Thomas Boesen

Structure of Eef3 and the Mechanism of Transfer RNA Release from the E-Site.

Elongation factor eEF3 is an ATPase that, in addition to the two canonical factors eEF1A and eEF2, serves an essential function in the translation cycle of fungi. eEF3 is required for the binding of the aminoacyl-tRNA–eEF1A–GTP ternary complex to the ribosomal A-site and has been suggested to facilitate the clearance of deacyl-tRNA from the E-site. Here we present the crystal structure of Saccharomyces cerevisiae eEF3, showing that it consists of an amino-terminal HEAT repeat domain, followed by a four-helix bundle and two ABC-type ATPase domains, with a chromodomain inserted in ABC2. Moreover, we present the cryo-electron microscopy structure of the ATP-bound form of eEF3 in complex with the post-translocational-state 80S ribosome from yeast. eEF3 uses an entirely new factor binding site near the ribosomal E-site, with the chromodomain likely to stabilize the ribosomal L1 stalk in an open conformation, thus allowing tRNA release.

Exotoxin A–eEF2 complex structure indicates ADP ribosylation by ribosome mimicry

The bacteria causing diphtheria, whooping cough, cholera and other diseases secrete mono-ADP-ribosylating toxins that modify intracellular proteins. Here, we describe four structures of a catalytically active complex between a fragment of Pseudomonas aeruginosa exotoxin A (ETA) and its protein substrate, translation elongation factor 2 (eEF2). The target residue in eEF2, diphthamide (a modified histidine), spans across a cleft and faces the two phosphates and a ribose of the non-hydrolysable NAD+ analogue, βTAD. This suggests that the diphthamide is involved in triggering NAD+ cleavage and interacting with the proposed oxacarbenium intermediate during the nucleophilic substitution reaction, explaining the requirement of diphthamide for ADP ribosylation. Diphtheria toxin may recognize eEF2 in a manner similar to ETA. Notably, the toxin-bound βTAD phosphates mimic the phosphate backbone of two nucleotides in a conformational switch of 18S rRNA, thereby achieving universal recognition of eEF2 by ETA.

Inhibition of demethylases by GSK-J1/J4

Arising from L. Kruidenier et al. Nature 488, 404–408 (2012); doi:10.1038/nature11262The recent publication of the first highly potent and specific inhibitor GSK-J1/J4 of the H3K27me3/me2-demethylases JMJD3/KDM6B and UTX/KDM6A provides a potential tool compound for this histone demethylase subfamily. This inhibitor was used in tissue culture assays to conclude that the catalytic activities of the KDM6 proteins are required in inflammatory responses; the generation of the inhibitor is intriguing, because it provides a strategy for generating sub-type-specific inhibitors of the 27-member Jumonji family and for the future treatment of various types of disease. Here we show that the inhibitor is not specific for the H3K27me3/me2-demethylase subfamily in vitro and in tissue culture assays. Thus, the inhibitor cannot be used alone for drawing conclusions regarding the specific role of H3K27me3/me2-demethylase activity in biological processes or disease. There is a Reply to this Brief Communications Arising by Kruidenier et al. Nature 514, http://dx.doi.org/10.1038/nature13689 (2014).

Curvature of Synthetic and Natural Surfaces Is an Important Target Feature in Classical Pathway Complement Activation

The binding of Abs to microbial surfaces followed by complement activation constitutes an important line of defense against infections. In this study, we have investigated the relationship between complement activation and the binding of human IgM Abs to surfaces with different curvatures. IgM Abs to dextran were shown to activate complement potently on dextran-coated particles having a diameter around 250 nm, whereas larger (600 nm) particles were less potent activators. This selectivity regarding particle dimension was also found for complement activation by colloidal substances of microbial origin. Peptidoglycan (PGN) is the major chemical component in the cell wall of Gram-positive bacteria. Fragments of purified PGN with sizes of ∼100 nm promoted complement activation effectively through the classical pathway. By contrast, larger or smaller fragments of PGN did not activate complement strongly. A careful analysis of PGN fragments released during planctonic growth of Staphylococcus aureus showed that these include curvatures that would permit strong IgM-mediated complement activation, whereas the curvature of intact cells would be less effective for such activation. Consistently, we found that the suspended PGN fragments were strong activators of complement through the classical pathway. We suggest that these fragments act as decoy targets for complement activation, providing protection for S. aureus against the host immune response to infection.

