Crystal structure of Vigna radiata cytokinin-specific binding protein in complex with zeatin

Abstract

The cytosolic fraction of Vigna radiata contains a 17-kD protein that binds plant hormones from the cytokinin group, such as zeatin. Using recombinant protein and isothermal titration calorimetry as well as fluorescence measurements coupled with ligand displacement, we have reexamined the K(d) values and show them to range from approximately 10(-6) M (for 4PU30) to 10(-4) M (for zeatin) for 1:1 stoichiometry complexes. In addition, we have crystallized this cytokinin-specific binding protein (Vr CSBP) in complex with zeatin and refined the structure to 1.2 A resolution. Structurally, Vr CSBP is similar to plant pathogenesis-related class 10 (PR-10) proteins, despite low sequence identity (<20%). This unusual fold conservation reinforces the notion that classic PR-10 proteins have evolved to bind small-molecule ligands. The fold consists of an antiparallel beta-sheet wrapped around a C-terminal alpha-helix, with two short alpha-helices closing a cavity formed within the protein core. In each of the four independent CSBP molecules, there is a zeatin ligand located deep in the cavity with conserved conformation and protein-ligand interactions. In three cases, an additional zeatin molecule is found in variable orientation but with excellent definition in electron density, which plugs the entrance to the binding pocket, sealing the inner molecule from contact with bulk solvent.

Figure 1.

Structures of Cytokinins. (A) Selected purine- and urea-type cytokinins. (B) A zeatin molecule (ZeaD1) as seen in the structure described here. The atom numbering, used throughout this article, is as in the Protein Data Bank entry 2FLH. The 2F_o-F_c electron density map is contoured at the 1.2σ level.

Figure 2.

ANS Binding to CSBP. Titration of 5 μM ANS in 10 mM MOPS buffer, pH 7.0, with Vr CSBP at 20°C. The line represents the best fit to Equation 1. The calculated K_d value is 32.5 ± 1.4 μM. ANS was excited at 360 nm, and emission at 480 nm was measured.

Figure 3.

Binding of Zeatin, 4PU30, 2iP, and Kinetin to Vr CSBP. Titration of 8.5 μM ANS complexed with 5.84 μM Vr CSBP in 50 mM Tris, pH 7.0, at 20°C, with zeatin (closed circles), 4PU30 (closed squares), 2iP (closed triangles), and kinetin (stars). (A) Experimental raw data. Lines are drawn to guide the eye. (B) Fitting of the experimental data to Equation 2. The K_d values were calculated using Equation 3 and are given in Table 1. ANS was excited at 360 nm, and emission at 480 nm was measured. The fluorescence signal was normalized to reflect 0 to 100% of ligand binding.

Figure 4.

Calorimetric Titration of Vr CSBP with Zeatin. The top panel shows raw heat data corrected for baseline drift obtained from 31 consecutive injections of 2.93 mM zeatin into the sample cell (1.05 mL) containing 0.148 mM Vr CSBP in 20 mM phosphate buffer, pH 6.5, at 20°C. The bottom panel shows the binding isotherm created by plotting the heat peak areas against the molar ratio of zeatin added to Vr CSBP present in the cell. The heats of mixing (dilution) were subtracted. The line represents the best fit to the model of n independent sites. The CSBP–zeatin binding is exothermic with 1:1 stoichiometry (n = 1.09), K_d of 106.8 μM, ΔH of −13.7 kJ·mol⁻¹, and ΔS of 29.1 J·mol⁻¹·K⁻¹.

Figure 5.

Overall Fold of the Vr CSBP with Annotation of Secondary Structure Elements. The molecule (D) is shown with its two zeatin ligands (ZeaD1, inner; ZeaD2, outer) in the binding pocket.

Figure 6.

Zeatin Ligands in the Binding Sites. (A) The ligand binding cavity in molecule A. This pattern is also seen in molecule D, with minor differences. Note the head-to-head (purine-to-purine) orientation of the zeatin ligands. (B) The same view for molecule B. Here, the outer zeatin molecule (ZeaB2) is rotated, with its isoprenoid tail pointing toward the purine ring (head-to-tail) of the inner ligand (ZeaB1). In (A) and (B), part of the protein (Phe-26 to Thr-52) has been omitted for clarity. For viewing into the binding pocket, the molecule in Figure 5 has been rotated −90° around the horizontal axis and then −45° around the vertical axis. (C) and (D) The inner (ZeaA1 [C]) and outer (ZeaA2 [D]) zeatin ligands of molecule A are shown in a 2F_o-F_c electron density map contoured at the 1.2σ level. Only one alternative is shown for residues modeled in dual conformation. (E) to (G) The close fit of the zeatin molecules in the internal cavities of molecules A, B, and C. The protein is shown in a cutaway surface representation with the main chain in a stick model. The ligands are represented by space-filling models with N atoms colored blue, O atoms colored red, and C atoms colored white or green for the inner or outer binding site, respectively. The cavity is filled by head-to-head–oriented ligands in molecule A (E), by head-to-tail–oriented ligands in molecule B (F), and by a single (inner) ligand in molecule C (G). The entrance to the cavity in molecule C is filled with ordered water molecules (red spheres).

Figure 7.

LIGPLOT (Wallace et al., 1995) Representations of the Interactions of the Zeatin Ligands in the Binding Pocket of the CSBP Molecules. (A) Molecule D with two zeatin ligands bound head-to-head (a nearly identical diagram for molecule A is not shown). (B) Molecule B with two zeatin ligands bound head-to-tail. (C) Molecule C with only one (inner) zeatin ligand in the binding pocket. Only interactions with protein atoms are shown. Water molecules forming additional hydrogen bonds with the zeatin molecules have been omitted for clarity.

Figure 8.

Conformations of the Zeatin Molecules. Two distinct conformations of the ligand molecules bound in the binding pocket can be distinguished. All of the inner zeatin molecules as well as the outer ones with head-to-head orientation populate the most abundant conformation. The outer ZeaB2 molecule (bound in head-to-tail mode) has a different value of the N10-C11-C12-C13 torsion angle. The purine rings of the zeatin molecules were superimposed in LSQKAB (Kabsch, 1976).

Figure 9.

Comparison of Vr CSBP with PR-10 Proteins. (A) Superposition of the Cα atoms of Vr CSBP (molecule A), Ll PR-10.1A (1A), Ll PR-10.1B (1B), Ll PR-10.2A (2A), and Betv1. The position of Tyr-149 (Tyr-148 in Ll PR-10.1 and Tyr-150 in Betv1) is indicated (stick model) to show the axial shift of helix α3 in Betv1. Calculations were made in ALIGN (Cohen, 1997). (B) Superposition of Vr CSBP molecule B (dark gray) with two zeatin molecules bound head-to-tail (black line) on Betv1 (light gray) in complex with two deoxycholate molecules (ball-and-stick model) (Markovic-Housley et al., 2003). Helix α3, covering the binding pocket in this view, has been removed for clarity. (C) Amino acid sequence alignment of selected CSBP and PR-10 proteins. Secondary structure elements are indicated as present in Vr CSBP. The level of conservation is expressed by the darkness of the lettering background. Note the conservation of the amino acid sequence in the Gly-rich loop L4 and the highly divergent sequence patterns at the end of loop L9 and at the N terminus of helix α3. Calculations were made in ClustalW (Thompson et al., 1994). Betv, Betula verrucosa; Gm, Glycine max; Ll, Lupinus luteus; Vr, Vigna radiata.