Mechanistic Insights into Pore Formation by an α-Pore Forming Toxin: Protein and Lipid Bilayer Interactions of Cytolysin A

Sathyanarayana, P and Visweswariah, SS and Ayappa, KG (2021) Mechanistic Insights into Pore Formation by an α-Pore Forming Toxin: Protein and Lipid Bilayer Interactions of Cytolysin A. In: Accounts of Chemical Research, 54 (1). pp. 120-131.

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Abstract

ConspectusPore forming toxins (PFTs) are the largest class of bacterial toxins playing a central role in bacterial pathogenesis. They are proteins specifically designed to form nanochannels in the membranes of target cells, ultimately resulting in cell death and establishing infection. PFTs are broadly classified as α- and β-PFTs, depending on secondary structures that form the transmembrane channel. A unique feature about this class of proteins is the drastic conformational changes and complex oligomerization pathways that occur upon exposure to the plasma membrane. A molecular understanding of pore formation has implications in designing novel intervention strategies to combat rising antimicrobial resistance, targeted-cancer therapy, as well as designing nanopores for specialized technologies. Central to unraveling the pore formation pathway is the availability of high resolution crystal structures. In this regard, β-toxins are better understood, when compared with α-toxins whose pore forming mechanisms are complicated by an incomplete knowledge of the driving forces for amphiphatic membrane-inserted helices to organize into functional pores. With the publication of the first crystal structure for an α-toxin, cytolysin A (ClyA), in 2009 we embarked on an extensive multiscale study to unravel its pore forming mechanism. This Account represents the collective mechanistic knowledge gained in our laboratories using a variety of experimental and theoretical techniques which include large scale molecular dynamics (MD) simulations, kinetic modeling studies, single-molecule fluorescence imaging, and super-resolution spectroscopy. We reported MD simulations of the ClyA protomer, oligomeric intermediates, and full pore complex in a lipid bilayer and mapped the conformational transitions that accompany membrane binding. Using single-molecule fluorescence imaging, the conformational transition was experimentally verified by analysis of various diffusion states of membrane bound ClyA. Importantly, we have uncovered a hitherto unknown putative cholesterol binding motif in the membrane-inserted helix of ClyA. Distinct binding pockets for cholesterol formed by adjacent membrane-inserted helices are revealed in MD simulations. Cholesterol appears to play a dual role by stabilizing both the membrane-inserted protomer as well as oligomeric intermediates. Molecular dynamics simulations and kinetic modeling studies suggest that the membrane-inserted arcs oligomerize reversibly to form the predominant transmembrane oligomeric intermediates during pore formation. We posit that this mechanistic understanding of the complex action of α-PFTs has implications in unraveling pore assembly across the wider family of bacterial toxins. With emerging antimicrobial resistance, alternate therapies may rely on disrupting pore functionality or oligomerization of these pathogenic determinants utilized by bacteria, and our study includes assessing the potential for dendrimers as pore blockers.

Item Type:	Journal Article
Publication:	Accounts of Chemical Research
Publisher:	American Chemical Society
Additional Information:	The copyright for this article belongs to American Chemical Society.
Keywords:	bacterial toxin; cholesterol; perforin, chemistry; Escherichia coli; kinetics; lipid bilayer; metabolism; molecular dynamics; protein tertiary structure, Bacterial Toxins; Cholesterol; Escherichia coli; Kinetics; Lipid Bilayers; Molecular Dynamics Simulation; Perforin; Protein Structure, Tertiary
Department/Centre:	Division of Biological Sciences > Molecular Reproduction, Development & Genetics Division of Interdisciplinary Sciences > Centre for Biosystems Science and Engineering Division of Mechanical Sciences > Chemical Engineering
Date Deposited:	24 Feb 2023 05:00
Last Modified:	24 Feb 2023 05:00
URI:	https://eprints.iisc.ac.in/id/eprint/80453

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