Báo cáo khoa học: ˚ Crystal structure at 3 A of mistletoe lectin I, a dimeric type-II ribosome-inactivating protein, complexed with galactose pdf

Crystal structure at 3 A
˚
of mistletoe lectin I, a dimeric type-II
ribosome-inactivating protein, complexed with galactose
Hideaki Niwa
1
, Alexander G. Tonevitsky
2
, Igor I. Agapov
3
, Steve Saward
1
, Uwe Pfu¨ ller
4
and Rex A. Palmer
1
1
School of Crystallography, Birkbeck College, University of London, Malet Street, London WC1E 7HX, UK;
2
Institute of
Transplantology and Artificial Organs, Moscow, Russia;
3
Institute of Genetics and Selection of Microorganisms, Moscow, Russia;
4
Institut fu
¨
r Phytochemie, Universita
¨
t Witten/Herdecke, Witten, Germany
The X-ray structure of mistletoe lectin I (MLI), a type-II
ribosome-inactivating protein (RIP), cocrystallized with

galactose is described. The model was refined at 3.0 A
˚
resolution to an R-factor of 19.9% using 21 899 reflections,
with R
free
24.0%. MLI forms a homodimer (A–B)
2
in the
crystal, as it does in solution at high concentration. The
dimer is formed through contacts between the N-terminal
domains of two B-chains involving weak polar and non-
polar interactions. Consequently, the overall arrangement of
sugar-binding sites in MLI differs from those in monomeric
type-II RIPs: two N-terminal sugar-binding sites are 15 A
˚
apart on one side of the dimer, and two C-terminal sugar-
binding sites are 87 A
˚
apart on the other side. Galactose
binding is achieved by common hydrogen bonds for the
two binding sites via hydroxy groups 3-OH and 4-OH and
hydrophobic contact by an aromatic ring. In addition, at the
N-terminal site 2-OH forms hydrogen bonds with Asp27
and Lys41, and at the C-terminal site 3-OH and 6-OH
undergo water-mediated interactions and C5 has a hydro-
phobic contact. MLI is a galactose-specific lectin and shows
little affinity for N-acetylgalactosamine. The reason for this
is discussed. Structural differences among the RIPs investi-
gated in this study (their quaternary structures, location of
sugar-binding sites, and fine sugar specificities of their

B-chains, which could have diverged through evolution from
a two-domain protein) may affect the binding sites, and
consequently the cellular transport processes and biological
responses of these toxins.
Keywords: lectin; mistletoe (Viscum album); ribosome-inac-
tivating protein; b)trefoil.
Theuseofthemistletoeplant(Viscum album), well known
in ancient religious ceremonies, is thought to have extended
to more medicinal purposes since ancient times [1]. More
recently, particularly since the beginning of the 20th century,
extracts of the plant have been applied in the treatment of
cancer, especially in continental Europe, although its
efficacy in this respect is not fully understood (http://
www.cancer.gov/cancerinfo/pdq/cam/mistletoe). It is now
considered that mistletoe lectin I (MLI) is the most
important component in this respect. MLI is a type-II
ribosome-inactivating protein (RIP) which consists of two
chains [1]: a catalytic A-chain, which inactivates protein
synthesis, and a lectin B-chain, which binds to carbohydrate
moieties of cell surfaces, triggering the internalization of
MLI into the cell. The two chains are linked by a disulfide
bond. Other type-II RIPs include the plant toxins ricin from
Ricinus communis and abrin from Abrus precatorius.
In type-I RIPs there is a single polypeptide chain which
shares sequence and structural homology with the A-chains
of type-II RIPs. The low cytotoxicity of type-I RIPs is
considered to be due to the lack of the cell internalizing
facility exerted by the B-chains of type-II RIPs [2–4].
After endocytosis of a type-II RIP, it is transported to the
Golgi network and then to the endoplasmic reticulum [5].

The toxin is finally translocated across the endoplasmic
reticulum membrane into the cytosol to exert its catalytic
action on ribosomes. Studies using MLI [6,7] indicate that
the disulfide bond that links the two chains is reduced and
the A-chain is unfolded before translocation into the
cytosol.
The A-chain inhibits protein synthesis by cleaving the
N-glycosidic bond in adenosine A4324 in 28S eukaryotic
rRNA by hydrolysis (EC 3.2.2.22) [8]. The structure of the
rRNA loop where this adenine exists has been studied using
synthetic nucleotides [9], and the location of the loop in a
ribosome was identified in a 5.0-A
˚
electron density map for
the crystal structure of a bacterial 50S ribosome subunit [10].
How the specific adenine is recognized by an RIP, the
mechanism of the catalytic action itself, and why the
depurination of the single adenine arrests protein synthesis
are not properly understood. It has also been demonstrated
that RIPs not only release adenine from rRNA, but in vitro,
both type-I and type-II RIPs release adenine from DNA, and
many type-I RIPs can release adenine from poly(A) [11].
Three type-II RIPs or mistletoe lectins, MLI, MLII and
MLIII, have been isolated from mistletoe extract [12], MLI
Correspondence to H. Niwa, School of Crystallography, Birkbeck
College, University of London, Malet Street, London WC1E 7HX,
UK. Fax: + 44 20 76316803, Tel.: + 44 20 76316800,
E-mail:
Abbreviations: MLI, MLII, MLIII, mistletoe lectin I, II, III;
RIP, ribosome-inactivating protein; MLA, MLI A-chain; RTA,

