Cholesterol Promotes Cytolysin a Activity by Stabilizing the Intermediates During Pore Formation

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) formation on cellular membranes. We therefore monitored rabbit on cholesterol-containing membranes. However, contrary to erythrocyte lysis following proteolytic cleavage of exposed cell cholesterol stimulation of ClyA activity, approximately fivefold surface proteins (Fig. 1A and SI Appendix,Fig.S2A) and contin- less ClyA was bound to cholesterol-containing membranes at ued to observe efficient and equivalent lysis. In contrast, eryth- equilibrium even though the initial rates of ClyA binding were rocytes pretreated with methyl β-cyclodextrin (MβCD), which similar (∼0.003 particles per μm2 per s), suggesting (approxi- selectively reduces the cholesterol content of the plasma mem- mately fivefold) higher rates of unbinding from cholesterol- brane (15, 16), displayed a drop in cell lysis by at least two orders containing membranes. However, the rapid binding of ClyA to in magnitude over a wide range of toxin concentrations (Fig. 1B). lipid membranes (seconds) contrasted with the much longer time Cholesterol-regulated ClyA activity was also observed in vesicle scales (t1/2 ∼ 2–7 min) for detecting cell lysis or vesicle leakage, dye leakage assays (6). For example, incorporation of cholesterol suggesting that ClyA binding to membranes was not the rate- in DOPC (1:1 mol %) vesicles resulted in a threefold enhance- limiting step in pore assembly. ment in ClyA lytic activity (Fig. 1C and SI Appendix,Fig.S2B). Diffusional Dynamics of ClyA on the Membrane. We asked if changes ClyA Binding to Artificial Bilayer Membranes. We first asked if the in the lateral mobility of the membrane-bound toxin explain the stimulation in activity in the presence of cholesterol was merely a observed stimulation of ClyA activity. Therefore, we examined consequence of enhanced binding. We examined ClyA in- the diffusional properties of single ClyA at low densities (25– teraction with lipid membranes by single-molecule particle 100 pM) on PEG-SLBs. We observed a large heterogeneity in tracking of fluorescently labeled ClyA on phospholipid bilayers the lateral displacements for ClyA particles on POPC and using total internal reflection fluorescence (TIRF) microscopy POPC:Chol membranes (Fig. 2B). On POPC PEG-SLBs, ClyA (17, 18). We established a single ClyA imaging assay on sup- molecules predominantly exhibited a single distribution of in- ported lipid bilayers using a functionally active, singly labeled stantaneous squared displacements (ISDs) with high mobility at

ABC

Fig. 1. Cholesterol stimulates the pore-forming activity of ClyA. (A) Turbidity assay to determine activity of ClyA is performed by measuring the optical

density of rabbit erythrocytes (black) with time after addition of ClyA. Proteolytic shaving of erythrocyte membranes (shaved RBC; red) did not change the t1/2 of the lysis (Inset) or the extent of lysis. Solid lines represent Boltzmann sigmoid fits to the data. (B) Partial removal of cholesterol from erythrocytes by

treatment with MβCD (red) increases the t1/2 for lysis in turbidity assays by more than 100-fold compared with untreated erythrocytes (black). (C) Vesicle dye leakage kinetics of ClyA for DOPC (green) and DOPC with 30% cholesterol (blue) and 50% cholesterol (gray) concentrations are shown. Solid lines represent

single exponential fits to the leakage data. The dye leakage t1/2 in presence of cholesterol is reduced by approximately threefold for 30–50 mol % cholesterol (Inset). Error bars represent SD of at least three experiments.

