Alexander Krah

Salmonella-Induced Mucosal Lectin RegIIIβ Kills Competing Gut Microbiota

Intestinal inflammation induces alterations of the gut microbiota and promotes overgrowth of the enteric pathogen Salmonella enterica by largely unknown mechanisms. Here, we identified a host factor involved in this process. Specifically, the C-type lectin RegIIIβ is strongly upregulated during mucosal infection and released into the gut lumen. In vitro, RegIIIβ kills diverse commensal gut bacteria but not Salmonella enterica subspecies I serovar Typhimurium (S. Typhimurium). Protection of the pathogen was attributable to its specific cell envelope structure. Co-infection experiments with an avirulent S. Typhimurium mutant and a RegIIIβ-sensitive commensal E. coli strain demonstrated that feeding of RegIIIβ was sufficient for suppressing commensals in the absence of all other changes inflicted by mucosal disease. These data suggest that RegIIIβ production by the host can promote S. Typhimurium infection by eliminating inhibitory gut microbiota.

Structure of the c10 ring of the yeast mitochondrial ATP synthase in the open conformation

The proton pore of the F1Fo ATP synthase consists of a ring of c subunits, which rotates, driven by downhill proton diffusion across the membrane. An essential carboxylate side chain in each subunit provides a proton-binding site. In all the structures of c-rings reported to date, these sites are in a closed, ion-locked state. Structures are here presented of the c10 ring from Saccharomyces cerevisiae determined at pH 8.3, 6.1 and 5.5, at resolutions of 2.0 Å, 2.5 Å and 2.0 Å, respectively. The overall structure of this mitochondrial c-ring is similar to known homologs, except that the essential carboxylate, Glu59, adopts an open extended conformation. Molecular dynamics simulations reveal that opening of the essential carboxylate is a consequence of the amphiphilic nature of the crystallization buffer. We propose that this new structure represents the functionally open form of the c subunit, which facilitates proton loading and release.

Complete Ion-Coordination Structure in the Rotor Ring of Na+-Dependent F-ATP Synthases

The membrane-embedded rotors of Na(+)-dependent F-ATP synthases comprise 11 c-subunits that form a ring, with 11 Na(+) binding sites in between adjacent subunits. Following an updated crystallographic analysis of the c-ring from Ilyobacter tartaricus, we report the complete ion-coordination structure of the Na(+) sites. In addition to the four residues previously identified, there exists a fifth ligand, namely, a buried structural water molecule. This water is itself coordinated by Thr67, which, sequence analysis reveals, is the only residue involved in binding that distinguishes Na(+) synthases from H(+)-ATP synthases known to date. Molecular dynamics simulations and free-energy calculations of the c-ring in a lipid membrane lend clear support to the notion that this fifth ligand is a water molecule, and illustrate its influence on the selectivity of the binding sites. Given the evolutionary ascendancy of sodium over proton bioenergetics, this structure uncovers an ancient strategy for selective ion coupling in ATP synthases.

Identification of candidate antigens for serologic detection of Helicobacter pylori-infected patients with gastric carcinoma.

Helicobacter pylori colonizes the stomach of almost half the world population and is a causative agent of gastric carcinomas and duodenal ulcers. Only a small fraction of infected people will develop these severe illnesses and a predictive test to identify people at high risk would greatly benefit disease management. Our study aimed to identify conserved bacterial antigens that may be useful for the development of such a diagnostic test. High‐resolution immunoproteomics by 2‐dimensional electrophoresis of H. pylori 26695 proteins was carried out with sera from infected patients with either duodenal ulcer (n=30) or gastric carcinoma (n=30), 2 clinically divergent conditions. According to their antigen recognition patterns clear groups of patients were identified. Although this classification did not correspond to the clinical status, it may be correlated to other bacterial or host factors that influence the outcome of infection. In general antigen recognition patterns were found to be highly variable, however by utilizing powerful image analysis and statistical tests the recognition of 14 antigenic protein species was found to differ significantly (p<0.01) between both diseases. Particular protein species of GroEL, HyuA, GroES and AtpA appear to be useful surrogate markers for gastric carcinoma detection and consequently should be considered for further prospective studies to assess their predictive value. For one protein species of AtpA, evidence was found that different post‐translational modifications may confer different immunogenicities.

Structural and energetic basis for H+ versus Na+ binding selectivity in ATP synthase Fo rotors

The functional mechanism of the F1Fo ATP synthase, like many membrane transporters and pumps, entails a conformational cycle that is coupled to the movement of H+ or Na+ ions across its transmembrane domain, down an electrochemical gradient. This coupling is an efficient means of energy transduction and regulation, provided that ion binding to the membrane domain, known as Fo, is appropriately selective. In this study we set out to establish the structural and energetic basis for the ion-binding selectivity of the membrane-embedded Fo rotors of two representative ATP synthases. First, we use a biochemical approach to demonstrate the inherent binding selectivity of these rotors, that is, independently from the rest of the enzyme. We then use atomically detailed computer simulations of wild-type and mutagenized rotors to calculate and rationalize their selectivity, on the basis of the structure, dynamics and coordination chemistry of the binding sites. We conclude that H+ selectivity is most likely a robust property of all Fo rotors, arising from the prominent presence of a conserved carboxylic acid and its intrinsic chemical propensity for protonation, as well as from the structural plasticity of the binding sites. In H+-coupled rotors, the incorporation of hydrophobic side chains to the binding sites enhances this inherent H+ selectivity. Size restriction may also favor H+ over Na+, but increasing size alone does not confer Na+ selectivity. Rather, the degree to which Fo rotors may exhibit Na+ coupling relies on the presence of a sufficient number of suitable coordinating side chains and/or structural water molecules. These ligands accomplish a shift in the relative binding energetics, which under some physiological conditions may be sufficient to provide Na+ dependence.