ricin toxin A-chain; ABA, abrin-a A-chain; MLB, MLI B-chain;
RTB, ricin toxin B-chain; ABB, abrin-a B-chain.
(Received 11 March 2003, revised 27 April 2003,
accepted 30 April 2003)
Eur. J. Biochem. 270, 2739–2749 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03646.x
being the most abundant. MLI exists as a noncovalently
associated dimer (A–B)
2
in high concentration [13,14] while
ricin, abrin, MLII and MLIII are monomeric toxins. The
importance of the fact that MLI is cytotoxic as a dimeric
type-II RIP has been emphasized, as it is known that some
type-II RIPs exist as dimers or tetramers and are not
cytotoxic to a whole cell system, even though their A-chains
can inhibit protein synthesis in a cell-free system [15]. These
include dimeric R. communis agglutinin [16], an isolectin of
ricin, and Sambucus nigra agglutinin V [17] from the elder
plant, and also a tetrameric S. nigra agglutinin I [18].
Type-II RIPs are usually Gal/GalNAc-specific lectins,
S. nigra agglutinin I alone having specific affinity for
terminal sialic acid sequences [19]. MLI also has some
affinity for terminal sialic acid sequences, which is another
major difference from ricin [20]. Among Gal/GalNAc
lectins, affinity for Gal and GalNAc differ. MLI is Gal
specific, showing little affinity for GalNAc [21], whereas
MLIII is GalNAc specific and MLII has similar affinity
for both [12]. Whereas ricin shows similar affinity for both
Gal and GalNAc, R. communis agglutinin exhibits little
affinity for GalNAc [22]. S. nigra agglutinin V has higher
affinity for GalNAc than Gal [17]. Why these differences

occur in homologous proteins or lectins is a subject for
further study.
Previous papers have described the crystallization of
MLI [23] and subsequently the structure of MLI at 3.7 A
˚
,
showing it to be a dimer in the solid state [24]. In this
paper, we present the structure of MLI with noncovalently
bound galactose. The present structure, including bound
galactose molecules, was refined at 3.0 A
˚
. The sugar-
binding sites are clearly defined and are discussed in detail.
The biological importance of MLI as a dimeric cytotoxic
type-II RIP is also discussed and details of the dimer
interface are analysed. The availability of the known
structure of MLI without a specific sugar [25] (pdb 1ce7,
2mll) has enabled us to make a comparison with the
present structure.
Weak diffraction is often associated with high solvent
content and a large unit cell. However, Krauspenhaar et al.
[26] recently reported the structure of MLI with adenine
bound in the A-chain active site. These crystals, grown in
a microgravity environment, exhibited improved diffraction
quality over other MLI complexes. The method of crystal-
lization may have contributed to the improvement in
diffracting power.
Materials and methods
Extraction and purification of MLI has been described
previously [7]. MLI crystals were obtained by the hanging

drop method. The protein solution at 18 mgÆmL
)1
concen-
tration contained 0.1
M
galactose and 0.01
M
acetate buffer
at pH 4.0. The reservoir solution contained 0.9
M
ammo-
nium sulfate and 0.1
M
glycine buffer at pH 3.4. The droplet
consisted of 1 lL of the protein solution and 1 lLofthe
reservoir solution. Hexagonal crystals grew to about
0.2 mm in a few weeks. Sequence data were obtained as
described previously [24] and in [27].
The X-ray data were collected at the Synchrotron
Radiation Source (SRS) at the Central Laboratory of the
Research Councils (CLRC) Daresbury Laboratory, UK,
on station 7.2, with wavelength 1.488 A
˚
and a 30-cm Mar
Research image plate detector. Before being mounted on a
loop, the crystal was soaked in a cryoprotectant solution
containing about 30% glycerol and was flash cooled to
100 K for data collection. Each image was collected with a
1.5 ° step and a 900-s exposure time. A total of 16 images for
the sweep range 24.0 ° were collected.