E7324 | www.pnas.org/cgi/doi/10.1073/pnas.1721228115 Sathyanarayana et al. Downloaded by guest on October 6, 2021 early time points (∼100 ms) (Fig. 2C). A linear correlation be- This was further validated by quantifying the rates of conversion tween MSD and the lag times indicated that the motion was between the diffusive states for individual ClyA molecules. Brownian for the initial fast species (SI Appendix, Fig. S6A). However, the fast diffusing population gradually diminished with Protein Conformational Transitions Underlie the Switch in Mobility a concomitant increase in a broad population of lower-mobility States. To determine the kinetics of transition between the ClyA states (Fig. 2C and SI Appendix, Fig. S6B). Global fitting of the mobility states, we employed hidden Markov model (HMM) cumulative distribution function (CDF) of instantaneous squared analysis of the particle trajectories and assigned three diffusive displacements yielded the presence of at least three diffusive states (22). We pooled all transitions across different time points 2 states: a fast (M; DM = 1.43 ± 0.07 μm /s), an intermediate (P1; for the two membrane compositions to generate a transition 2 DP1 = 0.24 ± 0.05 μm /s), and a slow mobility state (P2; DP2 = probability matrix (Fig. 3A). The cluster of transitions between the 0.040 ± 0.006 μm2/s) (Fig. 2 C, Inset and 2E and SI Appendix, SI mobility states largely overlap between POPC and POPC:Chol Results). The lower-mobility population did not demonstrate an membranes, indicating that ClyA sampled identical diffusive states increase in single-particle intensity compared with the initial fast in both POPC and POPC:Chol membranes. As observed in the ClyA species, hence arguing against oligomerization causing the CDF analysis (Fig. 2E), the majority of the transitions observed reduction in mobility (SI Appendix, Fig. S6 B and C). The ISD were between the M to P1 mobility states. Similar but less distribution reached equilibrium within 10 min, but ∼55% of the prominent transitions were observed between the P1 and P2 molecules remained in the fast mobility state (M) (Fig. 2 C states, with a large distribution of diffusion coefficients. We esti- and E). mated the mean diffusion coefficients of the three diffusive states On POPC:Chol bilayers, the protein initially displayed rapid to be 1.2–1.3 μm2/s, 0.06–0.12 μm2/s, and 0.02–0.03 μm2/s by diffusion (Fig. 2D), with diffusion coefficients comparable to those pooling all of the particle trajectories for HMM analysis (SI Ap- determined in POPC membranes (SI Appendix, Fig. S7A and pendix,TableS1). Comparable values obtained for the diffusion Table S1). However, ClyA quickly converted to the lower-mobility coefficients for each of the mobility states with cumulative ISD form with near-complete disappearance of the fast species fitting as well as HMM analysis support the existence of these (∼5 min), without any discernible increase in single-particle in- distinct diffusive states. The HMM analysis indicated that ClyA tensity (SI Appendix,Fig.S7B). The slow moving P2 fraction was could reversibly interconvert between these states. similar in both POPC and POPC:Chol membranes, indicating that The average forward and reverse rates from the HMM analysis major changes occurred between the M and P1 states (Fig. 2E). were computed (SI Appendix, Table S2). In POPC membranes,

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Fig. 2. Single-ClyA molecule dynamics on supported bilayers. (A) Binding kinetics of ClyA on POPC (green) and POPC:Chol (blue) SLB as determined by single exponential fit (line) to the increase in the number of ClyA particles observed on the membrane. (B) Representative single-particle trajectories (displacements from the origin) for ClyA molecules displaying low mobility (purple), high mobility (red), and heterogeneous behavior (black) on POPC (Top) and POPC:Chol

(Bottom) membranes are shown. (C) Time series of ISD distribution for ClyA (n = 1,000–5,000 for each set) on POPC membranes is shown (ntotal = 37,000). Solid lines represent fits to a Gaussian mixture model (GMM) with three species. (D) Corresponding time series of ISD distribution (n = 800–1,200 for each set) on

POPC:Chol bilayers are shown (ntotal = 18,000). The CDF of ISD is fit to three diffusive species for all time points (C and D, Insets). (E) Fraction of the ClyA diffusive species for both POPC (shades of green) and POPC:Chol (shades of blue) membranes is displayed for slow (P2, light), intermediate (P1, dark), and fast (M, darkest shade) populations at different time points after ClyA binding. Error bars represent SD.

