Physical, chemical and biological stress factors, such as microbial infection, upregulate the transcription levels of a number of plant genes, coding for the so‐called pathogenesis‐related (PR) proteins. For PR proteins of class‐10 (PR‐10), the biological function remains unclear, despite two decades of scientific research. PR‐10 proteins have a wide distribution throughout the plant kingdom and the class members share size and secondary structure organization. Throughout the years, we and other groups have determined the structures of a number of PR‐10 proteins, both in the crystalline state by X‐ray diffraction and in solution by NMR spectroscopy. Despite the accumulating structural information, our understanding of PR‐10 function is still limited. PR‐10 proteins are rather small (~ 160 amino acids) with a fold consisting of three α helices and seven antiparallel β strands. These structural elements enclose a large hydrophobic cavity that is most probably the key to their functional relevance. Also, the outer surface of these proteins is of extreme interest, as epitopes from a PR‐10 subclass cause allergic reactions in humans.

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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Plant pathogenesis‐related (PR) proteins of class 10 are the only group among the 17 PR protein families that are intracellular and cytosolic. Sequence conservation and the wide distribution of PR‐10 proteins throughout the plant kingdom are an indication of an indispensable function in plants, but their true biological role remains obscure. Crystal and solution structures for several homologues have shown a similar overall fold with a vast internal cavity which, together with structural similarities to the steroidogenic acute regulatory protein‐related lipid transfer domain and cytokinin‐specific binding proteins, strongly indicate a ligand‐binding role for the PR‐10 proteins. This article describes the structure of a complex between a classic PR‐10 protein [Lupinus luteus (yellow lupine) PR‐10 protein of subclass 2, LlPR‐10.2B] and N,N′‐diphenylurea, a synthetic cytokinin. Synthetic cytokinins have been shown in various bioassays to exhibit activity similar to that of natural cytokinins. The present 1.95 Å resolution crystallographic model reveals four N,N′‐diphenylurea molecules in the hydrophobic cavity of the protein and a degree of conformational changes accompanying ligand binding. The structural adaptability of LlPR‐10.2B and its ability to bind different cytokinins suggest that this protein, and perhaps other PR‐10 proteins as well, can act as a reservoir of cytokinin molecules in the aqueous environment of a plant cell.

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.

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