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 ;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.

The RNA Hydrolysis and the Cytokinin Binding Activities of PR-10 Proteins Are Differently Performed by Two Isoforms of the Pru p 1 Peach Major Allergen and Are Possibly Functionally Related

“…The calorimetric titration experiments described in this study showed that Pru p 1.01 binds one zeatin molecule per protein monomer with a dissociation constant of approximately 10 mM, while Pru p 1.06D does not show any binding capability under the same experimental conditions. Previous measurements using ITC indicated a specific interaction of VrCSBP with zeatin, with affinity .10 times lower than that observed for Pru p 1.01 (Pasternak et al, 2006). Such functional divergence could be due to structural differences of the binding pocket in Pru p 1.01 compared to its homolog.…”

“…Such functional divergence could be due to structural differences of the binding pocket in Pru p 1.01 compared to its homolog. Indeed, the available crystal structures of PR-10 proteins in complex with zeatin show that the ligand-protein interaction is not structurally conserved: VrCSBP was crystallized with one or two zeatin molecules in the binding pocket (Pasternak et al, 2006). Conversely, the lupin LlPR-10.2B crystal structure shows three zeatin molecules bound inside the pocket (Fernandes et al, 2008).…”

“…In this regard, it is interesting to note that, while all PR-10 proteins contain a highly conserved and stable b-sheet, they show structural variability in the a-helical content. In particular, the conformation of helix a3 determines the accessibility, the size, and the shape of the hydrophobic cavity that holds zeatin or other ligands (Biesiadka et al, 2002;Pasternak et al, 2006;Fernandes et al, 2008Fernandes et al, , 2009). …”

“…Additional roles of PR-10 proteins could be related to their capability to bind hydrophobic ligands, such as fatty acids, flavonoids, and steroids (Neudecker et al, 2001;Mogensen et al, 2002;Koistinen et al, 2005). PR-10 proteins are also capable of binding plant hormones, such as cytokinins (CKs; Fujimoto et al, 1998;Pasternak et al, 2006;Fernandes et al, 2008). CKs are signal elements that regulate plant growth and development, but which are also recently emerging as important components of the plant defense strategy repertoire (Chung et al, 2008).…”

“…This is revealed by the structures of a number of PR-10 proteins obtained by NMR and x-ray crystallography, such as Bet v 1 from birch (Betula spp. ; Gajhede et al, 1996;Markovic-Housley et al, 2003), four isoforms of LlPR10 from yellow lupin (Lupinus luteus; Biesiadka et al, 2002;Pasternak et al, 2005;Fernandes et al, 2008), VrCSBP (cytokininspecific binding protein) from mung bean (Vigna radiata; Pasternak et al, 2006), Pru av 1 from cherry (Prunus avium; Neudecker et al, 2001), and Ap g 1 from celery (Apium graveolens; Schirmer et al, 2005).…”