Lupinus luteus Pathogenesis-Related Protein as a Reservoir for Cytokinin Humberto Fernandes 1 , Oliwia Pasternak 1 , Grzegorz Bujacz 1,2 , Anna Bujacz 2 , Michal M. Sikorski 1 and Mariusz Jaskolski 1,3 ⁎ 1 Center for Biocrystallographic Research, Institute of Bioorganic Chemistry, Polish Academy of Sciences, 61-704 Poznan, Poland 2 Institute of Technical Biochemistry, Technical University of Lodz, 90-924 Lodz, Poland 3 Department of Crystallography, Faculty of Chemistry, A. Mickiewicz University, 60-780 Poznan, Poland Received 20 January 2008; received in revised form 7 March 2008; accepted 12 March 2008 Available online 19 March 2008 Plant pathogenesis-related (PR) proteins of class 10 (PR-10) are small and cytosolic. The main feature of their three-dimensional structure is a large cavity between a seven-stranded antiparallel β-sheet and a long C-terminal α-helix. Although PR-10 proteins are abundant in plants, their physiological role remains unknown. Recent data have indicated ligand binding as their possible biological function. The article describes the structure of a complex between a classic PR-10 protein (yellow lupine LlPR-10.2B) and the plant hormone, trans-zeatin. Previously, trans-zeatin binding has been reported in a structurally related cytokinin-specific binding protein, which has a distant sequence relation with classic PR-10 proteins. In the present 1.35 Å resolution crystallographic model, three perfectly ordered zeatin molecules are found in the binding cavity of the protein. The fact that three zeatin molecules are bound by the protein when only a fourfold molar excess of the ligand was used indicates an unusual type of affinity for this ligand and suggests that LlPR-10.2B, and perhaps other PR-10 proteins as well, acts as a reservoir of cytokinin molecules in the aqueous environment of the cell. © 2008 Elsevier Ltd. All rights reserved. Edited by R. Huber Keywords: plant hormones; cytokinin; trans-zeatin; plant PR-10 protein; yellow lupine Introduction Plants forced to grow in harsh conditions and exposed to pathogens have developed several means of defense. Besides establishing a physical barrier by strengthening their cell walls, plants also produce antibiotic compounds called phytoa- lexins and accelerate cell death to suppress the spread of infectious pathogens. The expression of a number of genes is induced by various types of pathogens or by chemicals such as ethylene and salicylic acid, which mimic the effect of pathogen infection and thus induce stress. 1 These genes code the so-called pathogenesis-related (PR) proteins. 2 PR proteins are grouped into 17 classes according to their biological activity or physicochemical properties and sequence homology. 2,3 According to accepted definition, they are synthesized de novo under stress conditions. Among them are chitinases, glucanases, enzymes of phenylpropanoid pathway, thionins, osmotins, proteases, proline-rich glycopro- teins, and PR class 10 (PR-10) proteins of unknown function, also known as intracellular pathogenesis- related (IPR) proteins. PR-10 proteins have been identified in more than 70 species belonging to both monocotyledonous and dicotyledonous plants. 4 They are small (155–163 residues), slightly acidic, resistant to proteases and are usually encoded by multigene families (for instance, 10 genes in yellow lupine, divided into two subclasses), 5 suggesting *Corresponding author. Department of Crystallography, Faculty of Chemistry, A. Mickiewicz University, Grunwaldzka 6, 68-780 Poland. E-mail address: mariuszj@amu.edu.pl. Abbreviations used: CSBP, cytokinin-specific binding protein; IPR, intracellular pathogenesis-related protein; LlPR-10.2B, Lupinus luteus (yellow lupine) PR-10 protein of subclass 2B; MLP, major latex protein; PR, pathogenesis-related; RMS, root-mean-square; STAR, steroidogenic acute regulatory protein; START, star-related lipid transfer. doi:10.1016/j.jmb.2008.03.027 J. Mol. Biol. (2008) 378, 1040–1051 Available online at www.sciencedirect.com 0022-2836/$ - see front matter © 2008 Elsevier Ltd. All rights reserved.