Data processing was carried out with
DENZO
and
SCALE-
PACK
[28], and subsequently with
SCALA
in the CCP4 suite
[29]. The space group was P6
5
22 as previously determined
[24], with unit cell parameters of a ¼ b ¼ 107.65 A
˚
,
c ¼ 311.92 A
˚
. The solvent content was calculated to be
71% for one molecule of MLI with molecular mass 62 kDa
per asymmetric unit [30]. The data were processed to 2.9 A
˚
,
providing 68 126 measured reflections, which reduced to
24 175 unique reflections. The overall completeness and
multiplicity (redundancy) to 2.9 A
˚
were 98.0% and 2.8,
respectively, and the overall R-merge was 6.9%.
Molecular replacement was performed with
AMORE
[31]

using the previous partial 3.7-A
˚
model [24], and the more
complete ricin model [32] (pdb 2aai) was used independ-
ently for checking purposes. The structure was refined
using
X
-
PLOR
[33] and in the later stages the
CNS
packages
[34], with manual intervention on graphics using O [35].
Refinement was carried out using data up to 3.0 A
˚
.(The
R-merge between 3.12 A
˚
and 3.0 A
˚
was 28.1%.) There
was a total of 21 899 reflections, of which 1117 (5%) were
kept separate to calculate R
free
. All reflections were used
for the refinement with a bulk-solvent correction proce-
dure. Individual isotropic B-factors were subsequently
employed. This reduced the R-factor and R
free
by 2.1%

and 1.9%, respectively, without introducing any unrea-
sonable B values. An anisotropic overall B-factor was also
refined.
The C-terminal residues after Gly248 of the A-chain
could not be located because the electron density was too
weak, presumably because of disorder. A total of six
glycosylating sugar units was included in the final model
as follows. For the four putative glycosylation sites of type
Asn-X-Thr/Ser in MLI (Asn112 in the A-chain, Asn61,
Asn96 and Asn136 in the B-chain), it was possible to
model the first glycosylating sugar as GlcNAc to all four
sites and in addition a second GlcNAc at Asn96 and
Asn136 in the B-chain. A model including four water
moleculeswasrefinedtoanR-factor20.6%withR
free
25.8% [36]. This model was further refined to include 47
water molecules, which were selected from the peaks
above 3.5 r in the F
o
–F
c
map by examining the geometry
and the electron density. The final R-factor and R
free
were
19.9% and 24.0%, respectively. Ramachandran plots
were calculated using
PROCHECK
[37]. For nonglycine
and nonproline residues, 87.6% were in the most favoured

regions, and 12.4% were in additional or generously
allowed regions.
Other important refinement statistics are summarized in
Table 1. Hydrogen bonds and hydrophobic contacts
between sugar molecules and protein, and between dimer
molecules were analysed using
HBPLUS
[38]. Figures 1 and
6–8 were drawn with
MOLSCRIPT
[39], and Figs 4 and 5 were
drawn with
SETOR
[40]. The refined coordinates and
structure factors have been deposited at PDB with the
accession code 1OQL.
2740 H. Niwa et al.(Eur. J. Biochem. 270) Ó FEBS 2003
Results
Overview and the A-chain
Figure 1 shows a ribbon diagram of the structure of MLI.
The overall structure of the MLI monomer is similar to two
other type-II RIPs, ricin [32] (pdb 2aai) and abrin-a [41]
(pdb 1abr), and when the current structure was super-
imposed on to the other type-II RIPs, the number of
matched Ca atoms for MLI and ricin was 469 (91.8% out of
511) with rmsd 0.92 A
˚
, and 479 (93.7%) with rmsd 1.05 A
˚
for MLI and abrin-a, using a 3.0-A

˚
cut-off distance. As
differences exist between the current and already published
sugar-free MLI [25] in terms of sequence and detailed
structure as described below, a structure-based sequence
alignment of the A-chains of MLI (MLA), ricin (RTA) and
abrin-a (ABA), together with a type-I RIP momordin [42]
(pdb 1mom) is shown in Fig. 2.
Although the structure of MLA determined here clearly
has some features in common with other RIPs, important
differences do occur, for example in solvent-exposed
regions, particularly in the region running from strand e
to helix B (residues 91–100 of MLA). In this region MLA
and ABA are structurally similar, whereas in RTA and
momordin this region is more extensive, with additional
amino-acid residues and consequent differences in the
structures. This region has been proposed [43] as being an
antigenic epitope site in RIPs, and, in fact, this is the site
where a monoclonal antibody against MLA has been found
to bind [7]. Another notably labile region is the sheet g–h.
Structural lability in this region is commonly observed in
both type-I RIPs and the A-chains of type-II RIPs, and is
the region in the present structure with the most consistently
high B-factors.
The active site is located in a prominent, centrally
located cleft of the A-chain. Six residues conserved among
all RIPs (Fig. 2; Tyr76, Tyr115, Glu165, Arg168, Trp199
and Ser203) are located here in a structurally highly
conserved hydrogen-bond network. Tyr76 alone in this
region exhibits various conformations in known RIP

