Volume 113, Issue 9, Pages 2029-2036 (November 2017) Lysenin Toxin Membrane Insertion Is pH-Dependent but Independent of Neighboring Lysenins  Ignacio L.B. Munguira, Hirohide Takahashi, Ignacio Casuso, Simon Scheuring  Biophysical Journal  Volume 113, Issue 9, Pages 2029-2036 (November 2017) DOI: 10.1016/j.bpj.2017.08.056 Copyright © 2017 Biophysical Society Terms and Conditions

Figure 1 Lysenin toxin membrane insertion is pH dependent. (a) HS-AFM movie frames of lysenin on an SM/Chol (1:1) bilayer (Movie S1). During HS-AFM movie acquisition, the pH is changed from 7.5 to <6.0, leading to an increased number of prepore lysenins (bright circles, protruding 5 nm) that insert into the membrane to become pore lysenins (dark circles, protruding ∼2.5 nm). (b) Graph of the number of prepores (i.e., image area occupied by pixels higher than the 3.75 nm threshold) as a function of movie acquisition time (black circles). The precise number of prepores and pores has been analyzed by cross-correlation searches at six specific time points (dashed lines) corresponding to the frames shown in (a). The average value of the SD maps shown in (c) reports about the topographical changes, i.e., the lateral mobility between subsequent frames, throughout movie acquisition. (c) SD maps over five consecutive frames (t − 4 s to t, where t is the time of recording the topography frames shown in a). To see this figure in color, go online. Biophysical Journal 2017 113, 2029-2036DOI: (10.1016/j.bpj.2017.08.056) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 2 Neighbors of lysenin pores. (a) HS-AFM frame displaying a mixture of prepore (bright circles) and pore (dark circles) lysenins. (b) Voronoi tessellation of the lysenins distributed in the area outlined by a dashed rectangle in (a). The Voronoi cells that are occupied by pore lysenins are in blue and those occupied by prepores are in red. (c) Detailed view of the Voronoi tessellation in the area outlined by a dashed rectangle in (b). Each Voronoi cell is characterized by three parameters: 1) its identity as a pore (blue, “pore”) or prepore (red, “pre”), 2) the number of pore neighbors (top number), and 3) the number of total neighbors (bottom number). (d) Three high-resolution HS-AFM image frames (at t = 439, t = 614, and t = 911 s) displaying an increasing number of pores, a decreasing number of prepores, and a decreasing total number of lysenins in the preserved image area. (e) Histograms of the number of pore neighbors for each pore that has five (blue), six (red), or seven (green) total neighbors. The three panels from left to right correspond to the three analyzed frames shown in (d). The black circles and lines correspond to binomial distributions with the same number of pores and prepores. (f) Summary of the results showing the experimental and binomial average numbers of pore neighbors of pores. To see this figure in color, go online. Biophysical Journal 2017 113, 2029-2036DOI: (10.1016/j.bpj.2017.08.056) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 3 Lysenin prepore and pore structures and position of the putative pH sensor. (a) Top views of the lysenin prepore and pore structures and the corresponding molecular surfaces (smaller images). The prepore model shown here was assembled from the monomeric soluble structure (PDB: 3ZXD) aligned to the pore structure (PDB: 5GAQ) by their receptor-binding domains (residues 160–297), which are essentially identical (root mean-square deviation, 0.47 Å; 908 atoms). (b) Side views of (a). The difference in height between the models of the prepore and the pore is ∼1.5 nm ((9)). In the HS-AFM topographs, the height difference is ∼2.5 nm, indicating that the membrane insertion might be slightly different from what the detergent shell in the cryo-EM structure suggests (9). (c) View of the prepore model superimposed on the pore aligned by their structurally unchanged receptor-binding domains. Top inset: The pH sensor, the small helix comprising Glu92, Glu94, and Glu97, is exposed to the inner cylinder face of the prepore structure. Bottom inset: In the pore structure, Glu92, Glu94, and Glu97 are located about in the middle of the 97-Å-long β-barrel. To see this figure in color, go online. Biophysical Journal 2017 113, 2029-2036DOI: (10.1016/j.bpj.2017.08.056) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 4 EQ-lysenin is pH insensitive. (a) HS-AFM movie frames of EQ-lysenin on an SM/Chol (1:1) bilayer (Movie S3). During HS-AFM movie acquisition, the pH was changed from 7.5 to 4.3, without a significant effect on the membrane insertion of the EQ-lysenin prepores. (b) Graph of the number of prepores (i.e., image area occupied by pixels higher than the 3.75 nm threshold) as a function of movie acquisition time. The precise number of prepores and pores has been analyzed by cross-correlation searches at six specific time points (dashed lines) corresponding to the frames shown in (a). To see this figure in color, go online. Biophysical Journal 2017 113, 2029-2036DOI: (10.1016/j.bpj.2017.08.056) Copyright © 2017 Biophysical Society Terms and Conditions