Cytokinin-induced structural adaptability of a Lupinus luteus PR-10 protein Humberto Fernandes 1 , Anna Bujacz 2 , Grzegorz Bujacz 1,2 , Filip Jelen 3 , Michal Jasinski 1 , Piotr Kachlicki 4 , Jacek Otlewski 3 , Michal M. Sikorski 1 and Mariusz Jaskolski 1,5 1 Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland 2 Institute of Technical Biochemistry, Faculty of Biotechnology and Food Sciences, Technical University of Lodz, Poland 3 Department of Protein Engineering, Faculty of Biotechnology, University of Wroclaw, Poland 4 Institute of Plant Genetics, Polish Academy of Sciences, Poznan, Poland 5 Department of Crystallography, Faculty of Chemistry, A. Mickiewicz University, Poznan, Poland On detection of various types of pathogens, such as viruses, bacteria and fungi, or chemicals, such as ethyl- ene or salicylic acid, which mimic the effect of pathogen infection and thus induce stress [1], plants mount an efficient defence programme in which a number of genes are induced and expressed. Among them are genes coding the so-called pathogenesis-related (PR) proteins [2], which have been grouped into 17 classes according to their biological activity or physicochemical properties and sequence homology [2,3]. PR proteins do not constitute a superfamily of proteins, but rather represent a collection of unrelated protein families which function as part of the plant defence system [4]. Most PR proteins are either secreted or localized in the vacuoles. In contrast, PR proteins of class 10 (PR-10) are the only group that is intracellular and cytosolic [5]. Keywords cytokinin; N,N¢-diphenylurea; plant hormones; PR-10 proteins; yellow lupine Correspondence M. Jaskolski, Department of Crystallography, Faculty of Chemistry, A. Mickiewicz University, Grunwaldzka 6, 60–780 Poznan, Poland Fax: +48 61 829 1505 Tel: +48 61 829 1274 E-mail: mariuszj@amu.edu.pl (Received 3 December 2008, revised 8 January 2009, accepted 9 January 2009) doi:10.1111/j.1742-4658.2009.06892.x 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 func- tion 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 com- plex 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 cyto- kinin. Synthetic cytokinins have been shown in various bioassays to exhibit activity similar to that of natural cytokinins. The present 1.95 A ˚ 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. Abbreviations CPPU, N-phenyl-N¢-(2-chloro-4-pyridyl)urea; CSBP, cytokinin-specific binding protein; Hyp-1, phenolic oxidative coupling protein from Hypericum perforatum; ITC, isothermal titration calorimetry; LlPR-10.2B, Lupinus luteus (yellow lupine) PR-10 protein of subclass 2; N,N¢-DPU, N,N¢-diphenylurea; NCS, (S)-norcoclaurine synthase; PR, pathogenesis-related; StAR, steroidogenic acute regulatory protein; START, StAR-related lipid transfer. 1596 FEBS Journal 276 (2009) 1596–1609 ª 2009 The Authors Journal compilation ª 2009 FEBS PR-10 proteins, first identified in cultured parsley cells [6], are small (155–163 residues), slightly acidic and show resistance to proteases. They are usually encoded by multigene families (for instance, in yellow lupine, there are 10 known genes encoding PR-10 pro- teins [7]), suggesting that a number of different protein homologues can be expressed in various plant organs under different conditions. This makes the study of the function of PR-10 proteins very complex. It is believed that PR-10 proteins are an essential component of a plant defence programme because their genes are usually induced by the attack of vari- ous pathogens and by environmental stress. Some pr-10 genes are, however, expressed constitutively, suggesting a more general biological role of PR-10 proteins in plant development [8–10]. Several other lines of evidence have implicated PR-10 proteins, or at least proteins that are sequence-related to the PR-10 class, in enzymatic functions, such as RNA hydrolysis [11] and the synthesis of (S)-norcoclaurine [12] or hypericin [13]. However, it is obvious that these functions are not universal among all PR-10 proteins. Flores et al. [14] have also postulated a stor- age function for the PR-10 class. Other functions sometimes attributed to the PR-10 proteins include antimicrobial activities, i.e. antifungal [5,14–16], anti- bacterial [14] and antiviral [15] activity. Another prop- osition postulates a hydrophobic ligand-binding role for PR-10 class members. Crystal and solution structures are known for sev- eral PR-10 homologues [17–25]. They all reveal the same general fold consisting of a seven-stranded anti- parallel b-sheet wrapped around a long C-terminal a-helix. These two elements, together with two short helices, form a hydrophobic cavity, the volume of which is disproportionately large for proteins of this size. The abnormal size of the cavity has been taken as an indication of an important role in hydrophobic ligand binding. Supporting evidence for such a ligand-binding role is provided by the following observations: (a) the structural similarity