title: Purification and three-dimensional electron microscopy structure of the Neisseria meningitidis type IV pilus biogenesis protein PilG.
abstract: Type IV pili are surface-exposed retractable fibers which play a key role in the pathogenesis of Neisseria meningitidis and other gram-negative pathogens. PilG is an integral inner membrane protein and a component of the type IV pilus biogenesis system. It is related by sequence to the extensive GspF family of secretory proteins, which are involved in type II secretion processes. PilG was overexpressed and purified from Escherichia coli membranes by detergent extraction and metal ion affinity chromatography. Analysis of the purified protein by perfluoro-octanoic acid polyacrylamide gel electrophoresis showed that PilG formed dimers and tetramers. A three-dimensional (3-D) electron microscopy structure of the PilG multimer was determined using single-particle averaging applied to samples visualized by negative staining. Symmetry analysis of the unsymmetrized 3-D volume provided further evidence that the PilG multimer is a tetramer. The reconstruction also revealed an asymmetric bilobed structure approximately 125 A in length and 80 A in width. The larger lobe within the structure was identified as the N terminus by location of Ni-nitrilotriacetic acid nanogold particles to the N-terminal polyhistidine tag. We propose that the smaller lobe corresponds to the periplasmic domain of the protein, with the narrower "waist" region being the transmembrane section. This constitutes the first report of a 3-D structure of a member of the GspF family and suggests a physical basis for the role of the protein in linking cytoplasmic and periplasmic protein components of the type II secretion and type IV pilus biogenesis systems.
authors.affiliation: Faculties of Life Sciences and Engineering/Physical Sciences, Manchester Interdisciplinary Biocentre, University of Manchester, 131 Princess Street, Manchester, United Kingdom.
authors.forename: Richard F
authors.initials: RF
authors.lastname: Collins
authors.forename: Muhammad
authors.initials: M
authors.lastname: Saleem
authors.forename: Jeremy P
authors.initials: JP
authors.lastname: Derrick
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000648:ultrastructure*; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D036781:Fimbriae Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000648:ultrastructure*; D015870:Gene Expression; D007091:Image Processing, Computer-Assisted; D046529:Microscopy, Electron, Transmission; D008958:Models, Molecular; D009243:NAD / Q000378:metabolism; D016624:Negative Staining; D009345:Neisseria meningitidis / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D019289:Pyruvic Acid / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000648:ultrastructure
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D009243:NAD; D036781:Fimbriae Proteins; D019289:Pyruvic Acid