Cholesterol promotes Cytolysin A activity by stabilizing the intermediates during pore formation Pradeep Sathyanarayanaa, Satyaghosh Mauryab, Amit Beherab, Monisha Ravichandranb, Sandhya S. Visweswariaha,c, K. Ganapathy Ayappaa,b, and Rahul Roya,b,d,1 aCentre for BioSystems Science and Engineering, Indian Institute of Science, 560012 Bangalore, India; bDepartment of Chemical Engineering, Indian Institute of Science, 560012 Bangalore, India; cDepartment of Molecular Reproduction, Development and Genetics, Indian Institute of Science, 560012 Bangalore, India; and dMolecular Biophysics Unit, Indian Institute of Science, 560012 Bangalore, India Edited by Michael L. Klein, Temple University, Philadelphia, PA, and approved June 25, 2018 (received for review December 6, 2017) Pore-forming toxins (PFTs) form nanoscale pores across target eral motion of the individual protein molecules on the mem- membranes causing cell death. Cytolysin A (ClyA) from Escherichia brane surface. However, little is known about toxin dynamics on coli is a prototypical α-helical toxin that contributes to cytolytic the lipid membrane and its implication on the formation of phenotype of several pathogenic strains. It is produced as a mono- higher-order structures. mer and, upon membrane exposure, undergoes conformational Here we address these questions using a prototypical α-PFT, changes and finally oligomerizes to form a dodecameric pore, Cytolysin A, which is produced by strains of Escherichia coli, thereby causing ion imbalance and finally cell death. However, Shigella, and Salmonella (6). The water-soluble ClyA monomer our current understanding of this assembly process is limited to produced by these bacteria is composed of a five–α-helix bundle studies in detergents, which do not capture the physicochemical and a hydrophobic β-hairpin (β-tongue) (7) (SI Appendix, Fig. properties of biological membranes. Here, using single-molecule S1). The protein assembles into a dodecameric cation-selective imaging and molecular dynamics simulations, we study the ClyA pore complex upon membrane binding. The structure of the assembly pathway on phospholipid bilayers. We report that cho- ClyA pore (assembled in detergents) displayed large structural lesterol stimulates pore formation, not by enhancing initial ClyA alterations in protomers of the pore, compared with the mono- binding to the membrane but by selectively stabilizing a protomer- meric water-soluble form (8). The β-tongue in the monomer like conformation. This was mediated by specific interactions by transforms into a helix–loop–helix, and reorganization of cholesterol-interacting residues in the N-terminal helix. Addition- α-helices causes the N-terminal helix to switch orientation by ally, cholesterol stabilized the oligomeric structure using bridging ∼ interactions in the protomer–protomer interfaces, thereby result- 180°, to form the inner lumen of the pore. ing in enhanced ClyA oligomerization. This dual stabilization of Detergent-induced ClyA oligomerization experiments have distinct intermediates by cholesterol suggests a possible molecular indicated that conformational transition in ClyA is the rate- mechanism by which ClyA achieves selective membrane rupture of limiting step in the assembly pathway (9, 10). However, ClyA eukaryotic cell membranes. Topological similarity to eukaryotic assembly driven by surfactants results in significantly slower ki- membrane proteins suggests evolution of a bacterial α-toxin to netics compared with membrane rupture and leakage (11). Apart adopt eukaryotic motifs for its activation. Broad mechanistic corre- from kinetic modeling studies (12), little is understood about the spondence between pore-forming toxins hints at a wider prevalence kinetics of this process in phospholipid bilayers. More impor- of similar protein membrane insertion mechanisms. tantly, it is not clear how pore-like ClyA complexes as observed on bacterial outer membranes vesicles (OMVs) can form without pore-forming toxin | membrane | cholesterol | single-molecule imaging | molecular dynamics Significance ore-forming toxins (PFTs) are cell membrane-rupturing Pore-forming toxins (PFTs) are the largest class of bacterial Pproteins and form the largest class of toxins that mediate exotoxins mediating virulence. Soluble toxin monomers oli- bacterial virulence (1–3). PFTs are secreted as water-soluble gomerize upon binding to cellular membrane and convert to monomers that bind strongly to the lipid membrane of eukary- stable membrane-integrated pores, causing cell death. This otic cells by adopting structures that traverse the membrane via conversion to an active form occurs in absence of extrinsic helices (α-PFT) or sheets (β-PFT). This allows the passage of factors and is