Crystal structure of the global regulator FlhD from Escherichia coli at 1.8 angstrom resolution

FlhD is a 13.3 kDa transcriptional activator protein of flagellar genes and a global regulator. FlhD activates the transcription of class II operons i n 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 an d, to this end, we have determined the crystal structure of the isolated Fl hD protein. The structure was solved by substituting seleno-methionine for natural sulphur-methionine in FlhD, crystallizing the protein and determini ng the structure factor phases by the method of multiple-energy anomalous d ispersion (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 alpha -helical. The C-termini of both FlhD monomers (residues 83-116) are completely disrupted by crysta l packing, implying that this region of FlhD is highly flexible. However, p art of the C-terminus structure in chain A (residues 83-98) was modelled us ing a native FlhD crystal. What is seen in chain A suggests a classic DNA-b inding, 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 tr ue, it might explain how FlhD exhibits plasticity in its DNA-binding specif icity, as each partner protein with which it forms a complex could alloster ically affect the binding specificity of its HTH motif. A disulphide bridge is seen between the unique cysteine residues (Cys-65) of FlhD native homod imers. Alanine substitution at Cys-65 does not affect FlhD transcription ac tivator activity, suggesting that the disulphide bond is not necessary for either dimer stability or this function of FlhD. Electrostatic potential an alysis indicates that dimeric FlhD has a negatively charged surface.