One of the mechanisms contributing to the protection by breast-feeding of the newborn against enteric diseases is related to the ability of human milk oligosaccharides to prevent the attachment of pathogenic bacteria to the duodenual epithelium. Indeed, a variety of fucosylated oligosaccharides, specific to human milk, form part of the innate immune system. In the present study, we demonstrate the specific blocking of PA-IIL, a fucose-binding lectin of the human pathogen Pseudomonas aeruginosa, by milk oligosaccharides. Two fucosylated epitopes, Lewis a and 3-fucosyl-lactose (Lewis x glucose analogue) bind to the lectin with dissociation constants of 2.2x10(-7) M and 3.6x10(-7) M respectively. Thermodynamic studies indicate that these interactions are dominated by enthalpy. The entropy contribution is slightly favourable when binding to fucose and to the highest-affinity ligand, Lewis a. The high-resolution X-ray structures of two complexes of PA-IIL with milk oligosaccharides allow the precise determination of the conformation of a trisaccharide and a pentasaccharide. The different types of interaction between the oligosaccharides and the protein involve not only hydrogen bonding, but also calcium- and water-bridged contacts, allowing a rationalization of the thermodynamic data. This study provides important structural information about compounds that could be of general application in new therapeutic strategies against bacterial infections.

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The role of the outer-membrane iron transporter FhuA as a potential receptor for the antimicrobial peptide MccJ25 (microcin J25) was studied through a series of in vivo and in vitro experiments. The requirement for both FhuA and the inner-membrane TonB-ExbB-ExbD complex was demonstrated by antibacterial assays using complementation of an fhuA − strain and by using isogenic strains mutated in genes encoding the protein complex respectively.

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The Escherichia coli outer membrane ferrichrome transporter FhuA was purified chromatographically in a neutral detergent (octyl glucoside or dodecyl maltoside). The amount of dodecyl maltoside bound to the protein (1.2 +/- 0.15 g/g of FhuA) and the Stokes radius of the FhuA-dodecyl maltoside complex (Rs = 4.2 nm) were determined using size exclusion chromatography. Sedimentation equilibrium and velocity experiments indicated that the FhuA preparation was monodisperse and that the protein was monomeric. The value found for the frictional coefficient of the protein-detergent complex (1.18) suggested a globular shape for the complex. Sedimentation experiments gave values for the molecular mass of the FhuA-dodecyl maltoside complex (180 kDa) and for the Stokes radius in complete agreement with those calculated from size exclusion chromatography. The circular dichroism spectrum indicated a 51% beta-sheet content. Functionality of the purified protein was assessed from fluorescence measurements using the DNA probe YO-PRO-1. Interaction of nM concentrations of FhuA with bacteriophage T5 resulted in the release of 90 +/- 8% of the phage DNA. The limiting step in DNA ejection was binding of the phage to its receptor. Release of DNA took place in a few seconds. Ferrichrome (0.8 microM) competed with the phage for binding to FhuA and prevented DNA ejection.

Tyrosine-containing model peptides were oxidized by horseradish peroxidase (HRP). This led to a peptide polymerization via condensation of the aromatic rings. Dimers, trimers, and tetramers (depending on the peptide length and on the position of the tyrosine in the sequence) were identified by electron spray mass spectroscopy. The second-order rate constants of the second step of the HRP reduction (CII --> E) was decreased by the presence of a positively charged amino group in the vicinity of the aromatic ring as determined by stopped flow measurements [k3 = 19 398 M-1 s-1 and k3 = 1016 M-1 s-1 for N-acetyltyrosine (NAT) and l-Tyr oxidations, respectively]. High-performance liquid chromatography was used to follow the kinetics of polymerization of some model peptides after their enzymatic oxidation. The first polymerization products exhibited a strong inhibitory effect toward further oxidation by HRP. This effect was not observed when using manganese-dependent peroxidase (MnP) which does not bind directly to the tyrosine residue but rather acts as a "distant catalyst". Saturation of the HRP was achieved with Pro-Gln-Gln-Pro-Tyr (kcat = 58 s-1, = 2.1 mM), NAT (kcat = 94 s-1, = 5.6 mM), and Gly-Tyr (kcat = 175 s-1, = 10.8 mM). Analysis of steady state kinetics of the reaction showed that the dimers formed initially behaved like competitive inhibitors. The value of the dissociation constant between HRP and dimers was 20 microM. A simplified model which accounts for these observations, including the formation of a Michaelis-Menten-like complex involving the donor and enzyme, is proposed and discussed.

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We previously designed a new family of artificial proteins named αRep based on a subgroup of thermostable helicoidal HEAT-like repeats. We have now assembled a large optimized αRep library. In this library, the side chains at each variable position are not fully randomized but instead encoded by a distribution of codons based on the natural frequency of side chains of the natural repeats family. The library construction is based on a polymerization of micro-genes and therefore results in a distribution of proteins with a variable number of repeats. We improved the library construction process using a “filtration” procedure to retain only fully coding modules that were recombined to recreate sequence diversity. The final library named Lib2.1 contains 1.7×109 independent clones. Here, we used phage display to select, from the previously described library or from the new library, new specific αRep proteins binding to four different non-related predefined protein targets. Specific binders were selected in each case. The results show that binders with various sizes are selected including relatively long sequences, with up to 7 repeats. ITC-measured affinities vary with Kd values ranging from micromolar to nanomolar ranges. The formation of complexes is associated with a significant thermal stabilization of the bound target protein. The crystal structures of two complexes between αRep and their cognate targets were solved and show that the new interfaces are established by the variable surfaces of the repeated modules, as well by the variable N-cap residues. These results suggest that αRep library is a new and versatile source of tight and specific binding proteins with favorable biophysical properties.