A New Type of Na+-Driven ATP Synthase Membrane Rotor with a Two-Carboxylate Ion-Coupling Motif

Multi-disciplinary methods reveal a novel type of ion binding in the rotor ring of the F1Fo-ATP synthase from the opportunistic pathogen Fusobacterium nucleatum.

On the Structure of the Proton-Binding Site in the Fo Rotor of Chloroplast ATP Synthases

The recently reported crystal structures of the membrane-embedded proton-dependent c-ring rotors of a cyanobacterial F(1)F(o) ATP synthase and a chloroplast F(1)F(o) ATP synthase have provided new insights into the mechanism of this essential enzyme. While the overall features of these c-rings are similar, a discrepancy in the structure and hydrogen-bonding interaction network of the H(+) sites suggests two distinct binding modes, potentially reflecting a mechanistic differentiation. Importantly, the conformation of the key glutamate side chain to which the proton binds is also altered. To investigate the nature of these differences, we use molecular dynamics simulations of both c-rings embedded in a phospholipid membrane. We observe that the structure of the c(15) ring from Spirulina platensis is unequivocally stable within the simulation time. By contrast, the proposed structure of the H(+) site in the chloroplast c(14) ring changes rapidly and consistently into that reported for the c(15) ring, indicating that the latter represents a common binding mode. To assess this hypothesis, we have remodeled the c(14) ring by molecular replacement using the published structure factors. The resulting structure provides clear evidence in support of a common binding site conformation and is also considerably improved statistically. These findings, taken together with a sequence analysis of c-subunits in the ATP synthase family, indicate that the so-called proton-locked conformation observed in the c(15) ring may be a common characteristic not only of light-driven systems such as chloroplasts and cyanobacteria but also of a selection of other bacterial species.

Analysis of Automatically Generated Peptide Mass Fingerprints of Cellular Proteins and Antigens from Helicobacter pylori 26695 Separated by Two-dimensional Electrophoresis

Helicobacter pylori is a causative agent of severe diseases of the gastric tract ranging from chronic gastritis to gastric cancer. Cellular proteins of H. pylori were separated by high resolution two-dimensional gel electrophoresis. A dataset of 384 spots was automatically picked, digested, spotted, and analyzed by matrix-assisted laser desorption ionization mass spectrometry peptide mass fingerprint in triple replicates. This procedure resulted in 960 evaluable mass spectra. Using a new version of our data analysis software MS-Screener we improved identification and tested reliability of automatically generated data by comparing with manually produced data. Antigenic proteins from H. pylori are candidates for vaccines and diagnostic tests. Previous immunoproteomics studies of our group revealed antigen candidates, and 24 of them were now closely analyzed using the MS-Screener software. Only in three spots minor components were found that may have influenced their antigenicities. These findings affirm the value of immunoproteomics as a hypothesis-free approach. Additionally, the protein species distribution of the known antigen GroEL was investigated, dimers of the protein alkyl hydroperoxide reductase were found, and the fragmentation of γ-glutamyltranspeptidase was demonstrated.

On the question of hydronium binding to ATP-synthase membrane rotors.

A recently determined atomic structure of an H(+)-coupled ATP-synthase membrane rotor has revived the long-standing question of whether protons may be bound to these structures in the form of a hydronium ion. Using both classical and quantum-mechanical simulations, we show that this notion is implausible. Ab initio molecular dynamics simulations of the binding site demonstrate that the putative H(3)O(+) deprotonates within femtoseconds. The bound proton is thus transferred irreversibly to the carboxylate side chain found in the ion-binding sites of all ATP-synthase rotors. This result is consistent with classical simulations of the rotor in a phospholipid membrane, on the 100-nanosecond timescale. These simulations show that the hydrogen-bond network seen in the crystal structure is incompatible with a bound hydronium. The observed coordination geometry is shown to correspond instead to a protonated carboxylate and a bound water molecule. In conclusion, this study underscores the notion that binding and transient storage of protons in the membrane rotors of ATP synthases occur through a common chemical mechanism, namely carboxylate protonation.

G117C MelB, a mutant melibiose permease with a changed conformational equilibrium

Replacement of the glycine at position 117 by a cysteine in the melibiose permease creates an interesting phenotype: while the mutant transporter shows still transport activity comparable to the wild type its pre steady-state kinetic properties are drastically altered. The transient charge displacements after substrate concentration jumps are strongly reduced and the fluorescence changes disappear. Together with its maintained transport activity this indicates that substrate translocation in G117C melibiose permease is not impaired but that the initial conformation of the mutant transporter differs from that of the wild type permease. A kinetic model for the G117C melibiose permease based on a rapid dynamic equilibrium of the substrate free transporter is proposed. Implications of the kinetic model for the transport mechanism of the wild type permease are discussed.