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PROTEINS: Structure, Function, and Genetics 28:268–284 (1997)
Role of Aromatic Amino Acids in Carbohydrate Binding
of Plant Lectins: Laser Photo Chemically Induced
Dynamic Nuclear Polarization Study of Hevein
Domain-Containing Lectins
Hans-Christian Siebert,1,2 Claus-Wilhelm von der Lieth,3 Robert Kaptein,2 Jaap J. Beintema,4 Klaas
Dijkstra,4 Nico van Nuland,4 Ukun M. S. Soedjanaatmadja,5 Ann Rice,6 Johannes F. G. Vliegenthart,2
Christine S. Wright,6 and Hans-Joachim Gabius1*
1Institut für Physiologische Chemie, Tierärztliche Fakultät, Ludwig-Maximilians-Universität, München, Germany
2Bijvoet Center for Biomolecular Research, University of Utrecht, Utrecht, The Netherlands
3Zentrale Spektroskopie, Deutsches Krebsforschungszentrum, Heidelberg, Germany
4Biochemisch Laboratorium and BIOSON Research Institute, Department of Biophysical Chemistry, University of
Groningen, Groningen, The Netherlands
5Laboratorium Biokimia, Jl. Singaperbangsa 2, Padjadjaran University, Bandung, Indonesia
6Department of Biochemistry and Molecular Biophysics, Virginia Commonwealth University, Richmond, Virginia
ABSTRACT
Carbohydrate recognition by
lectins often involves the side chains of tyrosine, tryptophan, and histidine residues. These
moieties are able to produce chemically induced dynamic nuclear polarization (CIDNP)
signals after laser irradiation in the presence
of a suitable radical pair-generating dye. Elicitation of such a response in proteins implies
accessibility of the respective groups to the
light-absorbing dye. In principle, this technique is suitable to monitor surface properties
of a receptor and the effect of ligand binding if
CIDNP-reactive amino acids are affected. The
application of this method in glycosciences can
provide insights into the protein-carbohydrate
interaction process, as illustrated in this initial study. It focuses on a series of N-acetylglucosamine-binding plant lectins of increasing
structural complexity (hevein, pseudohevein,
Urtica dioica agglutinin and wheat germ agglutinin and its domain B), for which structural
NMR- or X-ray crystallographic data permit a
decision of the validity of the CIDNP methodderived conclusions. On the other hand, the
CIDNP data presented in this study can be
used for a rating of our molecular models of
hevein, pseudohevein, and domain B obtained
by various modeling techniques. Experimentally, the shape and intensity of CIDNP signals
are determined in the absence and in the presence of specific glycoligands. When the carbohydrate ligand is bound, CIDNP signals of side
chain protons of tyrosine, tryptophan, or histidine residues are altered, for example, they are
broadened and of reduced intensity or disappear completely. In the case of UDA, the appearance of a new tryptophan signal upon ligand
binding was interpreted as an indication for a
r 1997 WILEY-LISS, INC.
conformational change of the corresponding
indole ring. Therefore, CIDNP represents a
suitable tool to study protein-carbohydrate interactions in solution, complementing methods such as X-ray crystallography, high-resolution multidimensional nuclear magnetic
resonance, transferred nuclear Overhauser effect experiments, and molecular modeling. Proteins 28:268–284, 1997 r 1997 Wiley-Liss, Inc.
Key words: lectins; agglutinins; chemically induced dynamic nuclear polarization (CIDNP); nuclear magnetic
resonance (NMR); molecular modeling
INTRODUCTION
The recognition of carbohydrate determinants by
lectins plays an important role in mediation of
intercellular binding and elicitation of biosignaling
processes.1–4 Structural aspects of this glycobiological interplay can, in principle, be visualized by
multidimensional nuclear magnetic resonance (NMR)
experiments and/or X-ray crystallography. However,
these techniques can suffer from a number of inherent restrictions, as, for example, molecular mass
limitations in multidimensional NMR studies and in
X-ray crystallography a lack of information about
the dynamic behavior of molecules in solution. There-
Contract grant sponsor: Human Capital and Mobility Program of the European Community
*Correspondence to: Hans-Joachim Gabius, Institut für Physiologische Chemie, Tierärztliche Fakultät, Ludwig-MaximiliansUniversität, 80539 München, Germany.