between PR-10 members and the steroidogenic acute regula- tory protein (StAR)-related lipid transfer (START) domain of human MLN64 protein [26], which is a steroid-binding domain related to StAR, involved in cholesterol translocation in human placenta and brain; (b) the crystal structure of Betv1 (a birch- pollen PR-10 protein) complexed with deoxycholate [21]; (c) the structural similarity with cytokinin-specific binding protein (CSBP) [24]; and (d) the crystal struc- ture of a yellow lupine (Lupinus luteus) homologue, LlPR-10.2B, complexed with the adenine-type cyto- kinin hormone trans-zeatin [25]. In this study, we have investigated whether the same LlPR-10.2B protein also has the ability to bind artifi- cial cytokinin molecules, chemically synthesized as urea derivatives. Despite the apparent lack of chemical simi- larity between the adenine- and urea-type molecules, their cytokinin-type effect is very similar [27,28]. The synthetic cytokinin investigated in the present work is N,N¢-diphenylurea (N,N¢-DPU). The crystal structure of the LlPR-10.2B protein preincubated with N,N¢- DPU shows that the protein is indeed able to store several N,N¢-DPU molecules in the internal cavity. The protein’s ability to bind N,N¢-DPU and other cyt- okinins has also been studied by isothermal titration calorimetry (ITC). In addition, the antifungal activity of the LlPR-10.2B protein has been investigated. Results Asymmetric unit contents The crystal asymmetric unit contains one LlPR-10.2B protein molecule complexed with four N,N¢-DPU mol- ecules, 134 modelled water molecules and two sodium cations. The metal cations have an octahedral coordi- nation close to loop L3 (Na1), where the ligands are three carbonyl O atoms (Pro31, Val34, Ile37) and three water molecules, and close to loop L9 (Na2), where the ligands are two carbonyl O atoms (Thr121, Gly123), the Oc1 atom of Thr121 and three water molecules. The correctness of the interpretation of the metal sites as sodium is confirmed by the satisfactory refinement of the B factors (34.4 and 41.0 A ˚ 2 for Na1 and Na2, respectively), by the final Na + ÆÆÆO distances (2.1–2.9 A ˚ ) and by the bond-valence test [29]. Model quality and overall folding The refined 1.95 A ˚ resolution crystallographic model of the LlPR-10.2B protein has good overall geometry and Ramachandran statistics (Table 1). The quality of the electron density maps is high and there are only a few less clear areas in the loop regions and at the C-terminus. The recombinant protein lacks the N-terminal methionine, excised during expression by Escherichia coli methionylaminopeptidase [30], whose catalytic efficiency is inversely proportional to the size of the side-chain of the amino acid in the penultimate position (Gly in the LlPR-10.2B sequence). The LlPR-10.2B molecule has an overall fold consisting, as in other PR-10 class proteins, of three a-helices (1–3) and seven antiparallel b-strands (1–7), giving rise to an a + b fold, whose most pronounced features are a b-grip over the C-terminal a3-helix and H. Fernandes et al. PR-10 protein–N,N ¢-DPU complex FEBS Journal 276 (2009) 1596–1609 ª 2009 The Authors Journal compilation ª 2009 FEBS 1597 a vast cavity bounded by all the helices and the con- cave face of the b-sheet. The strands of the b-sheet are connected by b-hairpins, except for the connection between the b1 and b2 edges of the sheet, which are joined by a right-handed crossover involving the a1 and a2-helices. There are seven b-bulges, which distort the regularity of the b-sheet, endowing it with a highly curved shape (Fig. 1). The C-terminal a3-helix is structurally and sequen- tially the most divergent element of the PR-10 struc- ture (Figs 2 and 3A,B). This divergence is higher in the N-terminal half of the helix. The present protein, LlPR-10.2B, is a close homologue of LlPR-10.2A, characterized structurally by Pasternak et al. [22]. Although the two sequences are nearly identical (91% sequence identity), and there are no differences in the N-terminal part of the a3-helix (Fig. 2), the strong inward collapse in the middle of the a3-helix, reported for the ligand-free LlPR-10.2A molecule, is not observed in the LlPR-10.2B protein complexed with cytokinins, including the present structure (Fig. 3A,B). The straightening of the a3-helix significantly increases the volume of the internal cavity. One of the loops, L4, shows extraordinary rigidity despite the presence of four Gly residues in its sequence. This Gly-rich loop is characterized by excel- lent electron density, low B factors and the highest degree of PR-10 sequence conservation (Fig. 2). The sequence signature xEGxGGxGTx and structural conservation of the Gly-rich loop are observed even in distant homologues, such as CSBP. The rigidity of the loop is maintained by a pattern of three hydrogen bonds between the Oc1 atom of Thr51 and the main- chain groups of Gly47 (N–H) and Gly48 (N–H and C=O). The electron density maps for the four N,N¢-DPU ligands found in the internal