Molecular Dissection of Bacterial Nanowires

ABSTRACT The discovery of bacterial conductive structures, termed nanowires, has intrigued scientists for almost a decade. Nanowires enable bacteria to transfer electrons over micrometer distances to extracellular electron acceptors such as insoluble metal oxides or electrodes. Nanowires are pilus based and in Geobacter sulfurreducens are composed of the type IV pilin subunit PilA. Multiheme c-type cytochromes have been shown to attach to nanowire pili. Two hypotheses have been proposed for electron conduction in nanowires. The first (termed the metal-like conductivity or MLC hypothesis) claims that the pilus itself has the electron-conductive properties and the attached cytochromes mediate transfer to the final electron acceptor, whereas the second hypothesis (termed the superexchange conductivity or SEC hypothesis) suggests that electrons are “hopping” between heme groups in cytochromes closely aligned with the pilus as a scaffold. In their recent article in mBio, Vargas et al. [M. Vargas, N. S. Malvankar, P.-L. Tremblay, C. Leang, J. A. Smith, P. Patel, O. Snoeyenbos-West, K. P. Nevin, and D. R. Lovley, mBio 4(2):e00210-13, 2013] address this ambiguity through an analysis of strain Aro-5, a G. sulfurreducens PilA mutant lacking aromatic residues in the nonconserved portion of PilA. These residues were suspected of involvement in electron transport according to the MLC hypothesis. The G. sulfurreducens mutant had reduced conductive properties, lending important support to the MLC hypothesis. The data also highlight the need for further and more conclusive evidence for one or the other hypothesis.

The Virus-Encoded Chemokine vMIP-II Inhibits Virus-Induced Tc1-Driven Inflammation

ABSTRACT The human herpesvirus 8-encoded protein vMIP-II is a potent in vitro antagonist of many chemokine receptors believed to be associated with attraction of T cells with a type 1 cytokine profile. For the present report we have studied the in vivo potential of this viral chemokine antagonist to inhibit virus-induced T-cell-mediated inflammation. This was done by use of the well-established model system murine lymphocytic choriomeningitis virus infection. Mice were infected in the footpad, and the induced CD8+ T-cell-dependent inflammation was evaluated in mice subjected to treatment with vMIP-II. We found that inflammation was markedly inhibited in mice treated during the efferent phase of the antiviral immune response. In vitro studies revealed that vMIP-II inhibited chemokine-induced migration of activated CD8+ T cells, but not T-cell-target cell contact, granule exocytosis, or cytokine release. Consistent with these in vitro findings treatment with vMIP-II inhibited the adoptive transfer of a virus-specific delayed-type hypersensitivity response in vivo, but only when antigen-primed donor cells were transferred via the intravenous route and required to migrate actively, not when the cells were injected directly into the test site. In contrast to the marked inhibition of the effector phase, the presence of vMIP-II during the afferent phase of the immune response did not result in significant suppression of virus-induced inflammation. Taken together, these results indicate that chemokine-induced signals are pivotal in directing antiviral effector cells toward virus-infected organ sites and that vMIP-II is a potent inhibitor of type 1 T-cell-mediated inflammation.

