title: Measuring kinetic drivers of pneumolysin pore structure.
abstract: Most membrane attack complex-perforin/cholesterol-dependent cytolysin (MACPF/CDC) proteins are thought to form pores in target membranes by assembling into pre-pore oligomers before undergoing a pre-pore to pore transition. Assembly during pore formation is into both full rings of subunits and incomplete rings (arcs). The balance between arcs and full rings is determined by a mechanism dependent on protein concentration in which arc pores arise due to kinetic trapping of the pre-pore forms by the depletion of free protein subunits during oligomerization. Here we describe the use of a kinetic assay to study pore formation in red blood cells by the MACPF/CDC pneumolysin from Streptococcus pneumoniae. We show that cell lysis displays two kinds of dependence on protein concentration. At lower concentrations, it is dependent on the pre-pore to pore transition of arc oligomers, which we show to be a cooperative process. At higher concentrations, it is dependent on the amount of pneumolysin bound to the membrane and reflects the affinity of the protein for its receptor, cholesterol. A lag occurs before cell lysis begins; this is dependent on oligomerization of pneumolysin. Kinetic dissection of cell lysis by pneumolysin demonstrates the capacity of MACPF/CDCs to generate pore-forming oligomeric structures of variable size with, most likely, different functional roles in biology.
authors.affiliation: Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford, OX3 7BN, UK. gilbert@strubi.ox.ac.uk.
authors.forename: Robert J C
authors.initials: RJ
authors.lastname: Gilbert
authors.affiliation: European Molecular Biology Laboratory, Structural and Computational Biology Unit, Meyerhofstraße 1, 69117, Heidelberg, Germany.
authors.forename: Andreas F-P
authors.initials: AF
authors.lastname: Sonnen
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D004912:Erythrocytes / Q000378:metabolism; D007700:Kinetics; D016062:Porosity; D012756:Sheep; D013301:Streptolysins / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D013301:Streptolysins; C032290:plY protein, Streptococcus pneumoniae
keywords: Kinetics; MACPF/CDC; Membrane structure; Oligomerization; Pore formation; Toroidal pore