cavity are of rather poor quality, contrasting with the excellent definition of the zeatin molecules in the LlPR-10.2B–zeatin complex [25]. In the present complex, the N,N¢-DPU molecules were modelled in elongated ‘tubes’ of 2F o ) F c electron density, which did not have sufficient features for unequivocal assignment of the ligand atoms (Fig. 4A). The evident disorder of the N,N¢-DPU molecules is the result of two factors: (a) the high degree of rotational freedom of the terminal phenyl rings (Fig. 4B); and (b) the lack of specific interactions that would anchor the N,N¢-DPU molecules to the protein framework. Table 1. Data collection and refinement statistics. Data collection Space group C222 1 Cell parameters (A ˚ ) a = 34.4, b = 73.2, c = 100.0 Resolution limits (last shell) (A ˚ ) 40.0–1.95 (2.02–1.95) Radiation source DESY, X11 (EMBL) Wavelength (A ˚ ) 0.8100 Temperature (K) 100 No. measured reflections 54070 No. unique reflections 9539 R int (last shell) 0.075 (0.398) Completeness (last shell) (%) 99.4 (96.8) Redundancy 5.7 <I ⁄ r(I)> (last shell) 20.8 (3.8) Refinement statistics Program used REFMAC5 Resolution limits (A ˚ ) 15.0–1.95 No. reflections 8631 No. reflections in test set 860 No. atoms Protein 1169 Ligand 64 Metal 2 (Na) Water molecules 134 R ⁄ R free 0.193 ⁄ 0.246 ÆBæ (A ˚ 2 ) Protein atoms 39.6 Ligand atoms 36.0 Water molecules 45.2 Rmsd from ideal Bond lengths (A ˚ ) 0.018 Bond angles (deg) 1.88 Chiral volumes (A ˚ 3 ) 0.106 Ramachandran u ⁄ w angles (%) Most favoured 93.8 Additionally allowed 6.2 Fig. 1. Overall fold of the LlPR-10.2B protein molecule with annota- tion of secondary structural elements. The four N,N ¢-DPU mole- cules found inside the binding cavity are shown in a ball-and-stick representation. The binding cavity is represented as a mesh cast, calculated in VOIDOO [52]. Sodium cations are represented as spheres. This and all other structural illustrations have been prepared using PYMOL [53]. PR-10 protein–N,N ¢-DPU complex H. Fernandes et al. 1598 FEBS Journal 276 (2009) 1596–1609 ª 2009 The Authors Journal compilation ª 2009 FEBS The only interatomic contacts of the N,N¢-DPU ligands are three N–HÆÆÆO hydrogen bonds to water molecules that are loosely positioned within the bind- ing cavity (Fig. 5). In view of the crude modelling, one could question whether the inclusion of the four N,N¢- DPU molecules is correct. However, as the crystalliza- tion buffer contained only N,N¢-DPU molecules and citrate anions at high abundance, and as the ligand electron density is definitely not compatible with the branched structure of the hydrophilic citrate moiety, the assumption that the binding cavity is occupied by N,N¢-DPU molecules is the only logical conclusion. The binding cavity The cavity enclosed within the LlPR-10.2B protein core in the present N,N¢-DPU complex has a large vol- ume, calculated as 3600 A ˚ 3 by the surfnet program [31], and can be accessed by two openings. The larger opening is located between the a3-helix and loops L3, L5, L7 and L9, and is gated by a salt bridge between Arg138 (a3-helix) and Glu59 (b3) and by a water- bridged contact between the side-chains of Glu131 (a3-helix) and Thr93 (loop L7). The second opening is found between the b1-strand and the a3-helix. The additional entrances seen near b5 ⁄ L7 ⁄ b6 and (a small one) near L2 ⁄ L4 in the crystal structure of the LlPR- 10.2B–zeatin complex [25] are closed in the present N,N¢-DPU complex. These differences are a result of a major conformational rearrangement in loop L7 (Fig. 3C), and of smaller but significant changes in loops L2 and L4. Despite the closure of the b5 ⁄ L7 ⁄ b6 entrance in the present complex, a similar situation to that detected for the LlPR-10.2B–zeatin complex [25] exists, with a water molecule (W168 in the present complex) disrupting the b5–b6-sheet near loop L7 (Fig. 6). In addition to the different numbers of entrances, the cavities also have significantly different contents in the two complexes. In the present LlPR-10.2B–N,N¢- DPU complex, the cavity is occupied by four N,N¢- DPU molecules (DPU1–4) and eight water molecules (Fig. 5), whereas three zeatin molecules and 25 water molecules fill the cavity of the LlPR-10.2B–zeatin com- plex. In reality, there are less than four N,N¢-DPU molecules inside the cavity, as one of the phenyl rings of the DPU4 molecule, pointing towards the b1–a3 opening (Fig. 1), is outside the cavity. A remarkable feature of N,N¢-DPU binding in the hydrophobic cavity is the lack of any specific interatomic inter- Fig. 2. Sequence alignment, calculated in CLUSTALW [54], for representative PR-10 proteins, for which crystal structures complexed with small-molecule ligands (or in apo form for LlPR10.2A) have been determined. LlPR10.2B ⁄ DPU, the present structure of a yellow lupine pro- tein complex with N,N¢-DPU; LlPR10.2B ⁄ zea, the same protein complexed with trans-zeatin [25]; LlPR10.2A, a close homologue from yellow lupine [22]; VrCSBP ⁄ zea, mung bean CSBP complexed with zeatin [24]; Betv1 ⁄ deox, birch pollen PR-10 protein complexed with deoxycho- late [21]. Residues identical in all sequences are marked with an asterisk. The symbols ‘:’ and ‘.’ below the sequences indicate conservative and semiconservative substitutions, respectively. The differences between the two members of the yellow lupine PR-10.2 subfamily are marked with |. The boxes outline regions that are most conserved (full line) or most divergent (broken line). The residues marked in grey interact with the ligands via van der Waals’ contacts, and those in black via hydrogen bonds. The secondary structural elements correspond to the LlPR10.2B ⁄ DPU structure. Residues implicated in RNase activity in other PR-10 proteins are underlined in the LlPR10.2B sequence. H. Fernandes et al. PR-10 protein–N,N ¢-DPU complex FEBS Journal 276 (2009) 1596–1609 ª 2009 The Authors Journal compilation ª 2009 FEBS 1599 actions, such as hydrogen bonds, between the protein and the N,N¢-DPU molecules (Figs 2 and 5). The differences in stoichiometry as well as the orien- tation and binding mode of the ligand molecules in the two LlPR-10.2B complexes result in a rearrangement of a number of side-chains that are pointing into the interior of the cavity. Clearly, some of these rearrange- ments are simple translations following the shifts of the Ca atoms that are connected with a remodelling of the protein backbone (Phe5, Tyr19, Val23, Ile30, Val34, Thr36, Ile37, Val40, Ile94, Ile97, Phe99, Thr101, Val115, Pro127, Asn128). Other side-chains, however, clearly have different conformations in the two complexes, ranging from minimal changes for the residues Asp7, Tyr9, Leu22, Phe57, Glu59, Lys64, Val66, Tyr80, Tyr82, Leu103, Ile119 and Phe142, to major conformational changes for the residues Lys53, Ile84, Ile117, Glu131, Arg138 and Phe143. Another interesting aspect is the number of residues, with side-chains pointing into the cavity, that have been modelled in double conformation. Unlike the LlPR- 10.2B–zeatin complex, where only one residue (Val66) has dual conformation, in the present complex two cavity-forming residues (Leu55 and His68) are in double conformation. The LlPR-10.2B–N,N¢-DPU complex In the present complex crystal structure, four N,N¢- DPU molecules have been modelled in the electron density (Figs 1 and 4A) with full occupancies. The average B factors of the N,N¢-DPU molecules are 31.8, 33.1, 34.2 and 45.0 A ˚ 2 for DPU1, DPU2, DPU3 and DPU4, respectively. DPU1 is found deep in the cavity close to the a1 and a2-helices (Fig. 1). It is anchored to the protein by several van der Waals’ contacts involving Leu22, Val23, Leu55, His68, Tyr80 and Phe142. DPU2 is placed near the a3 ⁄ L3 ⁄ L5 ⁄ L7 ⁄ L9 opening of the cavity (Fig. 1) and establishes van der Waals’ contacts with Ile37, Phe57, Ile58 and Arg138. DPU3 is aligned with the a3-helix near the b1 ⁄ a3 opening (Fig. 1) in an ori- entation that is roughly parallel to the DPU1 mole- cule. It is anchored to the protein via van der Waals’ contacts with Tyr9, Leu22, Phe99, Thr101, Leu103, Gly113, Val115, Gly139 and Phe143. DPU3 also α3 A BC D L9 L7 Fig. 3. Superposition of PR-10 structures, calculated with Lsqkab [42]. (A) Superposi- tion of four proteins with the PR-10 fold. Colour code: yellow, LlPR-10.2B–N,N ¢-DPU (Protein Data Bank code 3e85), this work; red, LlPR-10.2B–zeatin (2qim); magenta, LlPR-10.2A molecule A (1xdf); blue, CSBP– zeatin molecule B (2flh). The orientation emphasizes the differences in loops L7 and L9, and the C-terminal a3-helix. (B) ‘Sausage’ representation of the deviations between the Ca atoms of LlPR-10.2B–N,N¢- DPU and LlPR-10.2A. (C) ‘Sausage’ repre- sentation of the deviations between the Ca atoms of LlPR-10.2B–N,N ¢-DPU and LlPR- 10.2B–zeatin. (D) Stereoview of a superposi- tion of LlPR-10.2B–N,N¢-DPU (yellow ⁄ green) and LlPR-10.2B–zeatin (red ⁄ aqua blue) com- plexes. The C-terminal a3-helix has been omitted for clarity. PR-10 protein–N,N ¢-DPU complex H. Fernandes et al. 1600 FEBS Journal 276 (2009) 1596–1609 ª 2009 The Authors Journal compilation ª 2009 FEBS A B Fig. 4. (A) The four N,N¢-DPU molecules present in the crystal structure of the LlPR-10.2B–N,N¢-DPU complex. The 2F o ) F c electron density maps are contoured at the 1.0r level. (B) Conformation of the N,N¢-DPU molecules shown in a superposition of the urea moiety. The atom numbering scheme of the N,N¢-DPU molecule is also shown. Fig. 5. Illustration of the four N,N¢-DPU molecules found in the protein cavity, adapted from a LIGPLOT representation [55]. Hydrogen bonds are indicated by broken lines and van der Waals’ contacts by radial lines. Water molecules are shown as small grey spheres. H. Fernandes et al. PR-10 protein–N,N ¢-DPU complex FEBS Journal 276 (2009) 1596–1609 ª 2009 The Authors Journal compilation ª 2009 FEBS 1601 makes an indirect hydrogen bond contact with the pro- tein (Tyr82), mediated by a water molecule (W197) (Fig. 5). DPU4 is placed at the N-terminal part of the a3-helix, with the two phenyl