Sathyanarayana et al. PNAS | vol. 115 | no. 31 | E7325 Downloaded by guest on October 6, 2021 2 2 AC(0.8–2 μm /s) and transmembrane proteins (0.02–0.2 μm /s) (23– 27). Indeed, frictional effects of a single transmembrane helix have been reported to result in >55% reduction in diffusivity of a peripheral membrane protein (23). This is comparable to the decrease in diffusivity between the M and P1 states in ClyA, attributed to the insertion of the N-terminal helix. In addition, the M and P1 structures were the two prominent membrane- interacting conformations observed by Giri Rao et al. (28), where structure-based models captured the conformational transitions of ClyA from the monomer to the membrane-inserted protomer. In the same study, the order of the appearance of M B and P1 states corresponded with those observed in our experiments. Although the identity of the P2 state is unclear at this stage, it could arise from the residual pinning of the transmembrane helix to the underlying glass surface or another structural state. The significantly enhanced rate of switching of M to P1 state in cholesterol-containing membranes suggested that the distinct conformation of the P1 state is stabilized by the interaction of cholesterol with ClyA. To test this, we incubated POPC:Chol membranes with ClyA till diffusive states reached equilibrium and then treated these membranes with MβCD. We observed the reappearance of the fast-moving M species to comparable levels as observed in cholesterol-free membranes confirming that the direct interaction of cholesterol with ClyA was responsible for Fig. 3. Kinetics of diffusion state transitions and correspondence to struc- mediating the changes in toxin mobility (Fig. 3C). tural states. (A) Transition probability matrix plotted from HMM-based as- signments of transitions between different diffusive states for all time points N-Terminal Helix Interacts with Membrane Cholesterol. = Attenuation for both POPC and POPC:Chol clusters is depicted (n 37,000 for POPC and in the rate of transition from the intramembrane state P1 to the n = 18,000 for POPC:Chol). The major clusters representing transitions from monomer, M, to intramembrane, P1, and between P1 and P2 states and vice M state in the absence of cholesterol suggested that the P1 state versa are marked. (B) Kinetic scheme of postulated ClyA conformations on was able to sample cholesterol in the membrane. Thus, we hy- the membrane with transition rates (s−1) is depicted. The two membrane pothesized that the membrane-interacting segments (β-tongue domains in the protein (dark gray), namely, N-terminal helix (magenta) and and N-terminal helix) of ClyA may harbor cholesterol interacting β-tongue (yellow), and their locations with respect to the membrane (light motif(s). Upon inspection of the transmembrane segment of the gray) are highlighted. (C) ClyA ISD distribution before (blue) and after N-terminal helix in the protomer form, we identified residues (green) MβCD treatment is shown with solid lines representing the GMM fits that bore strong resemblance to a previously characterized (shaded region indicates SD). cholesterol interaction motif [cholesterol recognition and consensus motif (CRAC)] (29, 30) (SI Appendix, Fig. S9 A and B). the kinetic rates for transition between monomer, M, and However, the loose definition of this consensus sequence ap- −6 −1 pears to result in a large repertoire of proteins comprising these P1 states (kM → P1, POPC = 0.86 ± 3.3 × 10 s and kP1 → M, POPC = −5 −1 residues, many of which may