Molecular biology of Chlamydia pneumoniae surface proteins and their role in immunopathogenicity

BACKGROUND The association of Chlamydia pneumoniae with the development of atherosclerosis is based on serology and on detection of C pneumoniae-specific DNA by polymerase chain reaction in the atheromas. METHODS AND RESULTS Because the humoral immune response frequently recognizes epitopes present on the surface of the bacteria, we analyzed what components are present on the C pneumoniae surface. We identified a family of proteins, the GGAI or Omp4-15 proteins, of which at least 3 are present on the surface of C pneumoniae. We immunized rabbits with recombinant GGAI proteins and used these antibodies in immunofluorescence microscopy of experimentally infected mice. In lung sections, a massive infiltration with polymorph nuclear neutrophil cells was observed. In the bronchial epithelial cells, C pneumoniae inclusions were seen. Evidence was found of differential expression of the GGAI proteins. CONCLUSIONS On the basis of surface localization, differential expression, and the fact that the proteins are recognized by the human humoral immune response, we speculate whether these proteins, in addition to the lipopolysaccharides, are of importance for the immunopathogenesis of C pneumoniae.

Shedding of Large Functionally Active CD11/CD18 Integrin Complexes from Leukocyte Membranes during Synovial Inflammation Distinguishes Three Types of Arthritis through Differential Epitope Exposure

CD18 integrins are adhesion molecules expressed on the cell surface of leukocytes and play a central role in the molecular mechanisms supporting leukocyte migration to zones of inflammation. Recently, it was discovered that CD11a/CD18 is shed from the leukocyte surface in models of inflammation. In this study, we show that shedding of human CD11/CD18 complexes is a part of synovial inflammation in rheumatoid arthritis and spondyloarthritis but not in osteoarthritis. In vivo and in vitro data suggest that the shedding is driven by TNF-α, which links the process to central events in the inflammatory response. The shed complexes contain multiple heterodimers of CD11/CD18, are variable in size, and differ according to the type of synovial inflammation. Furthermore, the differential structures determine the avidity of binding of the complexes to the ICAM-1. With the estimated concentrations of CD11/CD18 in plasma and synovial fluid a significant coverage of binding sites in ICAM-1 for CD18 integrins is expected. Based on cell adhesion experiments in vitro, we hypothesize that the large soluble complexes of CD11/CD18 act in vivo to buffer leukocyte adhesion by competing with the membrane-bound receptors for ICAM-1 binding sites. As reported here for synovial inflammation changes in the concentration or structure of these complexes should be considered as likely contributors to disease activity.

The Mycoplasma hominis vaa gene displays a mosaic gene structure

Mycoplasma hominis contains a variable adherence‐associated (vaa) gene. To classify variants of the vaa genes, we examined 42 M. hominis isolates by PCR, DNA sequencing and immunoblotting. This uncovered the existence of five gene categories. Comparison of the gene types revealed a modular composition of the Vaa proteins. The proteins constituted a conserved N‐terminal part followed by a varying number of interchangeable cassettes encoding approximately 110 amino acids with conserved sequence boxes flanking the cassettes. The interchangeable cassettes showed a high mutual homology and a conserved leucine zipper motif. The smallest product contained only one cassette and the largest five. Additionally, two types of stop mutations caused by substitutions resulting in the expression of truncated Vaa proteins were observed. Our results expand the known potential of the Vaa system in generating antigen variation.

Molecular design of Mycoplasma hominis Vaa adhesin

The variable adherence‐associated (Vaa) adhesin of the opportunistic human pathogen Mycoplasma hominis is a surface‐exposed, membrane‐associated protein involved in the attachment of the bacterium to host cells. The molecular masses of recombinant 1 and 2 cassette forms of the protein determined by a light‐scattering (LS) method were 23.9 kD and 36.5 kD, respectively, and corresponded to their monomeric forms. Circular dichroism (CD) spectroscopy of the full‐length forms indicated that the Vaa protein has an α‐helical content of ∼80%. Sequence analysis indicates the presence of coiled‐coil domains in both the conserved N‐terminal and antigenic variable C‐terminal part of the Vaa adhesin. Experimental results obtained with recombinant proteins corresponding to the N‐ or C‐terminal parts of the shortest one‐cassette form of the protein were consistent with the hypothesis of two distinct coiled‐coil regions. The one‐cassette Vaa monomer appears to be an elongated protein with a axial shape ratio of 1:10. Analysis of a two‐cassette Vaa type reveals a similar axial shape ratio. The results are interpreted in terms of the topological organization of the Vaa protein indicating the localization of the adherence‐mediating structure.