytic processing would not occur until after translocation of the CPD and effector domains is completed. In the context of the holotoxin, catalytic residue Cys 3568 was found to be essential for the toxin to induce efficient actin cross-linking by the ACD and Rho inactivation by the RID, demonstrating that autoprocessing is essential for MARTX to induce cell rounding (8).While it is clear that InsP 6 activates the CPD and that auto...

Inositol Hexakisphosphate-dependent Processing of Clostridium sordellii Lethal Toxin and Clostridium novyi α-Toxin

“…Notably, both CPDs share conserved lysine residues, which are involved in InsP 6 binding. However, the conformation of the bound allosteric activator is largely different in the CPDs of toxin A and MARTX (21,22,36). Because the InsP 6 -binding properties of the isolated CPDs of toxin B, ␣-toxin, and lethal toxin are very similar, we do not suggest that major structural differences exist in the CPDs of these toxins.…”

Human Defensins Facilitate Local Unfolding of Thermodynamically Unstable Regions of Bacterial Protein Toxins

“…To test whether the inhibitory activity of human α-defensin HNP1 extends to MARTX Vc toxin, we assessed its effects on autoprocessing activity of MARTX cysteine protease domain (CPD Vc ) (Prochazkova et al, 2009; Shen et al, 2009) and catalytic activity of the actin crosslinking domain (ACD Vc ) (Kudryashov et al, 2008; Kudryashova et al, 2012). In the presence of HNP1, the initial reaction rate of actin crosslinking by ACD Vc was inhibited by 5.8 ± 1.2 fold as monitored by reduced accumulation of covalently crosslinked actin species (Figure 1A,B).…”

Identification of a His-Asp-Cys Catalytic Triad Essential for Function of the Rho Inactivation Domain (RID) of Vibrio cholerae MARTX Toxin

“…It is related to the glutamine synthetase family of enzymes and irreversibly connects G-actin monomers via isopeptide bonds between residues Lys-50 and Glu-270 in the presence of ATP and Mg 2ϩ / Mn 2ϩ (11)(12)(13)(14)(15). Another effector domain, the ␣␤-hydrolase domain, has been identified by analysis of CPD processing sites (7,16) and by sequence homology to ␣␤-hydrolase family members (17), although its mechanism of action has not been assessed experimentally.…”

“…MARTX toxins are large exotoxins of 350 -560 kDa characterized by the presence of amino acid (aa) repeats at the N and C termini that are thought to form a pore-like structure necessary for translocation of the central portion of the toxin across the eukaryotic plasma cell membrane (4). The translocated portion of the toxin includes a cysteine protease domain (CPD) that is necessary for inositol hexakisphosphate-induced autoproteolysis after Leu residues in unstructured regions of the holotoxin to release effector domains into the host cell cytosol (5)(6)(7)(8). Depending on the bacterial strain or species, a MARTX toxin carries an assortment of one to five effector domains that can be arrayed in different combinations and are exchanged between bacterial species by homologous recombination (4,9,10).…”

“…1A) is the Rho inactivation domain (RID Vc ) (7,16), which induces a 90% decrease in cellular levels of active GTP-bound RhoA (RhoA-GTP) as well as reduced activation of related GTPases Cdc42 and Rac, the net results of the loss of active RhoA GTPases is depolymerization of the actin cytoskeleton (18). The RID Vc consists of two subdomains one of which is a four-helical bundle membrane localization domain (4HBM) that targets RID Vc to the eukaryotic plasma membrane enriched at sites of cell-cell contacts (19,20).…”