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

Abstract

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.

Refining the Binding of the Escherichia coli Flagellar Master Regulator, FlhD 4 C 2 , on a Base-Specific Level

“…This implies that IHF recognizes DNA conformation (specifically, the minor groove) rather than the sequence specificity of the DNA (44). While the crystal structure of FlhD 4 C 2 has not been completely determined, the current data suggest that the FlhD 4 C 2 complex has dyad symmetry (7,63). The lack of data about FlhD 4 C 2 structure makes understanding how the Prior to the current study, much of what we knew about the FlhD 4 C 2 binding site was derived from bioinformatic approaches, e.g., sequence alignments, frequency statistics, and matrix searches of genomes that were applied to identify a consensus binding sequence.…”

“…In this context, it is noteworthy that FlhD 4 C 2 produces a large footprint (ϳ50 bp) and bends the DNA that it binds to by 111° (7,63). In most bacterial transcriptional regulatory proteins, a large DNA footprint (Ͼ30 bp) usually results from binding of multiple identical subunits, which in turn cause the target DNA to bend (6,8,34,52).…”

“…Lee and P. Matsumura, unpublished data). However, results from photo-cross-linking, alanine scanning mutagenesis, and heparin affinity of protein variants suggest that FlhD also contributes to DNA binding through Ser-82, Arg-83, and Val-84, located in the C terminus of the protein (7,13).…”

Bright Lights, Abundant Operons—Fluorescence and Genomic Technologies Advance Studies of Bacterial Locomotion and Signal Transduction: Review of the BLAST Meeting, Cuernavaca, Mexico, 14 to 19 January 2001

“…Two molecules of FlhD (gold and purple) form a tightly associated dimer which, when complexed with another protein, FlhC, activates transcription of flagellar genes. Residues in FlhD that define a putative binding surface for FlhC, as identified by mutagenesis experiments (19), are shown in ball-and-stick representation. (D) Crystal structure of a flagellin monomer from S. enterica (75).…”

Transcriptional Regulation inYersinia: An UpdateYersinia: An Update

“…Mutations within flhDC completely abolish swimming and swarming motilities. FlhD and FlhC form a heterotetrameric (C2D2) complex in which FlhC may act as an allosteric activator of FlhD, the DNA-binding subunit (Campos and Matsumura, 2001). Very recent reports strongly suggest that this regulatory complex is not only a 158 Marceau motility-specific activator (as initially thought) but probably also a global regulator.…”