rings pointing at the two different entrances to the cavity, namely b1 ⁄ a3 and a3 ⁄ L3 ⁄ L5 ⁄ L7 ⁄ L9 (Fig. 1). It is anchored to the protein via van der Waals’ contacts with Phe5, Gly89, Phe99, Ile117, Gly132, Ala135 and Arg138. The N,N¢-DPU ligands also establish van der Waals’ contacts between themselves, namely within the pairs DPU1–DPU2 and DPU1–DPU3. The N,N¢-DPU molecules have a rigid and planar carbonyl diamide group with two lateral phenyl substi- tutes at different orientations (Fig. 4B). In their elon- gated electron density, the N,N¢-DPU molecules can move along their long axes. This is consistent with the lack of direct hydrogen bond interactions with the protein scaffold. The N- and C-termini In the PR-10 protein topology, the amino terminus forms the b1-strand of the b-sheet and is typically highly ordered. In the present LlPR-10.2B–N,N¢-DPU complex, the presence of the sodium cation (Na2) coordinated in loop L9 pushes the first two amino acid residues of b1 away from the b-sheet. Because of the presence of the cation, the N-terminal NH 3 + group is not stabilized by the conserved hydrogen bonds to resi- dues in loop L9 [(Thr121)O, (Gly123)O, (Thr121)Oc1] seen in other yellow lupine PR-10 structures [20,22,25]. Nonetheless, the N-terminal part of the protein is not disordered as it has a very clear electron density. The presence of Na2 also induces a shift of loop L9 relative to the LlPR-10.2B–zeatin structure, where no cation was observed in this region (Fig. 3A,C). The sodium cation Na2 is thus responsible for conformational changes at both loop L9 and the N-terminus of the protein. However, the carboxyl terminus is less ordered, and no electron density is visible for the last three residues. Consequently, Pro154 is the last amino acid included in the model. Structural comparisons of PR-10 proteins All the known models of PR-10 proteins have a com- mon canonical fold, consisting of a seven-stranded antiparallel b-sheet wrapped around a long C-terminal a-helix. These elements, together with two short heli- ces, form a large hydrophobic cavity. Because of its unusual volume, particularly in view of the small size of the PR-10 proteins, the large cavity is assumed to have evolved for hydrophobic ligand binding. Despite the same overall fold, the superposition of the different PR-10 models reveals interesting struc- tural differences. The most significant is the strong inward kink of the C-terminal a3-helix observed for the ligand-free LlPR-10.2A protein [22], but not in the trans-zeatin complex of LlPR-10.2B [25]. As the sequences of the LlPR-10.2A and LlPR-10.2B homo- logues are 96% similar, and there are no differences in the sequence surrounding the point of bending (Phe142) (Fig. 2), the observed structural difference was assumed to be a manifestation of the adaptability of the PR-10 fold to the presence of the hormone ligands [25]. As a consequence of the straightening of the a3-helix, the cavity of LlPR-10.2B complexed with trans-zeatin has a volume of 4500 A ˚ 3 , in contrast with the volume of only 2000 A ˚ 3 observed in the ligand-free LlPR-10.2A structure. This ligand-induced adaptability hypothesis is reinforced by the present crystal structure of the LlPR-10.2B–N,N¢-DPU complex, which shows AB Fig. 6. Disruption of the antiparallel b-sheet in the region of the b 5–b6-strands by a water molecule. (A) Ball-and-stick represen- tation and 2F o ) F c electron density map contoured at the 1.3r level. (B) Cartoon representation of the protein with the region shown in (A) highlighted in dark blue. PR-10 protein–N,N ¢-DPU complex H. Fernandes et al. 1602 FEBS Journal 276 (2009) 1596–1609 ª 2009 The Authors Journal compilation ª 2009 FEBS the same difference, although of a smaller magnitude (Fig. 3A,C). The volume of the cavity in the present LlPR-10.2B–N,N¢-DPU complex is 3600 A ˚ 3 , i.e. it is smaller than in the zeatin complex, but still much larger then in apo LlPR-10.2A. These observations indicate an induced structural flexibility of yellow lupine PR-10 proteins, and perhaps also of homo- logues from other species. Comparison of all the struc- tures of PR-10 proteins demonstrates a huge variability in the volume of the cavity, which ranges from 4500 A ˚ 3 in the LlPR-10.2B–zeatin complex to 1100 A ˚ 3 in the CSBP–zeatin complex (molecule C). In addition to the difference in the cavity volume, the two complexes of the LlPR-10.2B protein, one with N,N¢-DPU and the other with trans-zeatin, show a dif- ference in the identity and coordination sites of metal ions. In contrast with the two sodium cations coordi- nated close to loops L3 and L9 in the present LlPR- 10.2B–N,N¢-DPU complex, in the LlPR-10.2B–zeatin complex only one metal site close to loop L3, and identified as calcium, was detected [25]. Metal cations were found in two other PR-10 protein structures, namely in LlPR-10.2A [22] and CSBP [24] (sodium in both cases), where they were coordinated by the L3 and L9 loops, respectively. Cytokinin-binding