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SummaryFlhD is a 13.3 kDa transcriptional activator protein of flagellar genes and a global regulator. FlhD activates the transcription of class II operons in the flagellar regulon when complexed with a second protein FlhC (21.5 kDa). FlhD also regulates other expression systems in Escherichia coli. We are seeking to understand this plasticity of FlhD's DNA-binding specificity and, to this end, we have determined the crystal structure of the isolated FlhD protein. The structure was solved by substituting seleno-methionine for natural sulphurmethionine in FlhD, crystallizing the protein and determining the structure factor phases by the method of multiple-energy anomalous dispersion (MAD). The FlhD protein is dimeric. The dimer is tightly coupled, with an intimate contact surface, implying that the dimer does not easily dissociate. The FlhD monomer is predominantly a-helical. The C-termini of both FlhD monomers (residues 83±116) are completely disrupted by crystal packing, implying that this region of FlhD is highly flexible. However, part of the C-terminus structure in chain A (residues 83±98) was modelled using a native FlhD crystal. What is seen in chain A suggests a classic DNA-binding, helix±turn±helix (HTH) motif. FlhD does not bind DNA by itself, so it may be that the DNA-binding HTH motif becomes rigidly defined only when FlhD forms a complex with some other protein, such as FlhC. If this were true, it might explain how FlhD exhibits plasticity in its DNA-binding specificity, as each partner protein with which it forms a complex could allosterically affect the binding specificity of its HTH motif. A disulphide bridge is seen between the unique cysteine residues (Cys-65) of FlhD native homodimers. Alanine substitution at Cys-65 does not affect FlhD transcription activator activity, suggesting that the disulphide bond is not necessary for either dimer stability or this function of FlhD. Electrostatic potential analysis indicates that dimeric FlhD has a negatively charged surface.

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Background: Only small effects on affinity are seen for seemingly critical lysines of LRP1 ligand RAP. Results: Lysines act in pairs and fully account for overall affinity to LRP1. Mutagenesis reduces binding up to 100,000-fold. Conclusion: Two pairs of paired lysine interactions can give high affinity binding of protein ligands to LRP1. Significance: Rules for identifying high affinity receptor binding sites are established.

SummaryFlhD and FlhC are the transcriptional activators of the flagellar regulon. The heterotetrameric complex formed by these two proteins activates the transcription of the class II flagellar genes. The flagellar regulon consists not only of flagellar genes, but also of the chemotactic genes and some receptor proteins. Recently, a connection between the flagellar regulon and some virulence genes has been found in some species. Furthermore, FlhD, but not FlhC, regulates another non-flagellar target. As a first attempt to understand the mechanism of the flagellar transcriptional activation by FlhD and FlhC, the structure of FlhD has been solved. In order to understand the mechanism of the action of FlhD when it regulates the flagellar genes, we conducted site-directed mutagenesis based on its three-dimensional structure. Six interaction surfaces in the FlhD dimer were mapped by alanine scanning mutagenesis. Two of them are surface clusters formed by residues His-2, Asp-28, Arg-35, Phe-34 and Asn-61 located at each side of the dimer core. The other four are located in the flexible arms of the dimer. The residues Ser-82, Arg-83, Val-84, His-91, Thr-92, Ile-94 and Leu-96 are located at this region. All these residues are involved in the FlhD/FlhC interaction with the exception of Ser-82, Arg-83 and Val-84. These three residues affect the DNA-binding ability of the complex. The three-dimensional topology of FlhD and the site-directed mutagenesis results support the hypothesis of FlhC as an allosteric effector that activates FlhD for the recognition of the DNA.

The inv gene of Yeminis enterocolitica codes for invasin, a member of the invasinhtimin-like protein family, which mediates the internalization of the bacterium into cultured epithelial cells. The putative inclusion of inv into a pathogenicity island was tested by investigating its flanking sequences. Indeed, the enteropathogenic Escherichia coli (EPEC) intimin, a member of the same family of proteins, is encoded by eaeA, a gene which belongs to a pathogenicity island. An ORF located upstream from inv was of particular interest since it appeared homologous both to the flagellar f/hA gene and to sepA, an EPEC gene lying inside the same pathogenicity island as eaeA. A mutant in this ORF was non-motile and non-flagellated while its invasion phenotype remained unaffected. These data indicated that the ORF corresponded to the flhA gene of Y. enterocolitica. Subsequently, the flhB and flhE genes, located respectively upstream and downstream from flhA, were identified. The three flh genes appear to be transcribed from a single operon called flhB, according to the nomenclature used for Salmonella typhimurium. lntergenic sequence between f/hE and inv includes a grey hole, with no recognizable function. Downstream from inv, we have detected the flagellar flgM operon as already reported. Finally, the incongruous localization of inv amidst the flagellar cluster is discussed; while transposition could explain this phenomenon, no trace of such an event was detected.

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