Received 9 September 1996; Accepted 3 January 1997
CARBOHYDRATE RECOGNITION BY LECTINS
fore, additional methods to reliably assess defined
structural aspects deserve attention. CIDNP experiments, which allow the detection of the amino acid
side chains of tyrosine, tryptophan, and histidine at
protein surfaces,5 offer such a possibility. They readily
provide information on their targets following
changes of external parameters such as temperature
and/or pH values, as demonstrated in studies of
several biomolecules, and they are not very timeconsuming.6,7 The CIDNP technique has previously
been used for comparative studies of nonspecific and
specific interactions between the lac-repressor headpiece and DNA,8,9 denatured states of lysozyme10 as
well as of glycoproteins in glycosylated and deglycosylated form11 or in sialylated and desialylated form12
in solution. So far, the CIDNP method has not been
applied to a carbohydrate-binding protein, although
it is known that side chains of tyrosine, tryptophan,
and histidine can play an essential role in ligand
binding of carbohydrate receptors apart from their
well-appreciated participation in stabilizing protein
conformation.13–18 Since only three types of amino
acids are under consideration for these measurements in contrast to conventional 1H-NMR experiments, the problem of overlapping signals is clearly
less stringent. When structural parameters from the
nuclear Overhauser effect (NOE)-dependent NMR
assessment or from X-ray crystallography are available, the calculation of surface accessibilities for the
respective types of side chains enables a useful
comparison of these datasets with the CIDNP results.
To determine the actual value of this method in the
field of glycosciences, several well-characterized Nacetylglucosamine-binding lectins from different species with increasing molecular complexity are selected as models. In detail, laser photo CIDNP
experiments of hevein, pseudohevein, Urtica dioica
agglutinin (UDA), wheat germ agglutinin (WGA),
and the B domain (domB) of WGA1 have been
carried out in the absence and in the presence of
their specific ligands. Hevein (43 amino acid residues) and pseudohevein (43 amino acid residues) are
small lectins of rubber trees (Hevea brasiliensis)
with ragged C-terminal sequences.19,20 In addition to
their assumed role in latex coagulation they are
considered to serve a protective function similar to
that of the related lectins WGA and UDA.21–24 The
ratio of expression of hevein to pseudohevein is 10:1,
and the amino acid sequences of the two proteins
differ only in six positions from each other.19 Among
these differences the replacement of Trp21, present
in hevein, by a tyrosine residue in pseudohevein, is
especially notable in the context of the CIDNP
experiments. Another member of the chitin-binding
family of plant lectins is UDA from stinging nettle,
whose structure consists of two heveinlike domains.
It has a single chain of 89 amino acids, comprising
two binding sites with different affinities for oligom-
269
Fig. 1. Laser photo CIDNP difference spectra (aromatic part)
of hevein and hevein–(GlcNAc)2 complexes at pD 4. a: Ligandfree hevein. b: 1 mmol hevein 1 1 mmol (GlcNAc)2.
270
H.-C. SIEBERT ET AL.
TABLE I. Surface Accessibilities [Å2] of CIDNP-Reactive Amino Acid Groups in the 8 Hevein Structures That
Represent Relative Local Energy Minima According to MD Simulations*
I
II
III
IV
V
VI
VII
VIII
x
160.9
87.6
32.5
75.0
148.7
71.1
20.1
57.0
162.8
76.1
19.6
92.6
143.0
101.8
22.7
94.4
167.9
72.3
32.0
106.9
121.1
90.8
23.0
111.1
151.4
83.7
25.3
89.9
125.9
75.4
28.2
55.3
125.2
70.2
3.2
50.9
137.4
70.4
0.0
85.3
144.6
103.0
0.0
88.2
149.7
82.4
26.6
98.4
113.4
98.6
0.0
105.4
133.9
79.8
11.5
78.1
a. Dot density: 1, sphere radius: 1.5 Å
Trp21
Trp23
Tyr30
His35
131.8
82.0
24.8
93.0
174.6
87.9
27.6
88.8
b. Dot density: 1, sphere radius: 4 Å
Trp21
Trp23
Tyr30
His35
122.1
68.4
17.8
65.6
153.1
69.8
15.9
75.5
*MD simulations were carried out in the GROMOS (Groningen molecular simulation) force field.44,45
TABLE II. Surface Accessibilities [Å2] of CIDNP-Reactive Amino Acid Groups in 7 NMR- and MD-Derived
Hevein Structures From the Protein Data Bank
I
II
III
IV
V
VI†
VII†
x
120.7
88.0
25.0
83.8
123.9
88.1
56.1
70.4
134.4
91.0
58.9
97.3
121.9
96.3
56.3
44.2
120.5
88.4
49.6
35.4
130.4
89.6
51.1
72.7
125.5
90.0
44.8
61.3
118.5
95.2
49.3
53.7
110.8
90.6
44.7
59.5
117.7
98.8
44.4
36.3
114.3
94.7
41.6
29.3
118.5
89.9
43.3
51.3
a. Dot density: 1, sphere radius: 1.5 Å
Trp21
Trp23
Tyr30
His35
150.4
91.5
46.9
81.6
141.1
83.6
65.0
96.1
b. Dot density: 1, sphere radius: 4 Å
Trp21
Trp23
Tyr30
His35
127.7
77.6
31.3
59.6
115.2
82.1
47.2
59.2
*MD simulations were carried out using the CHARMM (chemistry at Harvard macromolecular mechanics) force field.66
VI and VII are newly refined NMR structures of hevein and the respective information was kindly provided by Dr. N. H.
