ABSTRACT
Bovine V-ATPase from brain clathrin-coated vesicles was investigated by cryo-electron microscopy and single particle analysis. Our studies revealed great flexibility of the central linker region connecting V1 and V0. As a consequence, the two sub-complexes were processed separately and the resulting volumes were merged computationally. We present the first three-dimensional (3D) map of a V-ATPase obtained from cryo-electron micrographs. The overall resolution was estimated 34A by Fourier shell correlation (0.5 cutoff). Our 3D reconstruction shows a large peripheral stalk and a smaller, isolated peripheral density, suggesting a second, less well-resolved peripheral connection. The 3D map reveals new features of the large peripheral stator and of the collar-like density attached to the membrane domain. Our analyses of the membrane domain indicate the presence of six proteolipid subunits. In addition, we could localize the V0 subunit a flanking the large peripheral stalk.
Subject(s)
Brain/enzymology , Vacuolar Proton-Translocating ATPases/ultrastructure , Animals , Catalytic Domain , Cattle , Cryoelectron Microscopy , Holoenzymes/chemistry , Protein ConformationABSTRACT
The iron-limitation-inducible protein FrpB of Neisseria meningitidis is an outer-membrane-localized siderophore receptor. Because of its abundance and its capacity to elicit bactericidal antibodies, it is considered a vaccine candidate. Bactericidal antibodies against FrpB are, however, type-specific. Hence, an FrpB-based vaccine should comprise several FrpB variants to be capable of providing broad protection. To facilitate the development of a meningococcal subunit vaccine, we have established a procedure to obtain large quantities of the protein in a native-like conformation. The protein was expressed without its signal sequence in Escherichia coli, where it accumulated in inclusion bodies. After in vitro folding, the protein was biochemically, biophysically and biologically characterised. Our results show that in vitro folded FrpB assembles into oligomers, presumably dimers, and that it induces high levels of bactericidal antibodies in laboratory animals.
Subject(s)
Bacterial Outer Membrane Proteins/immunology , Neisseria meningitidis/immunology , Protein Folding , Receptors, Cell Surface/immunology , Animals , Antibodies, Bacterial/blood , Bacterial Outer Membrane Proteins/chemistry , Bacterial Outer Membrane Proteins/genetics , Bacterial Outer Membrane Proteins/isolation & purification , Circular Dichroism , Dimerization , Enzyme-Linked Immunosorbent Assay , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Inclusion Bodies , Mice , Microscopy, Electron, Transmission , Protein Conformation , Receptors, Cell Surface/chemistry , Receptors, Cell Surface/genetics , Receptors, Cell Surface/isolation & purification , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/immunologyABSTRACT
ClyA is a pore-forming toxin from virulent Escherichia coli and Salmonella enterica strains. Here, we show that the intrinsic hemolytic activity of ClyA is independent of its redox state, and that the assembly of both reduced and oxidized ClyA to the ring-shaped oligomer is triggered by contact with lipid or detergent. A rate-limiting conformational transition in membrane-bound ClyA monomers precedes their assembly to the functional pore. We obtained a three-dimensional model of the detergent-induced oligomeric complex at 12 A resolution by combining cryo- and negative stain electron microscopy with mass measurements by scanning transmission electron microscopy. The model reveals that 13 ClyA monomers assemble into a cylinder with a hydrophobic cap region, which may be critical for membrane insertion.
Subject(s)
Escherichia coli Proteins/chemistry , Hemolysin Proteins/chemistry , Cysteine/chemistry , Detergents/chemistry , Escherichia coli/chemistry , Escherichia coli/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli Proteins/ultrastructure , Hemolysin Proteins/metabolism , Hemolysin Proteins/ultrastructure , Lipids/chemistry , Microscopy, Electron , Models, Molecular , Oxidation-Reduction , Protein Structure, QuaternaryABSTRACT
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.
