Supporting Information Wade et al. 10.1073/pnas.1423754112 Materials and Methods alone (soluble) or after a 20-min incubation at 37 °C with 1-pal- Primary Sequence Alignment and Cluster Analysis. Sequence align- mitoyl-2-oleoyl-sn-glycero-3-phosphocholine/cholesterol liposomes ment of the CDC primary structures was performed using ClustalW (20 μL) to allow assembly of the oligomer. For all experiments, (1), and the phylogenetic trees were derived from a maximum emission scans of the unlabeled soluble or liposome-bound toxin parsimony analysis of the ClustalW-derived alignments, using were measured and subtracted from the experimental data to Mega6geneanalysissoftware(2). eliminate any changes in fluorescence not resulting from the NBD probe. An excitation wavelength of 480 nm and 4 nm bandpass Steady-State Fluorometry. Steady-state experiments were performed were used. Resolution was set to 1 nm with 0.1 s integration. on a Fluorolog-3 Spectrofluorometer with the FluorEssence soft- β4β5 disengagement was measured as previously described (3) ware. The polarity of the environment surrounding Cys-substituted for PFOV322C or PFOV322C:Y181A, where a 1:1 molar ratio of PFOE183C or PFOK336C (176 pmol total: 25% NBD labeled, 75% NBD-labeled to unlabeled protein was used to minimize NBD unlabeled) was determined by performing an NBD emission scan self-quenching in the oligomeric complex. Settings for measuring (500–600 nm) of proteins either in buffer (2.5 mL final volume) NBD emission were set as described above. 1. Larkin MA, et al. (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23(21): 3. Ramachandran R, Tweten RK, Johnson AE (2004) Membrane-dependent conforma- 2947–2948. tional changes initiate cholesterol-dependent cytolysin oligomerization and inter- 2. Tamura K, et al. (2011) MEGA5: Molecular evolutionary genetics analysis using maxi- subunit beta-strand alignment. Nat Struct Mol Biol 11(8):697–705. mum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28(10):2731–2739. Wade et al. www.pnas.org/cgi/content/short/1423754112 1of5 93 Bacillus anthracis (Anthrolysin) E K N Bacillus thuringensis (Thuringiolysin) E K N 100 Clostridium drakei E K N 90 Bacillus weihenstephanensis E K N Bacillus cereus (Cereolysin) E K N Brevibacillus brevis E K N Lysinibacillus sphaericus (Sphaericolysin) E K N 95 Paenibacillus alvei (Alveolysin) E K N Clostridium hydrogeniformans E K N Clostridium lundense E K N Clostridium perfringens (Perfringolysin O) E K N Peptostreptococcaceae bacterium VA2 E K N Clostridium bifermentans E K N 99 Streptococcus urinalis E K N 90 Streptococcus ictaluri E K N 100 Streptococcus didelphis E K N Streptococcus canis E K N 98 Streptococcus pyogenes (Streptolysin O) E K N 100 Streptococcus dysgalactiae subsp. equisimilis (Equisimilysin) E K N 92 Clostridium butyricum E K N Clostridium novyi NT (Novyiolysin I) D K G Clostridium tetani (Tetanolysin) D K G 99 Clostridium botulinum (Botulinolysin) D K G Clostridium novyi NT (Novyiolysin II) D K G Enterobacter lignolyticus* (Enterolysin) E K G Butyrivibrio sp. LB2008 S E D 98 92 99 Desulfobulbus propionicus* (Desulfolysin) S D G Austwickia chelonae E G G Listeria ivanovii (Ivanolysin) Q G G Listeria seeligeria (Seeligeriolysin) D G G 87 100 Listeria monocytogenes (Listeriolysin) D G G 91 100 Listeria innocua D G G Trueperella pyogenes (Pyolysin) E G G 100 Arcanobacterium haemolyticum (Arcanolysin) E G G Streptococcus suis (Suilysin) E G G 100 Streptococcus pneumoniae (Pneumolysin) K G G 100 Streptococcus mitis (Mitilysin) K G G Lactobacillus iners (Inerolysin) T G G Gemella bergeri E G G 100 Gemella cuniculi E G G Streptococcus intermedius (Intermedilysin)** S G G Gardnerella vaginosis (Vaginolysin)** S G G Streptococcus pseudopneumoniae** S G G 100 Streptococcus mitis (Lectinolysin)** S N G Streptococcus tigurinus** S E G Fig. S1. CDC phylogenetic tree. The evolutionary history of the CDCs was inferred using the maximum parsimony method. Tree 1 of the five most parsi- monious trees is shown (length = 3,065). The consistency index is 0.495063, the retention index is 0.673756, and the composite index is 0.336548 for all sites. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (200 replicates) is shown next to the branches (1). The maximum parsimony tree was obtained using the Subtree-Pruning-Regrafting algorithm (2), with search level 1, in which the initial trees were obtained by the random addition of sequences (10 replicates). The analysis involved 45 CDC primary structures. Positions containing gaps and missing data were eliminated. A total of 387 positions were in the final dataset. The alignment of their primary structures was carried out using ClustalW (3), and the evolutionary analyses were conducted in MEGA5 (4). The analogous residues for PFO residues E183, K336, and N197 are shown to the right of the phylogenetic tree. Note that Legend continued on following page Wade et al. www.pnas.org/cgi/content/short/1423754112 2of5 different strains of Streptococcus mitis have been shown to express a pneumolysin-like CDC (5) or a lectinolysin-like (6) CDC. *Gram-negative species. **Proven or predicted to use CD59 instead of cholesterol as their primary receptor based on the conserved YXYX14RS motif in domain 4 (6–8). 