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 and#946;-barrel pore-forming toxin (and#946;-PFT) that generates newlinetransmembrane oligomeric pores in the target cell membranes. VCC is secreted as a monomeric newlinewater-soluble protein and goes through a massive structural reorganization to accomplish the final newlineform as a transmembrane pore complex. Despite extensive investigation, the structural newlinereorganization of VCC and its regulation within the protein structure still remains unclear. The newlinemajor rearrangements include the restructuring of the pore-forming pre-stem motif and the newlinerelocation of the cradle loop. In the course of oligomeric pore-formation, the pre-stem motif gets newlinereleased from the hydrophobic protein core and inserts into the membrane to form the and#946;-barrel newlinepore architecture. The cradle loop clamps the pre-stem in the monomeric form and gets newlinerepositioned towards the inter-protomeric interface in the oligomeric form of VCC. Therefore, the newlinecradle loop may have critical implications in governing the reorganization of the pre-stem and newlineother neighboring domain(s)/motif(s). newlineIn the first part of our study, we show that the specific cradle loop residues govern the pore- newlineforming process of VCC by establishing crucial intra-molecular interactions responsible for the newlinesuccessful and sequential reorganization of the protein structure. Mutations of these residues alter newlinethe structural attributes of VCC and obstruct the insertion of the pre-stem motif into the target newlinemembrane without hampering the binding and oligomerization abilities of the toxin. These newlinemutations arrest VCC in a pre-pore-like oligomeric state, resulting in severely delayed pore- newlineforming kinetics, indicating an increased energy barrier associated with functional pore formation. newlineThe mutation of one of the residues disrupts the interactions of the cradle loop with the nearby and#946;- newlineprism domain and obstructs its rearrangement, causing pre-mature oligomerization of VCC newlinewithout membrane. In the presence of a membrane lipid bilayer, mutant shows severely newlinexxiiicompromised insertion of