governed solely by molecular determinants in the molecules from within the cell to the exterior, resulting in host protein and target membrane. Here we demonstrate the exis- cell lysis. The conformational transition of a PFT from a water- tence of cholesterol-binding motifs in ClyA, which stabilize soluble structure to a distinct membrane-associated protomer structural intermediates in the assembly pathway in presence form is not understood in mechanistic detail. For example, do of cholesterol. Our finding elucidates the basis for selective components in the eukaryotic cell membrane drive the confor- targeting of the toxin to eukaryotic membranes. Molecular mational transitions that result in an assembly competent state? engineering of these signatures could advance application of Does the membrane play an active role in stabilization of in- PFTs in cytolytic therapy. termediates that allow membrane insertion and pore formation? Membrane components that are essential in the β-PFT assembly Author contributions: P.S., S.M., A.B., S.S.V., K.G.A., and R.R. designed research; P.S., S.M., A.B., and M.R. performed research; P.S., S.M., A.B., and M.R. contributed new reagents/ BIOPHYSICS AND pathway have been well characterized and include protein re- analytic tools; P.S., S.M., A.B., and R.R. analyzed data; and P.S., S.M., A.B., S.S.V., K.G.A., ceptors, carbohydrates, or eukaryotic lipids such as cholesterol and R.R. wrote the paper. COMPUTATIONAL BIOLOGY and sphingomyelin (1). Determinants of membrane selectivity The authors declare no conflict of interest. are poorly understood in the case of α-PFTs with the exception This article is a PNAS Direct Submission. of some reports of sphingomyelin as a cofactor for toxin function Published under the PNAS license. for certain actinoporins (4, 5). Another crucial but less probed 1To whom correspondence should be addressed. Email: [email protected]. aspect of the assembly pathway is the role of the toxin’s lateral This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. motion on membranes. Toxin self-assembly is contingent on 1073/pnas.1721228115/-/DCSupplemental. establishing interprotomer contacts, which is influenced by lat- Published online July 16, 2018. www.pnas.org/cgi/doi/10.1073/pnas.1721228115 PNAS | vol. 115 | no. 31 | E7323–E7330 Downloaded by guest on October 6, 2021 altering bacterial viability, even upon overexpression, whereas (∼60% labeling efficiency) ClyA (Q56C) mutant (10). ClyA similar oligomeric complexes are cytotoxic in eukaryotic mem- appeared to be immobile on conventional membrane platforms branes (13, 14). (SI Appendix, Figs. S3 and S4A), possibly because of strong Here we characterize the initial steps of interaction of ClyA with surface effects of the underlying substrate (19, 20). Hence, model membranes and events that ultimately lead to the formation polymer-supported bilayers (PEG-SLBs) were used in all sub- of a pore. Using single-molecule fluorescence-based particle sequent experiments (SI Appendix, SI Results and Fig. S4 B and tracking and photobleaching analysis on supported lipid bilayers C). Toxin binding to the membrane was assessed by monitoring (SLBs), we identify distinct diffusive states that represent altered the appearance of fluorescent spots on the membrane surface as conformations of the toxin. The distribution of sampled states is ClyA (100 pM) was introduced into a microchannel containing sensitive to cholesterol content, and specific cholesterol-interacting PEG-SLBs. Unimodal intensity distribution and single-step residues selectively promote membrane insertion of the trans- photobleaching suggested monomeric ClyA as the dominant membrane segment of ClyA in presence of cholesterol. Further- membrane population (SI Appendix, Fig. S5). The binding of more, all-atom molecular dynamics simulations reveal specific ClyA reached equilibrium within tens of seconds for POPC cholesterol–protein interactions that mediate membrane binding PEG-SLBs and those containing 27.5% cholesterol (Fig. 2A). and oligomerization. Together, these cholesterol-stabilized ClyA We limited our experiments to these two membrane composi- intermediates bias the assembly pathway towards pore formation. tions (referred as POPC and POPC:Chol) because reports exist of membrane phase separation with higher concentrations of Results cholesterol either in POPC or in ternary lipid mixtures (21) that Cholesterol Enhances Membrane Rupture by ClyA. ClyA interacts might complicate data interpretation. directly with the lipid membranes (6), but it is not clear whether Binding of ClyA reached equilibrium faster by a factor of ∼3 − − any additional receptors are required for efficient ClyA pore (0.36 ± 0.015 s 1 in POPC:Chol vs. 0.095 ± 0.004 s 1 in POPC)