Mechanism of activation for transcription factor PhoB suggested by different modes of dimerization in the inactive and active states

Abstract

Response regulators (RRs), which undergo phosphorylation/dephosphorylation at aspartate residues, are highly prevalent in bacterial signal transduction. RRs typically contain an N-terminal receiver domain that regulates the activities of a C-terminal DNA binding domain in a phosphorylation-dependent manner. We present crystallography and solution NMR data for the receiver domain of Escherichia coli PhoB which show distinct 2-fold symmetric dimers in the inactive and active states. These structures, together with the previously determined structure of the C-terminal domain of PhoB bound to DNA, define the conformation of the active transcription factor and provide a model for the mechanism of activation in the OmpR/PhoB subfamily, the largest group of RRs. In the active state, the receiver domains dimerize with 2-fold rotational symmetry using their alpha4-beta5-alpha5 faces, while the effector domains bind to DNA direct repeats with tandem symmetry, implying a loss of intramolecular interactions.

Figure 1

Crystal Structures Show Different Dimers for Inactive and Active Regulatory Domains of PhoB. (A) Inactive PhoB_N dimerizes using the α1–α5 interface. Inactive PhoB_N is red; the dimer interface is beige. (B) Active PhoB_N dimerizes using the α4-β5-α5 interface. Active PhoB_N is green; the dimer interface is blue; and active site ligands are shown as spheres with magnesium in cyan, beryllium in pink, and fluorides in magenta. Figures were created using Pymol (http://pymol.sourceforge.net/).

Figure 2

H^N-¹⁵N HSQC Spectra of Inactive and Active Regulatory Domains of PhoB. The number of peaks observed in the HSQC spectra are 124 and 128 for inactive (red) and active (green) PhoB_N, respectively, both corresponding to monomers or two-fold symmetric dimers. At NMR concentrations, both proteins exist as symmetric dimers (see text). The large differences in the spectra of inactive and active PhoB_N suggest that the structures of the two dimers are significantly different.

Figure 3

Dilution Experiments of Inactive PhoB_N. Successive dilutions of inactive PhoB_N from 1.5 mM to 0.1 mM cause a progressive shift in some peaks indicating a fast monomer-dimer equilibrium on the NMR timescale (i.e. τ ≪ 100 ms). A section of the TROSY-HSQC spectrum is shown, with the inset zoomed on one of the resonances progressively shifting with decreasing concentration. Crosspeaks for 1.5 mM protein are shown in blue, for 0.75 mM in green, for 0.375 mM in yellow, and for 0.1 mM in red.

Figure 4

The Active Site of BeF₃⁻-activated PhoB_N. A stereo view of a difference electron density map (F_obs-F_calc) calculated at 2 Å with omission of the Mg²⁺, BeF₃⁻ and active site water molecules from the model is shown in blue, contoured at 3σ. Waters and the catalytic Mg²⁺ are shown as red and cyan spheres, respectively. The protein and BeF₃⁻ complex are shown in stick representation; beryllium is pink and fluorides are magenta (associations within the non-covalent BeF₃⁻ complex are shown as bonds for clarity).

Figure 5

Conformational Changes in PhoB_N upon Activation. The greatest conformational changes upon activation occur in the β4–α4 loop, the α4 helix and the α5-β5 loop. The important switch residues Thr83 and Tyr102, residues Ala84 and Arg85 in the β4–α4 loop, residue Asp90 in helix α4 and active site residue Asp53 are shown in stick representation. The inactive protein is red and the active protein is green. Active site ligands are shown in ball-and-stick representation; Mg²⁺ is cyan, beryllium is pink and fluorides are magenta.

Figure 6

Stereo View of the α4-β5-α5 Interface of Active PhoB_N. The residues involved in salt bridge and hydrophobic interactions at the interface are completely conserved in, and only in, the OmpR/PhoB subfamily of RRs. Residues involved in salt bridges are shown as sticks, and the hydrophobic residues are shown as spheres. The two protomers of the dimer are colored orange and green. Salt bridges are shown as dotted lines.

Figure 7

HetNOE Spectra of Inactive and Active PhoB_N. Negative resonances for H^Nεof arginines in the spectrum of inactive PhoB_N, indicating disordered arginines, become positive upon activation, indicating order, possibly due to involvement in salt bridges at the dimer interface of the active protein. (A) For inactive PhoB_N, positive resonances are colored red and negative resonances are colored blue; for active PhoB_N, positive resonances are colored green and no negative resonances are observed. (B) 1D slices through the arginine H^Nε region of the HetNOE spectrum of inactive PhoB_N show positive and negative resonances (top), whereas for activated PhoB_N, all arginine H^Nε resonances are positive (bottom).

Figure 8

Model of Phosphorylation-Induced Activation in the OmpR/PhoB Subfamily. In the active state, believed to be common to all OmpR/PhoB subfamily members, the receiver domains form a two-fold symmetric dimer while the DNA-binding domains bind to DNA with tandem symmetry. Inactive PhoB exists in equilibrium between a monomer and dimer. The inactive α1–α5 dimer, specific to PhoB, provides an additional means of inhibition by positioning the effector domains in opposite directions, incompatible with tandem DNA binding. Inactive PhoB_N is shown in red, active PhoB_N in green, the α1–α5 interface of the inactive dimer in beige, the α4-β5–α5 interface of the active dimer in blue, the DNA-binding domain in grey, and the recognition helix in pink. Full-length inactive PhoB was modeled by positioning the two domains of PhoB as they exist in the structure of DrrB.