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

Abstract

Metrics & MoreArticle Recommendations CONSPECTUS: Pore 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 αand 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.

Pore Forming Protein Induced Biomembrane Reorganization and Dynamics: A Focused Review

“…In the growing pore model, membrane insertion precedes oligomerization ( Figure 2B ). α -PFTs such as ClyA are thought to follow both pathways ( Giri Rao et al, 2016 ; Sathyanarayana et al, 2020 ). The membrane inserted oligomers can form complete ring-like structures or incomplete arc-like structures.…”

“…Pore formation is driven by the insertion of the hydrophobic β tongue ( Figure 3B ) into the membrane, accompanied by a large conformational change involving the swinging out of the N-terminus from the bundles of helices to form the membrane inserted pore state ( Benke et al, 2015 ; Giri Rao et al, 2016 ; Desikan et al, 2017 ). Although the pore forming pathways for ClyA have been extensively investigated ( Roderer and Glockshuber, 2017 ; Sathyanarayana et al, 2020 ) whether pore formation occurs via a prepore or growing pore model is a matter of debate. Evidence for partially inserted oligomeric states have been confirmed in MD simulations ( Desikan et al, 2017 ) and implicated in leakage kinetic ( Agrawal et al, 2017 ) models as well as CryoEM studies ( Peng et al, 2019 ).…”

“…In this section we turn our attention to ClyA which belongs to the class of α toxins with a distinctly different pore formation mechanism ( Sathyanarayana et al, 2020 ) when compared with the cholesterol dependent β cytolysin, LLO. We present our findings using confocal and STED-FCS measurements which probe membrane reorganization, lipid dynamics and domain preference during pore formation by ClyA.…”

A Generic Force Field for Simulating Native Protein Structures Using Dissipative Particle Dynamics

“…ClyA is an α pore-forming toxin secreted by Escherichia coli , which assembles on the target membrane to form a dodecameric transmembrane channel. 94,95 The structure of the ClyA monomer (PDB: 1QOY, 303 residues in Fig.1) is predominantly α helical containing the segments α A1 (residues 1-35), α A2 (residues 36-46), α B (residues 56-101), α C (residues 106-176), α F (residues 208-259) and α G (residues 269-291). The segment with residues 177-203 is referred to as the β -tongue.…”

Conformational flexibility is a key determinant of the lytic activity of the pore forming protein, Cytolysin A

“…On the other hand, the N-terminus helix detaches from the core helical bundle with a 126°outward movement to reposition itself to form the membrane inserted hydrophilic transmembrane pore [34]. Although, there have been several experimental and computational studies on ClyA since the crystal structure of the dodecameric pore complex was first elucidated [33, 40], several aspects of the pore forming mechanism are incompletely understood. Whether ClyA follows a pre-pore mechanism, where membrane assisted assembly precedes the formation of a lytic pore, or a growing pore mechanism where oligomerization is mediated through membrane inserted protomers is yet to be validated.…”