Abstract
Limited proteolysis, secondary structure and biochemical analyses, mass spectrometry, and mass measurements by scanning transmission electron microscopy were combined with cryo-electron microscopy to generate a three-dimensional model of the homomultimeric complex formed by the outer membrane secretin PulD, an essential channel-forming component of the type II secretion system from Klebsiella oxytoca. The complex is a dodecameric structure composed of two rings that sandwich a closed disc. The two rings form chambers on either side of a central plug that is part of the middle disc. The PulD polypeptide comprises two major, structurally quite distinct domains; an N domain, which forms the walls of one of the chambers, and a trypsin-resistant C domain, which contributes to the outer chamber, the central disc, and the plug. The C domain contains a lower proportion of potentially transmembrane beta-structure than classical outer membrane proteins, suggesting that only a small part of it is embedded within the outer membrane. Indeed, the C domain probably extends well beyond the confines of the outer membrane bilayer, forming a centrally plugged channel that penetrates both the peptidoglycan on the periplasmic side and the lipopolysaccharide and capsule layers on the cell surface. The inner chamber is proposed to constitute a docking site for the secreted exoprotein pullulanase, whereas the outer chamber could allow displacement of the plug to open the channel and permit the exoprotein to escape.
Highlights
Tel.: 33-145688494; Fax: 33-145688960; E-mail: max@pasteur.fr. 3 The abbreviations used are: T2SS, type II secretion systems; HPLC, high performance liquid chromatography; STEM, scanning transmission electron microscope; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. Tures similar to those of type IV pilins [7]) and an integral outer membrane protein called secretin. Besides their role in protein secretion by the T2SS (e.g. Klebsiella oxytoca protein PulD [8] and Pseudomonas aeruginosa protein XcpQ [9, 10]) and the type III secretion system (e.g. Yersinia enterocolitica protein YscC [10]), secretins are required for filamentous bacteriophage secretion (e.g. bacteriophage f1 protein pIV [11]) and type IV pilus assembly (e.g. Neisseria meningitidis and P. aeruginosa PilQ [9, 12])
A low resolution three-dimensional structure of a purified complex of the pullulanase T2SS secretin PulD and its pilotin, PulS, revealed a cylindrical complex with 12-fold symmetry and a central open channel of about 7 nm encircled by radial spokes that we originally presumed to be the pilotin, which is absent from pIV and PilQ [16]
The model we propose here for the secretin PulD complex is based on biochemical analysis and cryo-electron microscopy of particles adsorbed from zwitterionic detergent solution onto carbon films
Summary
The pullulanase T2SS of K. oxytoca is one of the most extensively studied secretons and has been completely reconstituted in E. coli. A low resolution three-dimensional structure of a purified complex of the pullulanase T2SS secretin PulD and its pilotin, PulS, revealed a cylindrical complex with 12-fold symmetry and a central open channel of about 7 nm encircled by radial spokes that we originally presumed to be the pilotin, which is absent from pIV and PilQ [16]. We report refined biochemical and structural analyses of intact and proteolyzed PulD multimers
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