Andersen and Dr. B. Cao.
†Structures
TABLE III. Surface Accessibilities [Å2] of CIDNPReactive Amino Acid Groups in 5 Hevein Structures
That Represent Relative Local Energy Minima
According to MD Simulations*
Trp21
Trp23
Tyr30
His35
I
II
III
IV
V
x
113.1
108.3
63.9
99.0
106.9
99.6
53.8
83.1
113.5
104.1
55.5
107.5
110.5
97.2
70.1
107.3
116.0
109.7
75.2
114.9
112.0
103.8
63.7
102.4
*Dot density: 1, sphere radius: 1.5 Å. MD simulations were
carried out in the CVFF (consistence valence force field).42,43
ers of N-acetylglucosamine (GlcNAc).25,26 Besides its
putative role in defense mechanisms in the plant, it
is interesting to note that it triggers immunomodulatory responses in mammalian cells such as carbohydrate-dependent cytokine release or T-cell subset
mitogenicity.27 These activities point to a potential
perspective for clinical application, as is currently
under investigation for the mistletoe lectin.28–30 The
isoforms of dimeric WGA, which originate from
hexaploid wheat (Triticum aestivum), also bind
GlcNAc oligomers. Each chain of 17 kDa is divided
into four domains with sequence repeats of 43 amino
acids in length, suggesting evolution by gene duplication and fusion.31–33 Their structures display increased structural complexity in comparison to hevein and UDA. In contrast to hevein, pseudohevein,
and UDA, WGA exhibits also a binding specificity for
N-acetylneuraminic acid (NeuNAc). Moreover, the
carbohydrate-binding sites of WGA are composed of
regions contributed by domains on different subunits
that are in contact with the subunit/subunit interface. Occupancy by either NeuNAc or GlcNAc oligosaccharides has been observed in crystal structures
at all these sites.14,34–37 With the help of recombinant
techniques it is possible to prepare single domains of
the lectin for assessment of their individual binding
properties. As the preferentially occupied site, domain B has been chosen for this purpose.13,38 In this
study, the single domain of the more complex lectin
WGA1 with its four covalently linked units is tested
to compare its behavior to that of the single-domain
proteins hevein as well as pseudohevein. These data
for one domain derived from WGA can also contrib-
CARBOHYDRATE RECOGNITION BY LECTINS
271
Fig. 2. Laser photo CIDNP difference spectra (aromatic part) of hevein and hevein–(GlcNAc)4
complexes at pD 3. a: Ligand-free hevein. b: 1 mmol hevein 1 1 mmol (GlcNAc)4. c: 1 mmol hevein 1
2 mmol (GlcNAc)4.
TABLE IV. Comparison of Surface Accessibilities
[Å2] of CIDNP-Reactive Amino Acid Groups
in Hevein Structures Obtained Either by
NMR Measurements in the Presence of the Ligand
or by Computer-Assisted Calculations of Hevein
in the Absence of the Ligand on the Basis
of These NMR Data*
Trp21
Trp23
Tyr30
His35
C
SD
H
SD
D
93.0
71.3
27.0
71.3
7.1
7.9
5.6
9.1
122.5
96.6
60.1
68.8
8.1
7.0
7.2
9.0
229.5
225.3
233.1
12.5
*Dot density: 1, sphere radius: 1.5 Å. C, hevein-ligand complex;
H, ligand-free hevein; SD, standard deviation.
ute to simplify the interpretation of the four hevein
domain-containing hololectin.
