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 ;10 ÿ6 M (for 4PU30) to 10 ÿ4 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 Å resolution. Structurally, Vr CSBP is similar to plant pathogenesisrelated 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 b-sheet wrapped around a C-terminal a-helix, with two short a-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.

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Pathogenesis-related (PR) proteins of class 10 are abundant in higher plants. Some of these proteins are induced under stress conditions as part of the plant defence mechanism. Other homologues are developmentally regulated and their expression varies in different plant organs. The PR-10 proteins are encoded by multigene families, have a weight of about 17 kDa and are found in the cytosol. In yellow lupin, nine different homologues have been identified and divided into two subclasses, LlPR-10.1 and LlPR-10.2. Within each subclass the sequence identity is about 75-91%, while across the subclasses it is only 59-60%. Here, the crystal structure of a yellow lupin PR-10 protein from the second subclass, LlPR-10.2A, is presented. The structure was solved by molecular replacement and refined to R = 0.205 using 1.9 A resolution data. The general fold of LlPR-10.2A resembles that of the other PR-10 proteins and consists of a long C-terminal alpha-helix surrounded by a seven-stranded antiparallel beta-sheet, with two shorter alpha-helices located between strands beta1 and beta2. The most variable part of the structure, the C-terminal helix, is strongly kinked towards the beta-sheet core in both LlPR-10.2A molecules present in the asymmetric unit. This unexpected feature reduces the size of the hydrophobic cavity observed in other PR-10 proteins that is reported to be the ligand-binding site. As in other PR-10 structures, a surface loop located near the entrance to the cavity shows very high structural conservation and stability despite the high glycine content in its sequence.

Abbreviations:CSBP, cytokinin-specific binding protein; MAD, multiwavelength anomalous diffraction; PR-10, pathogenesis-related proteins of class 10; rms, root-mean-square;Running title: MAD phasing using Ta 6 Br 12 PDB reference: 3C0V Synopsis: The first case of authentic structure determination by MAD phasing using high resolution data for a ( Ta 6 Br 12 ) 2+ derivative is analyzed to provide practical hints for the application of this useful phasing agent. 2The crystal structure of Cytokinin-Specific Binding Protein (CSBP) containing four independent molecules with 4x155=620 residues in the asymmetric unit of the P6 4 unit cell, has been solved by three-wavelength MAD, using 1. should be specifically bound and ordered. Good binding at protein surface is facilitated by the presence of acidic groups, indicating higher pH as preferable buffer conditions. In addition, the water channels in the crystal should be sufficiently wide (at least 11 Å) to allow free diffusion of the (Ta 6 Br 12 ) 2+ ions on soaking. A retrospective look at the initial molecular replacement calculations provides interesting insights about how the peculiar packing mode and strong bias of the MR-phased electron density maps had hindered a successful solution of the structure by this method.

Cytokinins, or plant growth hormones, bind with very high affinity to cytokinin-specific binding proteins (CSBPs). Recombinant mung bean CSBP has been overexpressed in Escherichia coli and crystallized in complex with zeatin, a natural plant growth hormone. The crystals belong to the hexagonal system, space group P6(2) or P6(4), with unit-cell parameters a = 113.62, c = 86.85 A, contain two to five copies of the protein in the asymmetric unit and diffract X-rays to 1.25 A resolution.

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The high-resolution (1.2 Å) crystal structure a cytokinin-specific binding protein from mung bean (VrCSBP) complexed with zeatin reveals that the protein, structurally resembles plant pathogenesis related proteins of class 10 (PR-10), despite a low sequence conservation (below 20%). The four VrCSBP molecules present in the asymmetric unit assemble into two dimers. Between the concave face of the molecular-sheet and the C-terminal helix, a binding pocket is formed where the zeatin molecules are located. Surprisingly, in three (out of the four) binding pockets two zeatin molecules are found, with excellent definition in the electron density maps. In one of the binding sites (observed also in the forth, single-site, VrCSBP molecule), the ligand molecules, located deep in the cavity, have identical conformation and hydrogen-bonding pattern. In the second binding site, at the entrance to the internal cavity, the ligand molecules show variable, but clearly defined, binding modes.

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