not participate in specific interac- 1.08 ± 1.3 × 10 s ) and interconversion between the P1 and −5 −1 tions with cholesterol. Therefore, site-directed mutagenesis was P2 states were fast (kP1 → P2, POPC = 1.2 ± 1.5 × 10 s and −5 −1 performed to mutate two residues that are reported as critical to k → = 0.7 ± 1.0 × 10 s ). However, low rates of P2 P1, POPC cholesterol interaction in the CRAC motif. ClyA Y27AK29A direct conversion between M and P2 implied that M ↔ P1 ↔ P2 is was severely compromised in cell lysis activity and vesicle leakage the dominant pathway (Fig. 3 A and B and SI Appendix,TableS2). ↔ (Fig. 4A and SI Appendix, Fig. S9C). The distribution of ISDs of Therefore, the M P2 transitions were ignored in further analysis. ClyA Y27AK29A on POPC:Chol membranes displayed a sig- Inthepresenceofcholesterol,therateforP1→ Mconversion = ± × −6 −1 nificant fraction of fast moving molecules resembling the profile decreased approximately sixfold (kP1 → M, Chol 0.18 2 10 s ) observed with wild-type ClyA in membranes devoid of choles- without significant changes in the rates of conversion between the terol (Fig. 4B). Lysis experiments with single mutations of other states. The cumulative and most significant effect of these ClyAK29A, Y27A, and Y27F established that central Tyr-27 was ∼ changes in rates is a net increase ( 10-fold) in the formation of the the key determinant for cholesterol interaction (SI Appendix, Fig. P1 state in the presence of cholesterol. Thus, cholesterol affects the S9D). Therefore, the N-terminal helix interacted with cholesterol kinetics of transitions between the ClyA states by stabilizing the first via specific residues, which stabilized the P1 state, and P1 and M intermediate (P1), postmembrane binding. mobility species were indeed two distinct conformations, with or At the low concentrations used, ClyA did not oligomerize, nor without a membrane-inserted N-terminal helix, respectively (Fig. were there changes in membrane fluidity or the presence of 3B). Because the conformational transition of ClyA has been membrane domains in the two membrane systems used (SI Ap- demonstrated to be the rate-limiting step in the assembly path- pendix, Fig. S8), ruling them out as factors inducing changes in way even at much higher concentrations of the protein (10, 28), ClyA mobility states. We therefore surmised that the lowered cholesterol-induced structural stabilization will operate at phys- mobility state (P1) was a consequence of specific protein–lipid iological concentrations of ClyA and promote lytic activity. interactions. Based on order of appearance of the diffusive states To further ascertain the nature and extent of interaction be- upon membrane binding and consistent with the structural data tween ClyA and cholesterol, we conducted all-atom, molecular (8), we assigned the diffusive states M and P1 as arising from two dynamics (MD) simulations of the single ClyA protomer mod- distinct conformations of ClyA, i.e., a β-tongue only and a eled from the full pore in 70% DOPC and 30% cholesterol bi- β-tongue with N-terminal helix membrane-inserted forms (Fig. layers with explicit water for a duration of 0.9 μs(SI Appendix, 3B). M and P1 states structurally resembled peripheral and Fig. S10A). Cholesterol molecules showed a distinctly high oc- transmembrane protein conformations, and their mobilities cupancy around the CRAC motif residues in contrast to the rest compared well with those reported for peripheral membrane of the membrane-inserted α-helix (Fig. 4C and SI Appendix, Fig.