MATERIALS AND METHODS
Carbohydrate Ligands
N,N8-Diacetylchitobiose, N,N8,N9-triacetylchitotriose, N,N8,N9,N--tetraacetylchitotetraose, N-acetylneuraminic acid(a2-3)lactose, N-acetylneuraminic
acid(a2-6)lactose and 2-deoxy-2,3-didehydro-Nacetylneuraminic acid (Neu5Ac2en) were purchased
from Sigma (Munich, Germany). Deuterated water
(99.96%) was obtained from Merck, Sharp and Dohme
(Montreal, Canada). Flavin I mononucleotide was a
gift from Dr. F. Müller (Sandoz, Basel). All other
reagents were of analytical grade.
Carbohydrate-Binding Proteins
Hevein and pseudohevein were isolated from the
latex of Hevea brasiliensis, as described elsewhere.19
Rubber latex of clones GT.1, PR-261 and LCB-1320
of Hevea brasiliensis were collected in several plantations (PTP XII Jalupang and PTP XIII Wangun Reja)
in the area of Subang (Western Java, Indonesia),
cooled on ice and centrifuged at high speed to collect
the lutoid body fraction. Repeated freezing and thawing of this fraction followed by centrifugation yielded
B serum, which was suspended at 4°C in water (4
mg/ml) after lyophilization and processed according
to established procedures to supply hevein and pseudohevein.
272
H.-C. SIEBERT ET AL.
Fig. 3. Laser photo CIDNP difference spectra (aromatic part) of pseudohevein and pseudohevein–(GlcNAc)4 complexes. a: Ligand-free pseudohevein. b: 1 mmol pseudohevein 1 1 mmol
(GlcNAc)4. c: Signal intensity of Trp23e3 in dependence of the ligand concentration.
TABLE V. Surface Accessibilities [Å2] of CIDNP-Reactive Amino Acid Groups of 10 Pseudohevein NMR
Structures That Represent Relative Local Energy Minima According to MD Simulations*
Tyr21
Trp23
Tyr30
I
II
III
IV
V
VI
VII
VIII
IX
X
x
82.9
123.7
30.1
91.9
119.8
27.9
94.1
124.8
24.7
83.2
126.5
27.1
84.7
125.1
36.3
87.9
130.8
39.3
88.3
143.4
36.0
89.8
114.8
37.2
84.3
118.3
36.7
103.4
116.2
36.9
89.1
127.3
33.2
*Dot density: 1, sphere radius: 1.5 Å. MD simulations were carried out in the GROMOS (Groningen molecular simulation) force
field.44,45
The lectin from commercially available rhizomes
of Urtica dioica was purified by successive steps,
employing affinity chromatography on chitin, baseand acid-washed to remove an agglutinating substance that is associated with chitin, and subjected
to cation exchange chromatography on sulfopropylSephadex C-50. Experimental details have been
given elsewhere.26,39 Additionally, gel filtration on
Biogel P-10 was performed to remove any aggregates. The lectin was judged to be homogeneous by
SDS-PAGE analysis.
