Conformational dynamics are a key factor in signaling mediated by the receiver domain of a sensor histidine kinase from Arabidopsis thaliana

Abstract

Multistep phosphorelay (MSP) cascades mediate responses to a wide spectrum of stimuli, including plant hormonal signaling, but several aspects of MSP await elucidation. Here, we provide first insight into the key step of MSP-mediated phosphotransfer in a eukaryotic system, the phosphorylation of the receiver domain of the histidine kinase CYTOKININ-INDEPENDENT 1 (CKI1_RD) from Arabidopsis thaliana We observed that the crystal structures of free, Mg²⁺-bound, and beryllofluoridated CKI1_RD (a stable analogue of the labile phosphorylated form) were identical and similar to the active state of receiver domains of bacterial response regulators. However, the three CKI1_RD variants exhibited different conformational dynamics in solution. NMR studies revealed that Mg²⁺ binding and beryllofluoridation alter the conformational equilibrium of the β3-α3 loop close to the phosphorylation site. Mutations that perturbed the conformational behavior of the β3-α3 loop while keeping the active-site aspartate intact resulted in suppression of CKI1 function. Mechanistically, homology modeling indicated that the β3-α3 loop directly interacts with the ATP-binding site of the CKI1 histidine kinase domain. The functional relevance of the conformational dynamics observed in the β3-α3 loop of CKI1_RD was supported by a comparison with another A. thaliana histidine kinase, ETR1. In contrast to the highly dynamic β3-α3 loop of CKI1_RD, the corresponding loop of the ETR1 receiver domain (ETR1_RD) exhibited little conformational exchange and adopted a different orientation in crystals. Biochemical data indicated that ETR1_RD is involved in phosphorylation-independent signaling, implying a direct link between conformational behavior and the ability of eukaryotic receiver domains to participate in MSP.

Keywords: X-ray crystallography; histidine kinase; nuclear magnetic resonance (NMR); protein dynamic; protein phosphorylation; receiver domain; relaxation dispersion.

Figure 1.

A, secondary structure representation of the activated CKI1_RD structure. The helices (α1–α5, cyan), β-strands (β0–β5, magenta), and loops (β3–α3 in dark green, other loops in pink) are numbered sequentially. The active-site residues of CKI1_RD are shown in stick representation (showing the chemistry). Polar interactions are highlighted by dashed lines, including the coordination sphere of a Mg²⁺ ion (orange sphere) with water molecules (red spheres). B, detailed structural description of the active site of CKI1_RD (stick representation). The coordination sphere of the Mg²⁺ ion and the polar interactions of Lys-1105 with Asp-992 and F₃ of BeF₃⁻ are highlighted by dashed lines. C, active site of CKI1_RD defined by the omit (2mF_o − DF_c) electron density map at 1.0 σ.

Figure 2.

Structural alignment (secondary structure representation) of the metal-free (blue), Mg²⁺-bound (red), and beryllofluoridated (green) forms of CKI1_RD.

Figure 3.

A, active site (stick representation) of CKI1_RD wild-type in the metal-free (blue), Mg²⁺-bound (red), and beryllofluoridated (green) forms. B, D1050A mutant (gray). C, D1050E mutant (yellow). D, alignment of the mutant and beryllofluoridated structures. The residue colors represent the respective forms/mutants of CKI1_RD. The Mg²⁺ ion is shown as a brown sphere in the Mg²⁺-bound form and as an orange sphere in the beryllofluoridated form.

Figure 4.

Dynamics of CKI1_RD. Order parameters S² (A), S_S² (B), effective correlation times τ_e (C), and τ_s (D), plotted for free (blue), Mg²⁺-bound (red), and beryllofluoridated (green) CKI1_RD. Cyan and magenta bars and pink lines above the plots represent α-helices, β-sheets, and loops, respectively. The β3–α3 loop is highlighted in dark green. Prolines, unassigned residues, and residues that could not be analyzed quantitatively are indicated above the plots by letters “P”, crosses, and dots (color-coded form individual CKI1_RD forms), respectively. Parameters estimated with the relative error greater than 50% are not shown.

Figure 5.

Exchange contributions to the relaxation at 600 MHz (A), plotted for free (blue), Mg²⁺-bound (red), and beryllofluoridated (green) CKI1_RD, and results of CPMG experiments at 950 MHz (apparent rates plotted as a function of the CPMG frequency) shown for Met-1049 (B), Met-1053 (C), and Met-1056 (D) of free (blue), Mg²⁺-bound (red), and beryllofluoridated (green) wild type, and of the D1050A (gray), D1050E (yellow), and D1057E,G1058N (slate blue) mutants. Cyan and magenta bars and pink lines above the plots represent α-helices, β-sheets, and loops, respectively. The β3–α3 loop is highlighted in dark green. Prolines, unassigned residues, and residues that could not be analyzed quantitatively are indicated above the plots by letters “P”, crosses, and dots (color-coded form individual CKI1_RD forms), respectively.

Figure 6.

Comparison of a selected region of ¹H,¹⁵N NMR spectra of free (blue), Mg²⁺-bound (red), and beryllofluoridated (green) CKI1_RD wild type, and of the D1050A (gray), D1050E (yellow), and D1057E,G1058N (slate blue) CKI1_RD mutants.

Figure 7.

Radiograms showing incorporation of ³²P into ETR1_HK (HK_P),CKI1_RD (RD_P), and AHP2 (AHP2_P) in the presence (A) and absence (B) of AHP2. Coomassie Blue-stained gels showing the amount of loaded ETR1_HK (HK), CKI1_RD (RD), and AHP2 (AHP2) are displayed below the radiograms. PageRuler Prestained Protein Ladder (Thermo Fisher Scientific) was used as a molecular weight marker (M).

Figure 8.

Relative TCS/LUC/Renilla expression in Arabidopsis protoplasts upon CKI1 overexpression in the absence (open boxes) and presence (filled boxes) of cytokinin (trans-zeatin). Error bars denote standard deviations.

Figure 9.

Comparison of orientation of the β3–α3 loop in plant and bacterial RDs. A, metal-free ETR1_RD (PDB code 1DCF, blue) and Mg²⁺-bound AHK5_RD of A. thaliana in complex with AHP1 (PDB code 4EUK, red). B, metal-free (PDB code 1TMY, cyan) and Mn²⁺-bound (PDB code 3TMY, magenta) CheY of T. maritima. C, RR468 of T. maritima in metal-free (PDB code 3DGF, purple) and beryllofluoridated (PDB code 3GL9, orange) forms, and in metal-free complex with HK859 (PDB code 3DGE, yellow). Beryllofluoridated CKI1_RD of A. thaliana is shown in green in all panels (PDB code 5N2N).