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

Abstract

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.

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

“…CheY and CheZ localization is dependent on CheA, and CheA localization is dependent on CheW. Similar localization studies using a CheZ-GFP fusion were reported by Brian Cantwell (M. (20). Two molecules of FlhD (gold and purple) form a tightly associated dimer which, when complexed with another protein, FlhC, activates transcription of flagellar genes.…”

“…The first group consists exclusively of flhC and flhD, whose products form a heterotetramer that is required for the synthesis of subsequent classes of flagellar components. Andres Campos and colleagues (P. Matsumura's lab) described the crystal structure of the FlhD homodimer at 1.8-Å resolution (20) (Fig. 4C).…”

Activation of Motility by Sensing Short-Chain Fatty Acids via Two Steps in a Flagellar Gene Regulatory Cascade in Enterohemorrhagic Escherichia coli

“…Since the amount of FlhC protein in the lrp mutant was not increased by butyrate, it is unlikely that SCFAs affect the stability of FlhC and FlhD proteins. One possible mechanism is that SCFAs affect FlhD 4 FlhC 2 activity by changing disulfide bond formation (2) or zinc binding (32) or through an unknown modification. The response to SCFAs via the class II promoter seems to be important for the induction of flagellar gene expression in the intestine.…”

FliT Selectively Enhances Proteolysis of FlhC Subunit in FlhD4C2 Complex by an ATP-dependent Protease, ClpXP

“…The effect of FliT on degradation of individual FlhC by ClpXP in vitro was not determined because the FlhC subunit could not be purified as a soluble protein, whereas FlhC could be purified when complexed in FlhD 4 C 2 . Purification of individual FlhC has so far not been successful (27,28).…”