Structure of internalin,a major invasion protein of Listeria monocytogenes,in complex with its human receptor E-cadherin

Listeria monocytogenes, a food-borne bacterial pathogen, enters mammalian cells by inducing its own phagocytosis. The listerial protein internalin (InlA) mediates bacterial adhesion and invasion of epithelial cells in the human intestine through specific interaction with its host cell receptor E-cadherin. We present the crystal structures of the functional domain of InlA alone and in a complex with the extracellular, N-terminal domain of human E-cadherin (hEC1). The leucine rich repeat (LRR) domain of InlA surrounds and specifically recognizes hEC1. Individual interactions were probed by mutagenesis and analytical ultracentrifugation. These include Pro16 of hEC1, a major determinant for human susceptibility to L. monocytogenes infection that is essential for intermolecular recognition. Our studies reveal the structural basis for host tro-pism of this bacterium and the molecular deception L. monocytogenes employs to exploit the E-cadherin system.     

Molecular mobility and nucleocytoplasmic flux in hepatoma cells

Fluorescence microphotolysis (photobleaching) was used to measure, in single polyethylene glycol-induced polykaryons of hepatoma tissue culture cells, nucleocytoplasmic flux and intracellular mobility for a series of dextrans ranging in molecular mass from 3 to 150 kD and for bovine serum albumin. For the dextrans, the cytoplasmic and the nucleoplasmic translational diffusion coefficients amounted to approximately 9 and approximately 15%, respectively, of the value in dilute buffer. The diffusion coefficients depended inversely on molecular radius, suggesting that diffusion was dominated by viscosity effects. By application of the Stokes-Einstein equation, cytoplasmic and nucleoplasmic viscosities were derived to be 6.6 and 8.1 cP, respectively, at 23 degrees C. Between 10 and 37 degrees C nucleoplasmic diffusion coefficients increased by approximately 45-85%, whereas cytoplasmic diffusion coefficients were virtually independent of temperature. In contrast to that of the dextrans, diffusion of bovine serum albumin was more restricted. In the cytoplasm the diffusion coefficient was approximately 1.5% of the value in dilute buffer; in the nucleus albumin was largely immobile. This indicated that albumin mobility is dominated by association with immobile cellular structures. Nucleocytoplasmic flux of dextrans depended inversely on molecular mass with an exclusion limit between 17 and 41 kD. This agrees with previous measurements on primary hepatocytes (Peters, R., 1984, EMBO [Eur. Mol. Biol. Organ.] J. 3:1831-1836), suggesting that in both cell types the nuclear envelope has properties of a molecular sieve with a functional pore radius of approximately 55 A.     

Promoter recognition and activation by the global response regulator CbrB in Pseudomonas aeruginosa

In Pseudomonas aeruginosa, the CbrA/CbrB two-component system is instrumental in the maintenance of the carbon-nitrogen balance and for growth on carbon sources that are energetically less favorable than the preferred dicarboxylate substrates. The CbrA/CbrB system drives the expression of the small RNA CrcZ, which antagonizes the repressing effects of the catabolite repression control protein Crc, an RNA-binding protein. Dicarboxylates appear to cause carbon catabolite repression by inhibiting the activity of the CbrA/CbrB system, resulting in reduced crcZ expression. Here we have identified a conserved palindromic nucleotide sequence that is present in upstream activating sequences (UASs) of promoters under positive control by CbrB and σ(54) RNA polymerase, especially in the UAS of the crcZ promoter. Evidence for recognition of this palindromic sequence by CbrB was obtained in vivo from mutational analysis of the crcZ promoter and in vitro from electrophoretic mobility shift assays using crcZ promoter fragments and purified CbrB protein truncated at the N terminus. Integration host factor (IHF) was required for crcZ expression. CbrB also activated the lipA (lipase) promoter, albeit less effectively, apparently by interacting with a similar but less conserved palindromic sequence in the UAS of lipA. As expected, succinate caused CbrB-dependent catabolite repression of the lipA promoter. Based on these results and previously published data, a consensus CbrB recognition sequence is proposed. This sequence has similarity to the consensus NtrC recognition sequence, which is relevant for nitrogen control.     

Synthesis, structure and reactivity of complexes containing [M(S2)2] fragments (M=Ru, Os; (S2)=1,2-benzenedithiolate (2−))

Subsequent addition of 1,2-benzenedithiol (S₂-H₂) and nBuLi to a solution of [Ru(NO)Cl₃ · xMeOH] in THF afforded exclusively the monomeric species NBu₄[Ru^II(NO)(S₂)₂] (1). Formation of dimeric (NBu₄)₂[Ru^II(NO)(S₂)₂]₂ (2) has been confirmed when the deprotonated ligand S₂-Li₂ was added to [Ru(NO)Cl₃ · xMeOH] and allowed to stir for 30 h. The monomer 1 undergoes aerial oxidation to give (NBu₄)₂[Ru^IV(S₂)₃] (3). The reaction between RuCl₃ · xH₂O and S₂-H₂ in the presence of NaOMe, afforded the dinulear Ru^III species (NMe₄)₂[Ru^III(S₂)₂]₂ (4). A modified method for the preparation of 1 is being employed to synthesize the osmium analogue NBu₄[Os(NO)(S₂)₂] (5) effectively. The solid state structures of 1, 2 and 3 were determined by X-ray crystal structure analysis. A comparison of relevant bond distance data suggests that 1,2-benzenedithiolate acts as an “innocent” ligand.