Structural characterization of GntR/HutC family signaling domain

Abstract

The crystal structure of Escherichia coli PhnF C-terminal domain (C-PhnF) was solved at 1.7 A resolution by the single wavelength anomalous dispersion (SAD) method. The PhnF protein belongs to the HutC subfamily of the large GntR transcriptional regulator family. Members of this family share similar N-terminal DNA-binding domains, but are divided into four subfamilies according to their heterogenic C-terminal domains, which are involved in effector binding and oligomerization. The C-PhnF structure provides for the first time the scaffold of this domain for the HutC subfamily, which covers about 31% of GntR-like regulators. The structure represents a mixture of alpha-helices and beta-strands, with a six-stranded antiparallel beta-sheet at the core. C-PhnF monomers form a dimer by establishing interdomain eight-strand beta-sheets that include core antiparallel and N-terminal two-strand parallel beta-sheets from each monomer. C-PhnF shares strong structural similarity with the chorismate lyase fold, which features a buried active site locked behind two helix-turn-helix loops. The structural comparison of the C-PhnF and UbiC proteins allows us to propose that a similar site in the PhnF structure is adapted for effector binding.

Figure 1.

Multiple sequence alignment of PhnF against other members of the HutC family and FadR DNA binding domain. The FadR and C-PhnF secondary structure elements with arrows for β-strands and cylinders for α-helices are shown in red and green, respectively. The first residue of each of the three versions (81–, 85–, 88–241) of the C-PhnF domain is indicated with an arrow. The residues weakly conserved across chorismate lyase and HutC families and the residues conserved among PhnF orthologs are boxed.

Figure 2.

(A) Ribbon diagram of the C-PhnF (left) and UbiC (right) structures. The α-helices and β-strands are numbered and colored red and yellow, respectively. The β-D-fructopyranose molecule (blue) and 4-hydroxybenzoate (magenta) are shown as stick figures. (B) Ribbon diagram of the C-PhnF dimer with monomers in yellow and green. The L1 loop, which adopts a different conformation in each monomer, is highlighted in magenta.

Figure 3.

Close view of the potential PhnF effector-binding site in comparison with the active site of UbiC. C-PhnF (left) and UbiC (right) with bound 4-hydroxybenzoate are colored cyan and purple, respectively. The secondary structure elements are represented in ribbons, while side chains of UbiC active site residues and their counterparts in PhnF are highlighted in yellow. The largest cavity in C-PhnF and the active site cavity in UbiC are represented as molecular surface.