E7326 | www.pnas.org/cgi/doi/10.1073/pnas.1721228115 Sathyanarayana et al. Downloaded by guest on October 6, 2021 ABwere detected), the fraction of particles with more than one photobleaching step was significantly higher in the presence of cholesterol (Fig. 5A and SI Appendix, Fig. S11B). This showed that cholesterol played a role in promoting formation of higher- order ClyA oligomers. To examine the molecular interactions of ClyA with cholesterol that manifests in enhanced assembly, we conducted MD simulations (0.6–0.8 μs) of the dodecameric pore (Fig. 5B and SI Appendix, Fig. S12) and a ClyA dimer (SI Appendix, Fig. S13A)in the protomer conformation in cholesterol membranes. In-plane CDmobility maps revealed a dramatically reduced mobility (and hence stronger binding) for cholesterol in the immediate vicinity of the pore complex (Fig. 5C). This was surprising because the cholesterol-interacting residues in the N-terminal helix now formed part of the pore lumen and were inaccessible to cholesterol. This behavior was also observed for the ClyA dimer, suggesting a crucial role of cholesterol in stabilizing the ClyA oligomers (SI Appendix, Fig. S13B and Table S4). Cholesterol was now observed to interact with the pocket flanked by adjacent residues of the neighboring β-tongues (Fig. 5 B and D). The cholesterol occupancy plots with the β-tongues revealed the pres- Fig. 4. Cholesterol interactions with the amino terminus of ClyA. (A)Eryth- ence of a dominant binding site at the dimer interface that harbors rocyte lysis activity (OD620) for ClyA (500 nM, black) and ClyA Y27AK29A K206, D171, and K175 at the top of the pocket, a highly hydro- μ (500 nM, purple, and 10 M, red and *) with Boltzmann sigmoid fits (lines) is phobic cavity (lined with I198, I194, and L192 at the bottom shown. Cell lysis t1/2 for ClyA (500 nM) and ClyA Y27AK29A* (10 μM) is shown in Inset. Error bars represent SD. (B) GMM fit to ISD distribution (at 5 min) for and G201, V202, A179, G180, and A183 in the middle) with Y178 providing stacking interactions. The cholesterol moiety ClyA Y27AK29A mutant (solid brown line) and ClyA (dashed blue line) in POPC: ∼ Chol (shaded region represents SD). (C) Fractional cholesterol occupancy values spent 97% of the simulation time in the dimer pocket and from MD simulations of the ClyA protomer are shown for the transmembrane sampled two dominant orientations with shared interactions N-terminal helix (Right)aswellastheβ-tongue region (Left). (D) Cholesterol between the two adjacent β-tongues (Fig. 5D and SI Appendix, lateral mobility (from MD) calculated is plotted near the ClyA protomer (top Fig. S13C). Both the membrane-inserted protomer and dimer view). Only the membrane-residing αA1 N-terminal helix (magenta) and β-tongue were found to form a distinct transmembrane water channel region (yellow) segments are shown for clarity. solvating the hydrophilic residues of the N terminus (SI Ap- pendix,Fig.S14), signifying the onset of pore formation. S10B and Table S3). A preferred propensity was observed at Discussion residues D25 and K29 highlighted by cholesterol hydroxyl group e PFT assembly has been the subject of growing investigation to interactions with the carboxylate oxygen of D25 and -amino understand protein conversion from soluble forms to stable group of K29 side chains (Fig. 4C). In-plane mobility maps based membrane-integrated structures, especially considering the re- on the displacement of cholesterol molecules revealed a signifi- semblance of their mechanism of action to amyloid proteins (35). cantly reduced mobility of the cholesterol molecules in the vicinity Target membranes do not serve merely as passive scaffolds for of the N-terminal helix further supporting direct cholesterol adsorption of PFTs from solution but also contain determinants interaction (Fig. 4D). High cholesterol occupancy and reduced that are essential for their function. Investigating PFT assembly – mobility of cholesterol were also observed near the residues (E204 in detergent solution, although useful for determining oligo- – β K206) that lie close to the lipid water interface in the -tongue, merized structures, fails to assess both the modulatory role indicating the possibility of distinct secondary site for cholesterol played by lipids and the influence of protein dynamics on the interaction (Fig. 4 C and D and SI Appendix,Fig.S10C). membrane surface that impinge on the assembly and kinetic pathways. Here we elucidate the mechanistic basis for stimula- Assembly of the ClyA Pore Is Enhanced by Cholesterol. Because the tion of toxin activity by membrane cholesterol. dodecameric ClyA pores can be assembled in detergents and E. Membrane specificity in pore-forming activity can be achieved coli (14, 31), it is apparent that cholesterol is not essential for the by molecular determinants, interactions with heterogeneous ClyA pore assembly. Compared with kinetic analysis in deter- membrane domains, or global physicochemical properties of the gents (9), our results indicate an enhanced rate of pore forma- host membrane (1). Cholesterol, a major component in the tion in membranes, which is further stimulated by cholesterol plasma membranes of mammalian cells (36), plays complex roles (Fig. 1 B and C). Although the presence of membrane choles- that includes stabilizing the plasma membrane and enhancing terol is essential for stabilization of the transmembrane helix, it several membrane–protein interactions both as an individual may also affect oligomerization. We examined this by inducing cofactor and as part of selectively enriched microdomains (37, oligomerization of ClyA on the bilayers by employing concen- 38). Pathogens can employ interaction with eukaryotic mem- trations of the toxin (10 nM) comparable to those used for brane components like cholesterol in various ways for recogni-

