Elucidating the mode of action of Vibrio cholerae cytolysin (VCC) by understanding the regulation and inhibition of its membrane-damaging pore-forming activity

Abstract

Vibrio cholerae cytolysin (VCC) is a prototype β-barrel pore-forming toxin (β-PFT) that generates transmembrane oligomeric pores in the target cell membranes. VCC is secreted as a monomeric water-soluble protein and goes through a massive structural reorganization to accomplish the final form as a transmembrane pore complex. Despite extensive investigation, the structural reorganization of VCC and its regulation within the protein structure still remains unclear. The major rearrangements include the restructuring of the pore-forming pre-stem motif and the relocation of the cradle loop. In the course of oligomeric pore-formation, the pre-stem motif gets released from the hydrophobic protein core and inserts into the membrane to form the β-barrel pore architecture. The cradle loop clamps the pre-stem in the monomeric form and gets repositioned towards the inter-protomeric interface in the oligomeric form of VCC. Therefore, the cradle loop may have critical implications in governing the reorganization of the pre-stem and other neighboring domain(s)/motif(s). In the first part of our study, we show that the specific cradle loop residues govern the pore forming process of VCC by establishing crucial intra-molecular interactions responsible for the successful and sequential reorganization of the protein structure. Mutations of these residues alter the structural attributes of VCC and obstruct the insertion of the pre-stem motif into the target membrane without hampering the binding and oligomerization abilities of the toxin. These mutations arrest VCC in a pre-pore-like oligomeric state, resulting in severely delayed pore forming kinetics, indicating an increased energy barrier associated with functional pore formation. The mutation of one of the residues disrupts the interactions of the cradle loop with the nearby β prism domain and obstructs its rearrangement, causing pre-mature oligomerization of VCC without membrane. In the presence of a membrane lipid bilayer, mutant shows severely xxiii compromised insertion of the pre-stem motif into the membrane. Mutation of another cradle loop residue perturbs the interactions with the pre-stem motif and delays pore formation by impeding pre-stem insertion, leading to a prolonged pre-pore-like state. Further, we investigated the role of the cradle loop in mediating crucial interprotomer interactions and stabilization of the pore architecture in the oligomeric state. In the second part of our study, we investigated curcumin, as a natural inhibitor of the membrane-damaging pore-forming function of VCC, and explored the underlying mechanistic basis. We found that curcumin can strongly interact with VCC and significantly compromise its pore-forming activity. Further, we found that curcumin can interfere with the membrane-binding ability of VCC. Moreover, curcumin traps the membrane-bound toxin molecules in a membrane bound oligomeric state, preventing functional pore formation. Overall, our study elucidated the under-explored aspects of VCC pore-formation mechanism, including regulation of its structural reorganization by revealing the specific role of the cradle loop, and also established curcumin as a natural inhibitor of its membrane-damaging pore-formig activity