structures [42], and its conformational change is the main
difference in active-site geometry compared with the
A-chain of ricin, as reported in sugar-free MLI [25]. In
MLA, there is a glycosylation site at Asn112 at the edge
of the active-site cleft, which is discussed further in the
Discussion section.
B-chain
The B-chain of MLI (MLB) consists of two homologous
globular domains. Each domain has a diameter of  30 A
˚
and consists of three repetitive subdomains, which form
a pseudo-threefold symmetry around a hydrophobic core.
The fold of one such domain has been classified as the
b-trefoil fold [44]. Sequence alignment of the six subdomains
with those of ricin (RTB) and abrin-a (ABB) is shown in
Fig. 3. Three B-chains can be aligned without insertion or
deletion except for the N-terminal region. It should be noted
that the assignment of subdomains here differs from that
described for RTB [45]: in the RTB description, 1a
1
(the
suffix indicates a strand number in a subdomain shown in
Fig. 3) was not included in the repetitive subdomain, and
one unit consists of, for the first one for example, 1a
2
,1a
3
,
1a
4

and 1b
1
, if the strand designation in Fig. 3 is used,
resulting in 1c and 2c units being one strand short. Three
disulfide bonds are conserved in MLB, but one in the 1a
subdomain is lost as the result of a mutation from Cys to
Ser39. There are two glycosylation sites in RTB and ABB,
which are conserved in MLB. In addition, MLB has
Table 1. Refinement statistics.
Parameter Value
Resolution (A
˚
) 50.0–3.0
No. of reflections (total, test set) 21899, 1117
No. of atoms
Total, proteins 4132, 3977 (511 residues)
Covalently bound sugars 84 (6 GlcNAc)
Ligands (galactose) 24 (2 Gal)
Solvent (water) 47
R (%) 19.9
R
free
(%) 24.0
Average B-factors (A
˚
2
)
All atoms 40.8
A-chain 45.2
B-chain 35.7

Covalently bound sugars 60.3
Ligands (galactose) 65.0
Solvent (water) 30.0
Rmsd from ideal values
Bond lengths (A
˚
) 0.011
Bond angles (°) 1.72
Fig. 1. Ribbon representation of the structure of MLI. The A-chain is
located above and the B-chain is below. The disulfide bond between
the two chains is shown in yellow. The glycosylating sugars included in
the final structure are shown in brown. Galactose molecules are
depicted in ball-and-stick. Tyr76 and Tyr115 in the active site of the
A-chain, and Asp23 in the N-terminal sugar-binding site of the B-chain
are shown in red.
Ó FEBS 2003 Mistletoe lectin I with galactose (Eur. J. Biochem. 270) 2741
another glycosylation site at Asn61. The structure of one
subdomain may be summarized as: strand 1 fi turn fi
b-sheet (strands 2 + 3) fiW-loop (including a 3
10
-helical
region at the end) fi strand 4. Strands 1 and 4 also form a
b-sheet. An interesting feature of the fold is provided by the
repetitive Gln-X-Trp (QxW) sequences (Fig. 3) [46].
Sugar-binding sites
Sugar-binding sites in RTB exist in the subdomains 1a and
2c [45], and the residues involved in protein–sugar inter-
actions in RTB are also retained in these (and only these)
two subdomains in MLB (Fig. 3). Clear electron density for
binding sugar molecules was observed at each site (Fig. 4).

b-Galactose was modelled into both sites, the electron
density being complete for the C-terminal site, but lacking
for 1-OH at the N-terminal site. The electron density for the
side chain of Lys254 in the C-terminal site was also weak
and tenuous, indicating disorder. Water molecules bound to
galactose, three in the C-terminal site, were included in an
attempt to complete all possible sugar-binding interactions.
Each sugar-binding site exists in a shallow cleft formed by
Fig. 2. Sequence alignment of MLA, RTA, ABA and momordin (MOM). The secondary-structure designation follows that of ricin [32]. The
C-terminal residues not included in the refined structure are indicated in lower case. Identically conserved residues among these four proteins are
shown in bold: those that are also identically conserved among all RIPs are darkly shaded, and those highly conserved are lightly shaded. Conserved
residues in RIPs may be obtained from the Pfam database. Possible glycosylation sites are underlined.
2742 H. Niwa et al.(Eur. J. Biochem. 270) Ó FEBS 2003
contiguous stretches in the protein chain with some 20
residues in one subdomain, from strand 2, a 3
10
-helical kink,
strand 3 and an W-loop with a 3
10
-helical kink at the end,
asindicatedinFigs3and5.
Possible interactions between MLB and galactose are
summarized in Fig. 5 together with those between RTB and
lactose using the same criteria for comparison, and Fig. 6
provides a stereoview of the N-terminal sugar-binding site in
MLB. Protein–galactose interactions that are common in
both the N-terminal and C-terminal sugar-binding sites of
RTB [45] are also common to the two sites of MLB. These
are: (a) via hydroxy groups 3-OH and 4-OH of galactose,
donating hydrogen bonds to Od2andOd1ofaspartate