erythrocyte lysis. ClyA molecules initially bound homogeneously tion. For example, in the case of cholesterol-dependent cytolysin BIOPHYSICS AND to the supported bilayer and then reorganized to form brighter β

family of -PFTs, binding to a membrane cholesterol is ac- COMPUTATIONAL BIOLOGY spots and large punctate features (SI Appendix, Fig. S11A). complished by a conserved tryptophan-rich loop motif in the Diffraction-limited ClyA complexes were quantified for fluo- D4 domain which allosterically activates distal domains essential rophore tags present per spot, by estimating the number of for toxin assembly (39, 40). Our findings reveal the role of photobleaching steps (32–34). Compared with a single photo- cholesterol in modulating the diffusional dynamics, structural bleaching step observed for the ClyA monomer, the ClyA puncta states, and assembly for the amphipathic and lesser understood consistently displayed a higher number of photobleaching steps α-PFTs and thereby enhancing PFT activity. that increased with time, indicating formation of ClyA oligomers. Analysis of single-molecule diffusion kinetics on supported At equilibrium (∼45 min, beyond which no significant changes lipid bilayers allowed us to discern conformational heterogeneity

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Fig. 5. Role of cholesterol in ClyA oligomerization on bilayers. (A) Distribution of the photobleaching steps for ClyA monomer (gray, n = 5,000) and assembled ClyA particles on POPC (green, n = 16,000) and POPC:Chol (blue, n = 15,250) is plotted. The fraction of multiple (>1) photobleaching steps is shown in Inset (same colors). Error bars represent SD. (B) Side view of the pore in membrane. Blue and red spheres represent phosphocholine and cholesterol head groups, respectively. Selected cholesterol molecules (with atoms within 0.5 nm of β-tongue) in upper leaflet are represented with an orange space-filling model (colors are the same as in Fig. 4D). (C) Cholesterol mobility map of the full ClyA pore in DOPC with 30% cholesterol is superimposed with membrane segments of the ClyA (colors are the same as in Fig. 4D). (D) Snapshots of the cholesterol moiety (yellow, hydroxyl head group in red) in the protomer– protomer interface formed between the β-tongues (two subunits colored in orange and blue) in the two major conformations (Top, at 100 ns, and Bottom, 300 ns of simulation time) for the ClyA dimer are shown. The residues in cholesterol pocket defined by charged side chains (K206 and D171) at the top, a hydrophobic interior (V202, A179, and A183), and isoleucine-rich tail (I194 and I198) region as well as residue Y27 from the N-terminal helix are highlighted.

of an α-PFT, Cytolysin A, that arises due to large structural The specific interactions described above allow us to reconcile rearrangements on the membrane. Post-membrane binding (us- the observation of pore-like structures of ClyA on E. coli OMVs. ing the β-tongue), membrane insertion of the N-terminal helix The absence of cholesterol in bacterial membranes limits the in- reduces the lateral mobility of the ClyA protein. We propose that sertion of N-terminal helices, thereby resulting in complexes that do this heterogeneous diffusion may be a signature feature in α-PFTs, not puncture the membrane. This would explain the nontoxicity of due to the obligate requirement for conformational change be- the oligomeric structures toward E.coli (14).However,OMVs fore pore formation which has been previously reported for containing oligomeric ClyA have been shown to be highly toxic to equinatoxin II (41). Furthermore, presence of cholesterol led to eukaryotic cells (14), perhaps due to interaction with cholesterol in target cells upon fusing to host membrane. conformational selection of the membrane-inserted protomer-like Until now, the cholesterol binding motifs have been primarily form, a key structural intermediate in the assembly pathway. MD found to be associated with transmembrane helices of mammalian simulations demonstrated that this was mediated by interactions with integral membrane proteins. Presence of specific cholesterol in- a cholesterol recognition motif in the N-terminal helix of ClyA. This teraction sites in a bacterial toxin reinforces our argument that the bore strong similarities to CRAC motifs that have been widely ob- motif could have evolved as a mechanism for selective targeting served and characterized in many eukaryotic transmembrane proteins of eukaryotic membranes. Mechanistic similarities among PFTs as (42–46). A similar role of specific lipids in membrane partitioning of well as other membrane proteins, such as viral envelope proteins the N-terminal transmembrane helix for actinoporins, equinatoxin II, and amyloid proteins (1, 35, 49), suggests that similar lipid-specific and fragaceatoxin C (FraC) has been demonstrated (47, 48), sug- protein interactions, which stabilize transmembrane protein in- gesting conservation of membrane insertion mechanisms among the sertion and oligomerization, might serve as a broad strategy for α-PFT family of proteins. achieving cell selectivity. In addition, a detailed understanding of In addition to stabilizing the N-terminal inserted state, MD the pore formation process at the spatiotemporal scales of protein simulations revealed that cholesterol played an important role in assembly and intermolecular interactions could potentially provide the ensuing stages of oligomerization by preferentially binding to a a path for developing novel drug targets that compromise pore previously uncharacterized pocket formed between two adjacent formation on target membranes and a potential route to mitigating β-tongues. Therefore, cholesterol plays a dual role: first in confor- rising bacterial virulence. mational selection of the N-terminal helix-inserted form and second Materials and Methods in stabilization of the assembled oligomers. Bridging lipids were also Erythrocyte Lysis Assay. For kinetic measurements of erythrocyte lysis, ClyA observed to stabilize the pore structure of FraC (5), hinting at variants were incubated with 200 μL of 1% suspension (vol/vol) of rabbit conserved modes of dual stabilization of intermediates among erythrocytes in phosphate buffered saline (PBS) at indicated concentrations. members of the α-PFT class. Turbidity was monitored by measuring optical density at 620 nm using a

