Crystal structures of the receiver domain of the response regulator PhoP from Escherichia coli in the absence and presence of the phosphoryl analog beryllofluoride

Abstract

The response regulator PhoP is part of the PhoQ/PhoP two-component system involved in responses to depletion of extracellular Mg(2+). Here, we report the crystal structures of the receiver domain of Escherichia coli PhoP determined in the absence and presence of the phosphoryl analog beryllofluoride. In the presence of beryllofluoride, the active receiver domain forms a twofold symmetric dimer similar to that seen in structures of other regulatory domains from the OmpR/PhoB family, providing further evidence that members of this family utilize a common mode of dimerization in the active state. In the absence of activating agents, the PhoP receiver domain crystallizes with a similar structure, consistent with the previous observation that high concentrations can promote an active state of PhoP independent of phosphorylation.

FIG. 1.

BeF₃⁻-activated PhoP_N. (A) The regulatory-domain dimer. Activated PhoP_N dimerizes with twofold rotational symmetry, forming an α4-β5-α5 interface. A ribbon depiction of the dimer is shown in green with the α4-β5-α5 dimer interface highlighted in gold and repetitive secondary-structure elements labeled. The noncovalent BeF₃⁻ complex and Mg²⁺ within the active site are shown as pink and cyan spheres, respectively. (B) Stereo view of the active site. A difference electron density map (F_o − F_c) calculated at 1.9 Å with omission of the Mg²⁺ and BeF₃⁻ complex from the model is shown in blue, contoured at 3σ. Carbons, nitrogens, and oxygens of the protein are shown as green, blue, and red sticks, respectively. The noncovalent BeF₃⁻ complex is shown in stick format with beryllium and fluorides in light and dark purple, respectively. Water molecules and Mg²⁺ are shown as red and cyan spheres, respectively. The network of hydrogen bonds that coordinates BeF₃⁻ and Mg²⁺ is shown as dashed lines. All molecular images were generated using Pymol (http://www.pymol.org).

FIG. 2.

Differences in the backbone conformations of activated versus unactivated PhoP_N. The conformational changes of the switch residues Thr79 and Tyr98, when BeF₃⁻ is bound at the active site Asp51, are correlated with minor differences in the overall positioning of secondary-structural elements relative to the unactivated protein. The unactivated protomer (chain A) is shown in red, and the activated protein is shown in green. The BeF₃⁻ complex is shown in blue, and Mg²⁺ is shown in yellow. Tyr98 has an outward orientation in the unactivated molecule and an inward orientation in the activated molecule.

FIG. 3.

Electron density in the β4-α4-β5 region of activated and unactivated PhoP_N proteins. Stereo views of the σA-weighted 2F_o − F_c electron density maps contoured at 2.5σ and calculated at 1.9 Å for activated PhoP_N (A) and at 2.54 Å for (B) chain A and (C) chain B of unactivated PhoP_N. The protein model is shown in stick representation (carbon, blue; nitrogen, dark blue; oxygen, red), and the relevant secondary-structural elements and tyrosine 98 are labeled in panel A for reference.

FIG. 4.

Comparison of B factors of unactivated and activated PhoP_N. (A) Average main-chain B factors for unactivated PhoP_N chain A (pink), unactivated PhoP_N chain B (orange), and activated PhoP_N (green). (B) Differences between B factors of activated PhoP_N and unactivated PhoP_N chain A (pink) and unactivated PhoP_N chain B (orange). The secondary-structural elements are shown in blue at the top.

FIG. 5.

Size exclusion chromatography of unphosphorylated and phosphorylated PhoP_N and PhoP. (A) Elution profiles of PhoP_N (solid line) and phosphorylated PhoP_N (dotted line) on a TosoHaas G2000SW column. (B) Elution profiles of full-length PhoP (solid line) and phosphorylated PhoP (dotted line) on a TosoHaas G3000SW_XL column. The conditions for chromatography and the protein standards used for calibration of the column are described in Materials and Methods.