assays To verify the structurally derived data, we used direct measurement of the thermodynamic parameters of LlPR-10.2B–ligand interactions. The cytokinin-binding capability of LlPR-10.2B was tested by ITC for natu- ral adenine-type (trans-zeatin, kinetin) and artificial urea-type [N,N¢-DPU, N-phenyl-N¢-(2-chloro-4-pyr- idyl)urea (CPPU)] hormones. The ITC assays were performed using different protein concentrations and different experimental conditions, as summarized in Experimental procedures. Under the experimental con- ditions, limited by the low solubility of the ligands, the ITC data show that the LlPR-10.2B protein can bind all four cytokinins. The data from the calorimetric measurements were analysed by fitting independent- and multiple-site models, as well as a cooperative model, to the raw data. Only the independent-site model provided meaningful results. The LlPR-10.2B protein has one binding site characterized by a K d value in the micromolar range and a different stoichi- ometry depending on the ligand molecule. LlPR- 10.2B–N,N¢-DPU binding is exothermic with a 1 : 1 stoichiometry (n = 0.83) and a K d value of 4.2 ± 1.6 lm (Fig. 7). Titration with trans-zeatin revealed an additional binding site, with a K d value in the submil- limolar range. Thus, the LlPR-10.2B–zeatin complex is characterized by two binding sites, one with a K d value of 12.3 ± 5.1 lm and 1 : 1 stoichiometry, and the other with a K d value of 193 ± 43.7 lm and 8 : 1 stoi- chiometry. CPPU and kinetin show clear interaction with the LlPR-10.2B protein; however, the thermo- dynamic parameters could not be determined accu- rately because of the low solubility of both ligands. Antifungal assays Many PR proteins have long been known to have anti- fungal or antibacterial activities [32–34], but, until Fig. 7. Calorimetric titration of LlPR-10.2B with N,N¢-DPU. The top panel shows raw heat data corrected for baseline drift obtained from 23 consecutive injections of 3.02 m M N,N¢-DPU into the sample cell (750 lL) containing 0.37 m M LlPR-10.2B protein in 3m M citrate buffer, pH 6.3, at 20 °C. The bottom panel shows the binding isotherm created by plotting the heat peak areas against the molar ratio of N,N ¢-DPU added to LlPR-10.2B present in the sample cell. The heats of mixing (dilution) were subtracted. The line represents the best fit to a model of n independent sites. LlPR- 10.2B–N,N¢-DPU binding is exothermic with 1 : 1 stoichiometry (n = 0.83) and a K d value of 4.2 ± 1.6 lM. H. Fernandes et al. PR-10 protein–N,N ¢-DPU complex FEBS Journal 276 (2009) 1596–1609 ª 2009 The Authors Journal compilation ª 2009 FEBS 1603 recently, the PR-10 family was not included in this group. The antifungal activity of PR-10 proteins was first demonstrated in 2002 for ocatin, a PR-10 homo- logue from the Andean tuber crop oca (Oxalis tuberose Mol.) [14], later for the hot pepper (Capsicum annuum) CaPR-10 protein [15], and recently for the yellow-fruit nightshade (Solanum surattense) SsPR-10 protein [16] and the peanut (Arachis hypogaea) AhPR-10 protein [5]. In general, recombinant proteins were tested for their ability to inhibit specific fungal growth. The effect of recombinant LlPR-10.2B on the in vitro growth of pathogenic fungi has been investi- gated in this work. For the assays, a lupine-specific fungus and two fungi specific for other plants belong- ing to the same class (Magnoliopsida) were used, namely Colletotrichum lupini, Leptosphaeria maculans and Leptosphaeria biglobosa. Purified recombinant LlPR-10.2B protein did not inhibit the growth of any of the fungi under study (not shown). Discussion The search for the biological role of the PR-10 pro- teins has focused recently on the abnormal size of the internal cavity, which could function as a binding site ⁄ reservoir for hydrophobic ligands in the aqueous environment of the plant cell. This proposition finds support in the structural similarity between classic PR-10 proteins and ligand-binding proteins such as CSBP and the START domain [24,26]. In addition, several studies have demonstrated the ability of PR-10 proteins to bind steroids, cytokinins, fatty acids and flavonoids [21,35,36]. A different possible function of PR-10 proteins has emerged from several other studies, connected with the enzymatic biosynthesis of second- ary metabolites, such as (S)-norcoclaurine [12] or hypericin [13]. The enzymes catalysing the above reac- tions [(S)-norcoclaurine synthase (NCS) and phenolic oxidative coupling protein from Hypericum perforatum (Hyp-1), respectively] have been postulated to belong to the PR-10 structural class of proteins based only on sequence comparisons (sequence identity about 38% for NCS and 45% for Hyp-1). Recently, these predic- tions have been confirmed for NCS [37], but further work will be necessary to verify these assumptions for the Hyp-1 protein [38]. However, in view of their high level of expression, a universal catalytic function for all PR-10 proteins seems unlikely. The present work provides