Extensive alanine scanning reveals protein-protein and protein-DNA interaction surfaces in the global regulator FlhD from Escherichia coli

FlhD and FlhC are the transcriptional activators of the flagellar regulon, The heterotetrameric complex formed by these two proteins activates the tra nscription of the class II flagellar genes. The flagellar regulon consists not only of flagellar genes, but also of the chemotactic genes and some rec eptor proteins. Recently, a connection between the flagellar regulon and so me virulence genes has been found in some species. Furthermore, FlhD, but n ot FlhC, regulates another non-flagellar target. As a first attempt to unde rstand the mechanism of the flagellar transcriptional activation by FlhD an d FlhC, the structure of FlhD has been solved. In order to understand the m echanism of the action of FlhD when it regulates the flagellar genes, we co nducted 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-a, As p-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 t he 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.