1. Felsenstein J (1985) Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39:783–791. 2. Nei M, Kumar S (2000) Molecular Evolution and Phylogenetics (Oxford Univ. Press, New York). 3. Larkin MA, et al. (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23(21):2947–2948. 4. Tamura K, et al. (2011) MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28(10): 2731–2739. 5. Jefferies J, et al. (2007) Identification of a secreted cholesterol-dependent cytolysin (mitilysin) from Streptococcus mitis. J Bacteriol 189(2):627–632. 6. Farrand S, et al. (2008) Characterization of a streptococcal cholesterol-dependent cytolysin with a lewis y and b specific lectin domain. Biochemistry 47(27):7097–7107. 7. Giddings KS, Zhao J, Sims PJ, Tweten RK (2004) Human CD59 is a receptor for the cholesterol-dependent cytolysin intermedilysin. Nat Struct Mol Biol 11(12):1173–1178. 8. Wickham SE, et al. (2011) Mapping the intermedilysin-human CD59 receptor interface reveals a deep correspondence with the binding site on CD59 for complement binding proteins C8alpha and C9. J Biol Chem 286(23):20952–20962. E183C 8×105 Soluble Prepore 6×105 4×105 FI (au) FI 2×105 0 500 520 540 560 580 600 Wavelength (nm) K336C 8×105 Soluble Prepore 6×105 4×105 FI (au) FI 2×105 0 500 520 540 560 580 600 Wavelength (nm) Fig. S2. E183 and K336 make a polar-to-nonpolar transition on prepore formation. PFOE183C and PFOK336C were modified via their Cys sulfhydryls with the environmentally sensitive fluorescent probe NBD (1), and the fluorescence emission was measured for the soluble monomer and the membrane-bound prepore. Each mutant was examined individually with a 10-fold molar excess of the cognate unlabeled mutant to separate the probes in the oligomer (on average, by about 20 nm), thereby minimizing any contribution to the fluorescence change resulting from self-quenching of the NBD probe. 1. Shepard LA, et al. (1998) Identification of a membrane-spanning domain of the thiol-activated pore-forming toxin Clostridium perfringens perfringolysin O: An alpha-helical to beta- sheet transition identified by fluorescence spectroscopy. Biochemistry 37(41):14563–14574. Wade et al. www.pnas.org/cgi/content/short/1423754112 3of5 6 1×10 Soluble 8×105 Liposomes 6×105 5 FI (au) 4×10 2×105 0 500 520 540 560 580 600 Wavelength (nm) Soluble 6 1.2×10 Liposomes 8.0×105 FI (au) 4.0×105 0.0 500 520 540 560 580 600 Wavelength (nm) Fig. S3. Mutation of Y181 in PFO prevents the disengagement of β5 from β4. The disengagement of β5 from β4 can be monitored by the nonpolar-to-polar transition of an NBD probe coupled to the sulfhydryl of Cys-substituted Val-322, which is buried under the β5α1 loop in the PFO monomer (1) (Fig. 1A). As shown, an NBD probe on the Cys sulfhydryl of PFOV322C:Y181A does not undergo the nonpolar-to-polar transition (Bottom) that is observed with PFOV322C (Upper). 1. Ramachandran R, Tweten RK, Johnson AE (2004) Membrane-dependent conformational changes initiate cholesterol-dependent cytolysin oligomerization and intersubunit beta-strand alignment. Nat Struct Mol Biol 11(8):697–705. Wade et al. www.pnas.org/cgi/content/short/1423754112 4of5 D152 (monomer) D152 (oligomer) 2.2 Å 16.1 Å K110 (monomer) K110 (oligomer) Fig. S4. Staphylococcus aureus α-hemolysin exhibits a putative electrostatic trigger. (Left) Dimer from the α-hemolysin oligomeric structure (1) overlaid with the α-hemolysin monomer structure (2). The TMHs (cyan and coral) are shown as folded structures in the monomer and as extended structures in the oligomer. (Right) Close-up of the relative position of K110 in the overlay of soluble monomer with the oligomer pore. The relative position of D152 is also shown in the soluble monomer (green) and the pore complex (yellow). The distance separating K110 and D152 charged centroids (dashed lines) before pore formation is ∼16 Å and is only 2 Å in the oligomeric pore. Mutation of either residue traps α-hemolysin in a stable prepore structure (3, 4). The arrow shows the relative change in position (∼16 Å) of K110 in the overlaid monomer and pore complex. Ribbon structures were created and rendered with UCSF Chimera. 1. Song L, et al. (1996) Structure of staphylococcal alpha-hemolysin, a heptameric transmembrane pore. Science 274(5294):1859–1866. 2. Foletti D, et al.