Báo cáo khoa học: Cytokinin-induced structural adaptability of a Lupinus luteus PR-10 protein potx

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:
(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
protein cavity, although the ITC data indicate a 1 : 1

stoichiometry. This 1 : 1 ratio may be explained by
the crystal structure of the complex, where only one
N,N¢-DPU molecule, DPU3, forms a hydrogen bond,
albeit through a water molecule, with the protein. All
the other N,N¢-DPU molecules are attached to the
protein via a weak net of van der Waals’ interac-
tions. It is probable that the N,N¢-DPU molecule
involved in hydrogen bonding, DPU3 in the crystal
structure, is the only one contributing to the
observed enthalpy change of )9.8 kJÆmol
)1
on titra-
tion of LlPR-10.2B.
The discrepancy in the amount of bound molecules
observed in the crystallographic complexes and in the
ITC experiment arises most probably from the weak
ligand binding of the studied molecules. Thus, the
buffer composition (mainly pH, ionic strength, cryo-
protectant, organic solvents, etc.) and the relative
concentration of the two partners used in the crystalli-
zation process, and in in vitro binding studies, can
influence the amount of complexed species that is
observed. As the biological function of the PR-10 pro-
teins is still unknown, it is difficult to speculate which
technique is better for describing the real phenomenon.
The main purpose of the binding studies, both through
complex structure determination and ITC measure-
ments, is to determine what kind of ligands can be
bound by the LlPR-10.2B protein, and this goal has
been successfully achieved.

There is an increasing volume of data indicating a
ligand-binding function for PR-10 proteins. The stud-
ies carried out in order to investigate such a function
in more detail have shown that the PR-10 proteins
indeed bind hydrophobic ligands, and that, on bind-
ing, the protein undergoes structural adaptation.
Moreover, the flexible ligands are also subject to con-
formational 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 not only function as a sink for
hydrophobic ligands, but may also be very efficient in
the release of their cargo, for example, to the final
receptors.
Experimental procedures
Protein production and purification
Recombinant LlPR-10.2B protein was expressed and puri-
fied from E. coli cells as described previously [44]. The yield
of the recombinant protein after purification was nearly
47 mgÆL
)1
of liquid culture.
Crystallographic procedures
The LlPR-10.2B–N,N¢-DPU complex was formed by incu-
bating 11 mgÆmL
)1
protein (in 3 mm sodium citrate buffer,
pH 6.3) with N,N¢-DPU (100 mm, dissolved in acetone) in
a 1 : 5 molar ratio. The concentration of acetone in the

final crystallization solution was never higher than 4%
(v ⁄ v). Crystallization experiments were performed at room
temperature using the hanging-drop vapour-diffusion
method. The protein–ligand complex crystallized from
high-salt precipitants containing 1.4 m sodium citrate in
0.1 m citrate buffer, pH 6.0. Single crystals appeared after
5–7 days and grew to the dimensions of 0.35 · 0.08 ·
0.04 mm in around 3 weeks. X-Ray diffraction data extend-
ing to 1.95 A
˚
resolution were collected at the EMBL X11
beamline at the DORIS ring of the DESY synchrotron.
The crystal was flash vitrified at 100 K in a stream of cold
N
2
gas [45]. The high salt content of the mother liquor
provided sufficient cryoprotection. The data were indexed,
integrated and scaled using hkl2000 [46]. The final statistics
are reported in Table 1.
The protein crystallized in the C222
1
space group with
one protein molecule in the asymmetric unit, corresponding
to a Matthews volume of 1.86 A
˚
3
ÆDa
)1
. The structure was
solved by molecular replacement with the molrep program

[47] using the crystallographic model of the LlPR-10.2B
protein from its complex with zeatin (Protein Data Bank
ID 2qim) [25] as the probe. Most of the residues were
clearly visible in the electron density map phased by molec-
ular replacement. The remaining side-chains, the N,N¢-
DPU and water molecules, as well as the sodium cations,
were modelled in the electron density maps during interac-
tive cycles of modelling in xtalview [48] and coot [49] that
alternated with structure refinement in refmac5 [50]. The
refinement was performed against all unique data, except
for 860 randomly selected reflections that were used for
R
free
testing. Stereochemical restraints for the N,N¢-DPU
molecule were imposed as for the protein residues. A dictio-
nary of idealized stereochemical targets was prepared in
refmac5 [50]. Water molecules were added if the corre-
sponding F
o
) F
c
peaks were higher than 4r, and they were
retained if their B factors refined to < 70 A
˚
2
. The occupan-
cies of residues in double conformation were manually
adjusted based on the electron density maps and tempera-
ture factors. The final model includes 154 protein residues,
four N,N¢-DPU ligands, 134 water molecules and two

sodium ions (Table 1).
ITC
Before the experiment, the protein solutions were dialysed
extensively against 3 mm sodium citrate buffer, pH 6.3, or
10 mm Mops, pH 7.0, at 4 °C. The titrations were per-
formed using a Nano ITC 5300 instrument (Calorimetry
Sciences, Lindon, UT, USA). The ligands were dissolved in
PR-10 protein–N,N ¢-DPU complex H. Fernandes et al.
1606 FEBS Journal 276 (2009) 1596–1609 ª 2009 The Authors Journal compilation ª 2009 FEBS
dialysis buffer at concentrations of 16.92, 0.33, 3.02 and
23.99 mm for trans-zeatin, kinetin, N,N¢-DPU and CPPU,
respectively, and injected in 5–25 lL aliquots. ITC experi-
ments were performed at either 20 or 25 °C. The ITC data
were analysed with origin software to obtain the stoichi-
ometry (n) and dissociation constants (K
d
). The heats of
mixing were subtracted. The concentrations of the protein
and ligands were estimated spectrophotometrically.
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 · 10
6
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 ster-
ilized protein ranging from 5 to 50 lg were pipetted onto the
discs. The protein solutions and all buffers were sterilized

using 0.22 lm filters (Millipore, Bedford, MA, USA). As
a control, filter paper alone, water, buffer and protein
after thermal treatment (5 min at 100 °C) were used. The
plates were incubated in the dark at 28 °C. C. lupini,
L. maculans and L. biglobosa were from the collection of the
Institute of Plant Genetics, Polish Academy of Sciences.
Coordinates
Atomic coordinates and structure factors for the LlPR-
10.2B–N,N¢-DPU complex have been deposited in the
Protein Data Bank, accession code 3e85.
Acknowledgements
We thank Dr Luiza Handschuh for a batch of recombi-
nant LlPR-10.2B protein, Alina Kasperska for help
with protein purification and Pawel Lachowicz for help
with the ITC experiments. This work was supported by
grants from the State Committee for Scientific Research
to MMS (grants 6 P04B 004 21 and 2 P04A 053 27)
and MJ (grant N N204 2584 33). HF was supported by
a Marie Curie Fellowship from the European Union.
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