E7328 | www.pnas.org/cgi/doi/10.1073/pnas.1721228115 Sathyanarayana et al. Downloaded by guest on October 6, 2021 microplate reader (Tecan) with time, with intermittent orbital shaking at Fraction and mobility of discrete diffusive states that are sampled by single 37 °C. Lysis data obtained were fit to a Boltzmann sigmoid function to ex- molecules were obtained by analysis of their squared displacements (52).

tract half-life (t1/2). Instantaneous squared displacements (ISD) were calculated as Erythrocyte shaving of membrane surface exposed protein fragments was 2 2 2 achieved by incubation with 0.6 units of Proteinase K at 37 °C for 1 h. Re- rðτÞ = ðXt+τ − Xt Þ + ðYt+τ − Yt Þ , action was terminated by addition of PMSF to a final concentration of 2 mM, τ and the erythrocytes were washed with PBS and diluted to a final 1% (vol/vol) where is the lag time interval. 2 suspension for the hemolysis assay. Cholesterol depletion in rabbit erythro- The probability density distribution of squared displacements (r ) for time cytes was carried out by incubation of 1% (vol/vol) suspension of RBCs with interval, τ, for a Brownian particle motion with diffusion coefficient, D,is 1mMmethyl-β-cyclodextrin for 20 min at 25 °C. Cells were then washed with given by PBS and taken for lysis assay. All animal experiments were approved by the

Institutional Animal Ethics Committee of the Indian Institute of Science. − r2 r2 ðτÞ À Á e 0 f r2, τ dr2 = , 2ðτÞ PEG-Supported Lipid Bilayer. PEG-SLBs were synthesized as described earlier r0 (50) with some modifications. PEG-SUVs (SI Appendix, SI Methods) were di- where r2ðτÞ = 4Dτ. luted 1:1 in PBS (containing 3 mM CaCl2). Twenty μL of this vesicle solution 0 was introduced into the chamber using a micropipette. The channel as- Integration of the equation above yields the cumulative distribution sembly was incubated at 37 °C for 1 h in a humidifying chamber. The function channels were washed with PBS to remove unfused vesicles. Bilayer coverage and fluidity of PEG-SLBs were assessed by confocal imaging and fluorescence À Á − r2 r2 ðτÞ recovery after photobleaching (SI Appendix). F r2, τ = 1 − e 0 .