structural evidence that classic PR-10 proteins can bind N,N¢-DPU, the first synthetic cytokinin to be identified [27]. Cytokinins are structurally diverse and biologically versatile phytohor- mones, involved in the differentiation of shoot meri- stem and root tissues, leaf formation and senescence, chloroplast development, etc. [39]. The natural cytoki- nins are adenine derivatives and can be classified by the character of their N6 substituent as isoprenoid, aromatic or furfuryl cytokinins. Cytokinins with an unsaturated isoprenoid side-chain are by far the most prevalent, in particular those with a trans-hydroxylated substituent (trans-zeatin and its derivatives). Various phenylurea derivatives constitute a group of synthetic cytokinins, some of which are highly active, e.g. N,N¢- DPU, CPPU or thidiazuron [28]. The phenylurea derivatives have been shown to exhibit biological activ- ity very similar to that of N6-substituted adenine derivatives in various cytokinin bioassays. These com- pounds were developed for commercial use as defoli- ants in cotton and other crops, and are now widely used as cytokinins in higher plant tissue cultures and micropropagation protocols [40]. The similarity of the biological activity of two structurally unrelated classes of compounds has posed one of the more interesting problems in the study of cytokinin structure–function relationships [41]. The present LlPR-10.2B–N,N¢-DPU complex is the second example of a cytokinin complex of this protein. Recently, we reported the crystal structure of LlPR- 10.2B complexed with trans-zeatin [25]. The binding mode of the two cytokinins is different (Figs 2 and 3D) which, in view of the chemical difference between these ligands, is not surprising. The largest difference is in the stoichiometry of the complexes. In the present LlPR-10.2B–N,N¢-DPU complex, four N,N¢-DPU mol- ecules are accommodated in the hydrophobic cavity of the protein, in contrast with the 3 : 1 stoichiometry of the complex with trans-zeatin. This remarkable ability of the hydrophobic cavity of PR-10 proteins to hold a variable number of ligand molecules is highlighted when the CSBP–zeatin complex is considered, in which a single CSBP molecule can bind either one or two trans-zeatin ligands [24]. The second aspect that differentiates the two LlPR- 10.2B complexes is the protein–ligand interactions. Although, in the case of trans-zeatin, several hydrogen bonds anchor the ligand molecules to the protein [25] (Fig. 2), the N,N¢-DPU molecules interact with the protein virtually exclusively via van der Waals’ con- tacts (Figs 2 and 5). Thirdly, there is no direct corre- spondence between the ligand molecules in the two complexes. In general terms, the N,N¢-DPU molecules 1–3 and zeatin 1–3 occupy similar spatial positions inside the protein cavity, but without individual over- lap (Fig. 3D). DPU4 is placed at a distinct position that does not overlap with any zeatin molecule (Fig. 3D). PR-10 protein–N,N ¢-DPU complex H. Fernandes et al. 1604 FEBS Journal 276 (2009) 1596–1609 ª 2009 The Authors Journal compilation ª 2009 FEBS A structural superposition of the LlPR-10.2B mole- cules in the two complexes reveals that their Ca traces show small but significant differences, despite the same overall fold (Fig. 3A). The rmsd between their Ca coordinates is 0.77 A ˚ , with the major differences local- ized in loop L7 (maximum deviation of 9.3 A ˚ for the Ca atoms of Gly89) and loop L9 (maximum deviation of 4.7 A ˚ at Gly123). The conformational change of loop L9 is evidently caused by the presence of the sodium cation (Na2) coordinated in this area in the LlPR-10.2B–N,N¢-DPU structure. In the LlPR-10.2B– N,N¢-DPU complex, this cation disrupts the stabilizing hydrogen bond between the N-terminus and loop L9 that is observed in all other yellow lupine PR-10 struc- tures [20,22,25], as well as the typical b-sheet associa- tion with the b7-strand. This disruption, however, does not create any major disorder of the N-terminus as the residues have excellent definition in the electron density map. The other sodium cation (Na1) is coordinated by loop L3 at the same position as the calcium ion in the LlPR-10.2B–zeatin structure [25]. The change in metal identity is most probably caused by the high sodium concentration in the crystallization buffer. The sodium site at loop L9 is new in this yellow lupine PR-10 structure, but it is interesting to note that the same site was occupied by a metal cation in the crystal structure of mung bean CSBP [24]. As described above, the major folding differences between the two LlPR-10.2B models are localized at loop L7. This reshaping cannot be explained by the different packing modes (C222 1 and P6 5 for LlPR- 10.2B–N,N¢-DPU and LlPR-10.2B–zeatin, respec- tively), and thus it must be concluded that it results from the different ligand cargo present in the cavity. In both cases, the lattice interactions are weak, with only one salt bridge (Asp92ÆÆÆLys20) present in the LlPR-10.2B–N,N¢-DPU