(Asp23 in the N-terminal site, Asp235 in the C-terminal
Fig. 3. Sequence alignment of six subdomains in MLB, RTB and ABB. The strand regions are indicated by horizontally striped rectangles with the
strand number in the top row, and the common 3
10
-helical (kink) regions are indicated by obliquely striped rectangles. Identically conserved Ile and
Trp are shown in dark shading and the conserved hydrophobic residues in light shading. Cysteines that make disulfide bonds are in yellow, the key
residues involved in sugar binding are in magenta and possible glycosylation sites are marked in cyan. Repetitive QxW sequences are underlined.
Fig. 4. Stereoview of an electron density map of the C-terminal MLB sugar-binding site. Cyan: 2F
o
–F
c
+1.25r;blue:F
o
–F
c
+3.0r;red:
F
o
–F
c
) 3.0 r. Purple, F
o
–F
c
+3.0r, was calculated by deleting coordinates of galactose and water molecules.
Ó FEBS 2003 Mistletoe lectin I with galactose (Eur. J. Biochem. 270) 2743
Fig. 5. Schematic drawings of the sugar-binding sites of MLB and RTB. (A) MLB N-terminal site. (B) MLB C-terminal site. (C) RTB N-terminal
site. (D) RTB C-terminal site. Key residues, hydrogen bonds that are formed with bound sugar, and secondary-structure elements are shown.
Fig. 6. Stereoview of the N-terminal MLB sugar-binding site showing a hydrogen-bond network.
2744 H. Niwa et al.(Eur. J. Biochem. 270) Ó FEBS 2003

site); (b) Nd2 of asparagine (Asn47, Asn256) donating a
hydrogen bond to 3-OH of galactose; (c) an aromatic ring
(Trp38, Tyr249) making hydrophobic contact by stacking
its ring approximately parallel to the C3–C4–C5 plane of
galactose. These aspartates, asparagines and aromatic rings
are identically conserved residues in all B-chain sugar-
binding sites.
In addition to these common interactions, specific
contacts are made in each site, as described below in
comparison with RTB. In the N-terminal site, Nf of Lys41
interacts with 2-OH and 3-OH of galactose, and 2-OH
donates a hydrogen bond to Od2 of Asp27. In RTB,
although the lysine is conserved (Lys40), because of the
distance between the probable hydrogen of Nf and 2-OH or
3-OH being slightly greater than the criterion used (2.5 A
˚
),
it was not selected as a hydrogen bond here. Asp27 in MLB
corresponds to Gly26 in RTB, which consequently does not
form a hydrogen bond. In RTB, 6-OH forms hydrogen
bonds with Ne2 of Gln35 and a main-chain N, whereas in
MLB 6-OH is not involved in interactions with the protein.
The glutamine is conserved in the two proteins, however,
and differences in orientation of the CH
2
OH groups in
galactose are responsible for the changes in these interac-
tions. The main-chain N of Asp26 in MLB donates a
hydrogen bond to 4-OH, but in RTB a bond between N of
the corresponding residue (Asp25) and 6-OH is probable, as

mentioned above. In the C-terminal site of MLB, a water-
bridged hydrogen bond is made between 3-OH of galactose
and Oc1 of Thr252, which is equivalent to Lys41 at the
N-terminal site. In RTB, Thr252 is mutated to His251, of
which Ne2 directly donates a hydrogen bond to 3-OH. In
MLB, positive electron density was observed extending
from 6-OH of galactose to Arg245, where two hydrogen-
bonding water molecules were located (Fig. 4). Ile247,
which is equivalent in position to Gln36 of the N-terminal
site, makes hydrophobic contact with C6 of galactose, as is
also observed in RTB. The main-chain N of Gln238 donates
a hydrogen bond to 4-OH, as equivalent interactions are
observed in the N-terminal site of MLB (Asp26–4-OH, as
described above) and in the C-terminal site of RTB
(Ala237–4-OH). Although specific interactions with bound
sugar are not established, Asp26 in MLB with galactose
undergoes conformational change (v
1
¼ )164°)ascom-
pared with Asp25 in RTB with lactose (v
1
¼ )65°).
Hydrogen-bond networks of the sugar-binding sites
Oc2 of the identically conserved aspartate (Asp23, Asp235),
which accepts a hydrogen bond from 3-OH of galactose,
accepts another hydrogen bond from Ne2ofglutamine
(Gln48, Gln257) (Fig. 6). The Ne2andOe2ofthis
glutamine form hydrogen bonds with the main-chain atoms
in the W-loop. Nd2 of identically conserved asparagine
(Asn47, Asn256), which donates a hydrogen bond to 3-OH