Fluorescence Microscope Setup. The single-particle experiments were per- For a system with three distinct diffusive populations, the equation can be formed on an inverted microscope (Olympus IX81). A 532-nm laser (Sap- rewritten as phire; Coherent) was used to excite the Cy3-labeled ClyA Q56C molecules on a custom-built objective-type total internal reflection microscope. A À Á − r2 − r2 − r2 r2 ðτÞ r2 ðτÞ r2 ðτÞ combination of 25.4- and 300-mm biconvex lenses (Thor Laboratories) were F r2, τ = αe 1 + βe 2 + ½1 − ðα + βÞe 3 , used to expand the laser beam before a lens of focal length 150 mm was α β − α + β 2ðτÞ used to focus the (16-mW) laser beam on its back focal plane (BFP) of the where , , and [1 ( )] are the fractions of diffusive species with r1 , 2ðτÞ 2ðτÞ τ objective (UAPON 100× OTIRF; Olympus). The laser spot at the BFP was r2 , and r3 as their mean squared displacements at time lag, ,re- translated away from the optical axis to achieve total internal reflection. spectively. This expression was used to fit an empirical cumulative distribu- Fluorescence emission from 80 μm × 40 μm area was collected by the objective tion function of the squared displacements to obtain the mean squared and passed through a dichroic mirror (FF545/650-Di01-25 × 36; Semrock) and displacements and relative fractions of the three underlying components. All a long-pass filter (BLP02-561R-23.3-D; Semrock) before detection on an of the analyses were performed using custom scripts in MATLAB (Math- electron multiplying charge-coupled device (Andor ixon Ultra 897). Shutter works). Gaussian mixture modeling of the log transformed squared dis- (LS6; Vincent Associates) was used to control the laser illumination time. placements was carried out using custom scripts in MATLAB. A total of 1,000 optimization iterations were carried out, and full covariance matrices ClyA Interaction with SLB Membrane. For real-time binding to SLBs, ClyA was were specified for the modeling. In addition, regularization was performed diluted to the working concentration in PBS containing 0.1 mg/mL BSA and to prevent ill-conditioned covariance estimates. was introduced into the channel (containing the SLB) using a syringe pump (NewEra) at a flow rate of 50 μL/min. Image sequences (500–2,000 frames) Molecular Dynamics Simulations. Single-protomer and dimer structures were were acquired with a 25-ms exposure time immediately after flow into the extracted from the dodecameric structure of Cytolysin A pore (Protein Data microchannel and subsequently at different times over a time period of Bank id: 2WCD) (8), and the complete dodecamer was used as is, for carrying 30 min. All experiments were conducted at 25 °C. For experiments involving out MD simulations. Initial structures were generated by placing the pro- the depletion of cholesterol, ClyA was first incubated for a period of 5 min teins (protomer, dimer, and pore) in a DOPC/Cholesterol (70:30) lipid bilayer (to reach equilibrium) with POPC:Cholesterol SLBs. Subsequently, the bilayer using the Chemistry at Harvard Macromolecular Mechanics-Graphical User was incubated with 10 mM methyl-β-cylclodextrin at 25 °C for 40 min to Interface (CHARMM-GUI) membrane builder (53). The system was solvated deplete cholesterol followed by washes with PBS before imaging and ana- using TIP3P water, and sodium and chloride ions were added for electro- lyzing single-particle tracks. neutrality. The full pore simulation was carried out at 0.15 M salt concentration. AMBER99SB-ILDN force field (54) with φ corrections (55) was used to Particle Tracking and Analysis of Squared Displacements. Particle detection capture the dynamics of ClyA with the Slipids force fields for the DOPC and – and tracking was performed using u-track MATLAB package on the image cholesterol (56 58). Complete details of the simulations and parameters can sequences acquired (51). Briefly, a 2D Gaussian fit with an SD of one pixel be found in SI Appendix. was performed on the diffraction-limited particles to estimate the subpixel location and intensity (after background correction). A gap length of zero ACKNOWLEDGMENTS. We thank Benjamin Schuler for sharing the ClyA 56C frames was specified for tracking, and particles having a lifetime of fewer plasmid and Sunaina Banerjee, Subbarao Kanchi, Sreenath Balakrishnan, Ayush than three frames were discarded from analysis. Agrawal, Rajat Desikan, and Aravind Penmatsa for reagents, technical assis- tance, and discussions. This work was supported by a Department of Science and For calculating the rate of binding of ClyA to the SLBs, number of particles in Technology-Intensification of Research in High Priority Area (DST-IRHPA) grant μ 2 each frame as detected by u-track was normalized for a unit m area. This was (to K.G.A. and S.S.V.), Department of Biotechnology-Innovative Young Biotech- fit to a single exponential distribution for estimating the apparent rate of in- nologist Award (DBT-IYBA) and DST grants (to R.R.), and computational facilities teraction of ClyA with the membrane. The initial rate (slope at t = 0) was used to [Supercomputer Education and Research Centre (SERC)] at Indian Institute of calculate the rate of ClyA binding. Science (IISc). P.S., S.M., and A.B. are supported by fellowships from IISc.

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