structure, and one main-chain hydrogen bond (Gly89ÆÆÆVal2) in the LlPR-10.2B– zeatin structure. More revealing than a simple Ca alignment in this case of identical ligand-binding structures is an all- atom superposition of the protein scaffolds. Such a superposition, calculated in Lsqkab [42], is character- ized by an rmsd of 1.8 A ˚ . The largest difference of 13.5 A ˚ is found for the Cc2 atoms of Leu90. Several reports have shown recently that PR-10 pro- teins can exhibit antifungal properties [5,14–16]. Some authors have associated the antifungal activity of PR-10 proteins with their purported RNase activity. Chadha and Das [5] reported, for example, that these activities are linked in AhPR-10, a PR-10 protein from peanut. Previously, Moiseyev et al. [43] hypothesized that the residues Lys54, Glu96, Glu148 and Tyr150 (ginseng ribonuclease 1 sequence) are responsible for RNase activity, and thus can be expected to be vital for antifungal activity. As the indicated residues are fully conserved in the LlPR-10.2B protein sequence (Fig. 2), antifungal activity could be expected for this protein as well. However, in our experiments, no anti- fungal activity could be observed for any of the fungi tested (C. lupini, L. maculans and L. biglobosa). The fungus C. lupini is specific for lupine plants and infects the leaves. The remaining two fungi are pathogens of oilseed rape (Brassica napus). The two crystal structures of LlPR-10.2B show that it can serve as a ligand-binding protein. The structures provide evidence of the ability of the LlPR-10.2B pro- tein to bind cytokinins, either natural (trans-zeatin) or synthetic (N,N¢-DPU). Under the experimental condi- tions, limited by the low solubility of the ligands in water-based buffers, ITC binding affinity data for the four cytokinins were obtained. Two of them represent the synthetic group (N,N¢-DPU and CPPU) and the other two are natural cytokinins, with one example of isopentenyl (trans-zeatin) and one of furfuryl (kinetin) N6-substituted adenine. The three crystal structures of PR-10-type proteins complexed with cytokinins, namely CSBP–zeatin [24], LlPR-10.2B–zeatin [25] and LlPR-10.2B–N,N¢-DPU (present work), together with cytokinin-binding affinity studies for the CSBP [24] and LlPR-10.2B (present work) proteins, provide a broad view of the inter- actions of these plant hormones with PR-10 and PR-10-like folded proteins. One of the binding sites for trans-zeatin observed for the LlPR-10.2B protein has a similar K d value (193 lm) to that obtained for CSBP (106 lm) [24]. However, the ITC-determined stoichi- ometry is different. The other binding site observed for LlPR-10.2B with all four cytokinin ligands, character- ized by a K d value in the micromolar range, was not observed for CSBP. The CSBP–N,N¢-DPU complex has not been investigated, and so no comparison with LlPR-10.2B can be made. However, both proteins interact with kinetin and CPPU, suggesting that a CSBP–N,N¢-DPU complex is also possible. The different ligand-binding characteristics of the LlPR-10.2B and CSBP proteins may be a result of the large difference in the volume of the binding cavity (3600–4500 A ˚ 3 for LlPR-10.2B, 1100–1600 A ˚ 3 for CSBP), as measured by the surfnet program [31]. The smaller cavity volume of CSBP results from the C-terminal a3-helix being less separated from the b-grip, and the a1 and a2-helices being closer to the centre of the protein. The crystal structure of the LlPR-10.2B–N,N¢-DPU complex shows four N,N¢-DPU molecules inside the H. Fernandes et al. PR-10 protein–N,N ¢-DPU complex FEBS Journal 276 (2009) 1596–1609 ª 2009 The Authors Journal compilation ª 2009 FEBS 1605 [...]... that the PR-10 proteins indeed bind hydrophobic ligands, and that, on binding, the protein undergoes structural adaptation Moreover, the flexible ligands are also subject to conformational changes Such a situation, in which the binding partners are capable of mutual adjustments, together with an association that is characterized by relatively low binding constants, suggests that the PR-10 proteins may... 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Antifungal assays The antifungal activity of the LlPR-10.2B protein was tested by a radial growth inhibition assay adapted from the method of Schlumbaum et al [51] Conidial spore suspensions (50 lL, 2 · 106 spores per millilitre) were placed in the centre of potato dextrose agar plates, and sterilized paper discs were placed around them Subsequently, various amounts of sterilized protein ranging from... proteins from parsley indicates that intracellular pathogenesisrelated proteins are ribonucleases Planta 193, 470–472 12 Samanani N & Facchini PJ (2002) Purification and characterization of norcoclaurine synthase The first committed enzyme in benzylisoquinoline alkaloid biosynthesis in plants J Mol Chem 277, 33878–33883 13 Bais HP, Vepachedu R, Lawrence CB, Stermitz FR & Vivanco JM (2003) Molecular and... 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