of galactose, also donates a hydrogen bond to the main-
chain O of valine (Val24, Val236), while the main-chain N
of this valine donates a hydrogen bond to the main-chain O
of the asparagine, thus forming a bridge between the region
from strand 2 to the helical kink (left-hand side of a sugar-
binding site in Figs 5 and 6) and the helical kink at the end
of the binding site (right-hand side). The above valine and
glutamine are identically conserved in all B-chain sugar-
binding sites (Fig. 3). A hydrogen bond that corresponds to
the one from the valine N to asparagine O is correspond-
ingly observed in any subdomain of the RIP B-chain.
Dimer structure
In the MLI dimer, shown in Fig. 7, two A–B monomers are
related by crystallographic twofold symmetry and face each
other at the N-terminal domain of the B-chain, where the
three hairpin loops in the b-trefoil fold make major contacts
with those of the other in the following way: a « c¢, b « b¢,
c « a¢ [24]. When viewed perpendicularly to the twofold
axis, two oblong-shaped MLB molecules are seen to make
an angle of  160 °. The dimensions of the dimer are
157 · 63 · 48 A
˚
3
, and the contact area is 755 A
˚
2
per
monomer. The existence of a dimer structure is consistent
with an earlier electron microscope study of MLI [14], which
corroborates the idea that this is a real dimer form, not an

artefact of the crystal packing. The distances between sugar-
binding sites in one dimer, calculated as straight distances
between corresponding O4 atoms of galactose, are shown in
Fig. 7.
A number of both polar and nonpolar contacts are
observed at the dimer interface, as shown in Fig. 8. Residues
in the 1b subdomain (the side chain of Tyr68, the main-
chain atoms of Ala72 and Gly73, and Val74) make
hydrophobic contact with the equivalent region in the other
molecule. In addition, Og of Tyr68 makes a hydrogen bond
with the side chain of Gln122 in the 1c¢ subdomain. Several
hydrogen bonds are formed between residues in the 1a and
1c¢ subdomains, some of which are water mediated. In
addition, Ile114 makes hydrophobic contacts with carbon
atoms in the side chains of Arg25 and Asn26. Among the
residues that make polar interactions, Gln34, which makes
hydrogen bonds with the main-chain atoms of Thr118, and
Ser111, which makes water-mediated hydrogen bonds with
the O of Gly32, are not conserved in RTB and ABB.
However, Oc of Ser111 is located in a similar position to
Oc1 of Thr110 of RTB. Among the hydrophobic contacts,
Ilel14 of MLB is mutated to an asparagine in RTB and
a serine in ABB.
Comparison of MLI structures
Two MLI structures are currently available in the PDB,
namely 1ce7 and 2mll [25]. The coordinate sets for these
are identical and will be referred to only as 2MLL, which
is MLI without lectin-bound sugar. 2MLL was refined at
2.7 A
˚

to an R-factor ¼ 25.1% and R
free
¼ 31.9%, accord-
ing to the PDB file. The published model comprises 241
residues in the A-chain, 255 residues in the B-chain, 3
glycosylating GlcNAc sugars, and 215 water molecules.
Excluding the C-terminus of the A-chain, where residues
could not be located in either the current structure or
2MLL, and the N-terminus of the B-chain, where the
structure is not defined well in the current structure and
residues were not located in 2MLL, the numbers of
differences in sequence between the two structures are 39
in the A-chain and 31 in the B-chain. These differences arise
from: (a) differences in original sequences determined by
two methods [27,47]; (b) the fact that 2MLL contains
truncated residues in solvent-exposed loops and also a total
Ó FEBS 2003 Mistletoe lectin I with galactose (Eur. J. Biochem. 270) 2745
of 11 deleted residues compared with its original sequence
[47].
The A-chains of the present structure and 2MLL can be
superimposed, with rmsd 0.56 A
˚
for 232 matched residues,
and the B-chains with 0.45 A
˚
for 253 matched residues with
3.0-A
˚
cut-off distance. In the A-chains, structural differ-
ences exist in solvent-exposed regions, and there is a shift by

