Stepwise visualization of membrane pore formation by suilysin, a bacterial cholesterol-dependent cytolysin

Figure supplement 1. Symmetry of suilysin prepores and pores, determined by negative-stain EM. DOI:10.7554/eLife.04247.004 Figure supplement 2. Resolution curves for EM maps. DOI:10.7554/eLife.04247.005 Figure supplement 3. Electrostatic potential maps and interacting residues. DOI:10.7554/eLife.04247.006 Figure supplement 4. Comparison between prepore and crystal structure conformations. DOI:10.7554/eLife.04247.007 Accession codes for EM maps: prepore EMD-12698, pore EMD-12711. ; Membrane attack complex/perforin/cholesterol-dependent cytolysin (MACPF/CDC) proteins constitute a major superfamily of pore-forming proteins that act as bacterial virulence factors and effectors in immune defence. Upon binding to the membrane, they convert from the soluble monomeric form to oligomeric, membrane-inserted pores. Using real-time atomic force microscopy (AFM), electron microscopy (EM), and atomic structure fitting, we have mapped the structure and assembly pathways of a bacterial CDC in unprecedented detail and accuracy, focussing on suilysin from Streptococcus suis. We show that suilysin assembly is a noncooperative process that is terminated before the protein inserts into the membrane. The resulting ring-shaped pores and kinetically trapped arc-shaped assemblies are all seen to perforate the membrane, as also visible by the ejection of its lipids. Membrane insertion requires a concerted conformational change of the monomeric subunits, with a marked expansion in pore diameter due to large changes in subunit structure and packing. ; BBSRC (BB/G011729/1, BB/J005932/1, BB/J006254 and BB/ K01692X/1), the ERC (advanced grant 294408), and the Leverhulme Trust (RPG-2012-519). ; Peer-reviewed ; Publisher Version