one residue in the strand a at the N-terminus. However,
there is no significant discrepancy in the active site, including
the conformation of Tyr76 (Tyr75 in 2MLL), which differs
from that of ricin.
In the B-chains, three points relating to sugar binding are:
(a) in the N-terminal sugar-binding site, the locations of Oe1
and Ne2 of a glutamine (Gln36 in the current structure and
Gln32 in 2MLL) are interchanged; (b) in the C-terminal site,
2MLL has deletions at Gln238 and Ala239 of the current
Fig. 8. Stereoview of the MLI dimer interface.
Three hairpin loops (strand 2 and 3) of the
N-terminal domains of the B-chains are
shown. The dimer molecules are related by
twofold symmetry and the view is along the
twofold axis. Residues that make hydrophobic
contacts are in darker grey.
Fig. 7. Ribbon representations of MLI dimer.
(A)and(B)showorthogonalviewsofthe
whole dimer with bound galactose molecules.
(C) is the top view of (A), depicting only
B-chains, where in the N-terminal domains
only the hairpin loops (strand 2 and 3) of three
subdomains are depicted and their design-
ations are shown in the same colour. Distances
between galactose molecules (straight distan-
ces between two O4 atoms) are also shown.
2746 H. Niwa et al.(Eur. J. Biochem. 270) Ó FEBS 2003
structure and, as a result, the position of Ala239 in the
current structure is occupied by Asn232 in 2MLL, which
corresponds to Asn240 of MLI; (c) the conformation of

a lysine (Lys254 in the current structure and Lys246 in
2MLL) is different in the two structures; however, as
mentioned previously the electron density of this side chain
is weak in the current structure (and possibly also in 2MLL)
and this point does not justify further discussion.
Discussion
The refined structure of MLI complexed with galactose is
presented. The molecular structure shares common features
with other RIPs without extra or shortened main-chain
loops. MLA has a glycosylation site at the rim of the active-
site cleft, which is unique among RIPs. On superimposition
of the structure of the RTA–ApG complex [48] on to MLA,
a glycosylating MLA sugar at Asn112 was seen to occupy
(at least partially) the guanine-binding site of the RTA
substrate analogue. However, glycosylation appears not to
affect the catalytic activity because it is known that
recombinant MLA shows similar activity to that of plant-
derived MLA [27]. As molecular dynamics studies also
suggest [48,49], it is possible that, when the RNA substrate
loop binds to an A-chain of a type-II RIP, it adopts a
different conformation from that of the dinucleotide
substrate analogue. In fact, the conformation of the bound
ApG in ricin differs from that in the structure of the
ribosomal loop determined by NMR [50] (pdb 1scl) or by
X-ray crystallography [9] (pdb 430d, 483d).
The two sugar-binding sites of MLB exhibit common
features sometimes observed in sugar-binding sites in
proteins other than RIP B-chains: an extensive hydrogen-
bond network and hydrophobic stacking [51]. Sugar-affinity
studies have shown that 4-OH and then 3-OH are the

hydroxy groups of galactose that strongly affect sugar
binding for MLI [21] and ricin [52], and they are involved in
the common binding mode for the two sugar-binding sites
of both proteins.
It is known that GalNAc binds only to the C-terminal
sugar-binding site of ricin, which is the high-affinity site [53].
However, experimental results on the difference in sugar
specificity between the two binding sites are not available for
MLB. In interpreting sugar-binding specificity assays of
lectins, consideration should be paid to the actual number of
binding sites in each protein. The two sugar-binding sites
in type-II RIP B-chains are structurally and chemically very
similar, but not identical. As a dimer, MLI in fact possesses
four sugar-binding sites, and this property, in view of the
novel interbinding-site distances resulting from this dimeri-
zation, is likely to affect the toxin’s ability to bind to cell
surface sugars. Ambiguity exists when the sugar specificity
of a single binding site is examined using biochemical data
that are unavoidably from all of the available binding
sites. With respect to the specificity of MLI for GalNAc,
however, evidence derived from solid-phase assay [54]
indicates that each binding site in fact does not have high
GalNAc specificity.
There is insufficient space for the N-acetyl group of
GalNAc to be accommodated in the N-terminal site of
MLB, because it is blocked by the two residues that form
hydrogen bonds with 2-OH, namely Asp27 and Lys41.
As to the C-terminal site, there is a possibility that the
disordered side chain of Lys254 causes steric hindrance with
the N-acetyl group of GalNAc. However, because of the

disorder, whether it completely hinders sugar binding is not
conclusive. When a GalNAc is located at the C-terminal
sugar-binding site of RTB so that its pyranose ring is
superimposed on that of the galactose of bound lactose, it is
found that the oxygen in the acetyl group could form a
hydrogen bond with the hydroxy group of Ser238. In MLB
Ser238 in RTB is mutated to Ala239 and cannot make a
hydrogen bond with a sugar. It is proposed that this serine
in RTB may contribute to GalNAc binding.
In the present MLI structure, the electron density of
galactose in the N-terminal site of MLI was less pronounced
than that in the C-terminal site. This may suggest that, of
the two sites, the N-terminal site has the lower affinity for
galactose, as is also the case for ricin.
MLI crystallizes readily from ammonium sulfate in acidic
conditions (pH 3.4 for the actual crystal used for the current
structure analysis). Sugar affinity decreases under acidic
conditions for MLI [55] and ricin [56], therefore the electron
density of galactose shown in Fig. 4 may be regarded as
corresponding to a partially bound sugar. The cause of this
decrease in affinity is considered to be either due to
protonation of the aspartate in the sugar-binding site,
which can be a hydrogen-bond acceptor, or conformational
change. As there is little conformational change among the
crystal structures of MLI (pH 3.4), ricin (pH 4.75) [32] and
abrin-a (pH 8.0) [41], the decrease in sugar affinity in acidic
conditions is probably associated with protonation of the
identically conserved aspartate, which interacts with 3-OH
and 4-OH of bound galactose in these sugar-binding sites.
The MLI dimer involves several polar and hydrophobic

contacts through the N-terminal domains of two B-chains.
As the formation of the dimer is concentration dependent
[13,14], the overall interaction is weak. In the 1a subdomain
of the B-chains of some type-II RIPs, the hairpin loop is
stabilized by a disulfide bond, as in RTB and ABB. However,
this S-S bond does not exist in MLB because of the mutation
from a cysteine to Ser40, and the loop is more flexible.
Therefore, it may be speculated that the increased flexibility
caused by the loss of the disulfide bond in MLB may play a
crucial role in dimer formation [24]. As little shift of the 1a
loop is observed in the superimposition of MLB, RTB and
ABB, it appears that at least permanent dislocation of the
loop is not a requisite for dimer formation. It is not possible
to conclude, however, from this study alone how important
each of the various interactions described in the Results
section is individually for the dimer formation. Although
hydrophobic 1b hairpin loops do in fact contact each other,
this region is also hydrophobic in monomeric RTB and ABB,
as seen in the sequence in Fig. 3, therefore this hydrophobic
contact alone is not enough to cause MLI dimer formation.
Among the residues that make interactions, Gln34 and
Ile114 are unique to MLB. Mutation studies of these residues
may reveal their role in the dimer formation.
In contrast with some type-II RIPs mentioned in the
Introduction, the dimeric structure of MLI does not
interfere with its toxic activity. It may be, however, that
the quaternary structure does affect other aspects of
the biological processes. One factor that may be involved
is the relative disposition of the sugar-binding positions.
Ó FEBS 2003 Mistletoe lectin I with galactose (Eur. J. Biochem. 270) 2747

The distance between two sugar-binding sites in a monomer
is 47 A
˚
, whereas in the MLI dimer two N-terminal sugar-
binding sites are 15 A
˚
apart on one side of the dimer and
two C-terminal sites are 87 A
˚
apart on the other side of the
dimer (Fig. 7). All sugar-binding sites are centrally located
in the MLI dimer. Unless some quaternary structural
change occurs, the dimer must bind to the cell surface by
laying the whole (A–B)(B–A) structure along the cell surface
so that the binding sites are close enough to cell-surface
oligosaccharides.
As there is no significant difference in catalytic activity
between recombinant MLA and plant-origin MLA, it
may be argued that differences in cytotoxicity between
MLI and ricin are mainly influenced by the B-chains [27].
The actual binding sites of the toxins on cells, subsequent
transport processes, and biological responses are probably
affected by differences in fine sugar specificity and/or
quaternary structure with associated differences in the
arrangement of the sugar-binding sites. Recent studies
highlight differences in the membrane-binding sites of
MLIandricin[57].Ithasbeensuggested[58]thatricin
may in fact dimerize on binding to cell surface receptors.
However, the study was carried out on ricin with
chemically blocked sugar-binding sites, and the actual

dimer form was not specified.
There is evidence to suggest that RIP B-chains evolved
from a primordial peptide of about 40 residues: (a) by gene
duplications and fusions into a three-subdomain protein
(a b-trefoil domain); and (b) by further duplication into a
two-domain protein [59]. The fact that some bacteria have
proteins with a b-trefoil domain sharing sequence and
structural homology with RIP B-chains [60] suggests that
b-trefoil fold proteins existed in early evolutionary proces-
ses. When the six subdomains of MLB are aligned, it is seen
that the sequence identity between subdomains and also
between two domains is about 20%. However, overall
sequence identity between MLB, RTB and ABB is more
than 50% [27]. Hence, it is reasonable to speculate that RIP
B-chains diverged evolutionarily from a precursor protein
that already had a two-domain structure.
Acknowledgements
We thank members of the School of Crystallography at Birkbeck
College for their valuable discussions, and colleagues at Daresbury
Laboratory, UK for their help and support during the course of this
work.
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