WGA was isolated from raw wheat germ, as described previously.40
Clones of the 43-residue domain B of WGA were
generated by using a fusion protein construct for
convenient purification and detection.38 Briefly, two
overlapping synthetic oligonucleotides were de-
signed which encompass the complete coding region
of domain B, including cleavage sites for restriction
enzymes at each end, and the heveinlike terminal
sequence G E Q. These were amplified by the polymerase chain reaction and ligated into the plasmid
pUC 18, previously engineered to encode a truncated
sequence of hepatitis B antigen followed by an
arginine moiety for tryptic cleavage. The core-domB
fusion product was expressed in Escherichia coli and
purified by hydroxyapatite and Sepharose CL-4B
column chromatography in successive steps according to a described method.41 The fusion partners
were separated by trypsin digestion in Tris-buffered
saline, pH 8.0. Optimal yields of domB were obtained
in 2M guanidine-HCl at protein concentrations ,2.5
mg/ml. domB was isolated from the supernatant of
this digest after precipitation of core by either lower-
273
CARBOHYDRATE RECOGNITION BY LECTINS
TABLE VI. Surface Accessibilities [Å2] of CIDNP-Reactive Amino Acid Groups of 10 Pseudohevein NMR
Structures That Represent Relative Local Energy Minima According to MD Simulations*
I
II
III
IV
V
VI
VII
VIII
IX
X
x
82.2
132.4
44.1
78.8
122.2
30.7
79.0
131.9
40.7
129.0
100.8
83.8
133.3
128.9
81.2
122.4
106.3
89.0
a. Start structure: hevein structure, manual amino acid replacement. Dot density: 1, sphere radius: 1.5 Å
Tyr21
Trp23
Tyr30
86.1
122.9
43.6
82.4
122.6
47.6
83.2
134.8
33.5
74.7
132.3
35.8
73.5
141.9
36.9
68.2
139.7
43.7
81.8
137.7
44.5
78.7
132.8
46.3
b. Start structure: ProMod based on hevein structure. Dot density: 1, sphere radius: 1.5 Å
Tyr21
Trp23
Tyr30
107.2
96.9
73.3
111.8
110.7
79.7
104.4
104.5
90.3
113.8
106.5
72.1
133.3
104.5
95.1
119.2
102.8
110.8
125.7
107.1
103.3
146.3
100.2
100.8
*MD simulations were carried out in the CVFF (consistence valence force field).42,43
Fig. 4. Energy minimum conformation of pseudohevein, emphasizing the surface exposition of
Tyr21 and Trp23 in the upper part of the picture.
ing the pH to 4.5 or with 45% ammonium sulfate.
The purity and oligomeric state of domB were determined by using HPLC, P-30 gel filtration, amino acid
sequence analysis, and mass spectrometry. The ability of domB to bind GlcNAcN oligomers was verified
by chitin affinity chromatography, tryptophan fluorescence spectroscopy, and isothermal titration calorimetry.
CIDNP Method
CIDNP experiments were performed at 360 MHz
on a Bruker AM-360 NMR spectrometer. The light of
a continuous-wave argon ion laser (Spectra Physics,
Mountain View, USA) that operates in the multiline
mode with principal wavelengths of 488.0 and 514.5
nm was directed to the sample by an optical fibre and
274
H.-C. SIEBERT ET AL.
By alternately recording a light and four dark freeinduction decays, Fourier transformation and subtraction of the resulting light spectrum from the
dark spectrum, the CIDNP difference spectrum was
established, containing only lines of polarized residues. The tyrosine-dependent CIDNP effect corresponds to a spin-density distribution of the intermediate phenoxy radical with strong negative signals of
the e1, e2 protons and less intense positive signals for
the d1, d2 protons. The CIDNP spectrum of tryptophan is caused by an intermediate radical with
strong spin density at the d1, e3, and h2 positions of
the indole unit and very small spin density at the z2
and z3 positions, which all lead to positive CIDNP
signals. CIDNP difference spectra of histidine show
positive singlet signals for proton e1 and proton d2.6
Two-Dimensional NMR Experiments
Fig. 5. Laser photo CIDNP difference spectra (aromatic part)
of domB (WGA1) and domB (WGA1)–(GlcNAc)4 complexes. a: 0.2
mmol ligand-free domB (WGA1). b: 0.2 mmol domB (WGA1) 1
0.5 mmol (GlcNAc)4.
chopped by a mechanical shutter controlled by the
spectrometer. Typical operation conditions were 1 s
presaturation pulse for water suppression, 0.5second light pulse (5 W), 5-µs RF pulse (90° flip
angle), 1-second acquisition time, and 5-second delay. The duration of the light pulse is short enough to
prevent serious heating due to light absorption. 8,
16, or 32 light scans gave an adequate signal-tonoise ratio for the tested samples. CIDNP was
generated by using flavin I mononucleotide. Deuterated water was used as solvent for the assay mixture
containing 0.2 to 2 mmol lectin, 0.5 to 2 mmol ligand,
and 0.4 mmol flavin. The pD was calculated by
adding 0.4 to the reading of a conventional pH meter,
equipped with a glass electrode. Chemical shifts
were determined relative to the position of the
acetone signal (2.225 ppm). The broad signal in the
region 4.7–4.8 ppm arose from the partially suppressed resonance of residual HOD (signal of partly
deuterated water) at a ppm value of 4.76 ppm (pD 5
5.8, T 5 300 K).
Briefly, the individual measurements were carried
out in the following order of steps, as outlined in
detail elsewhere.6 After the addition of a flavin dye to
the protein solution, the sample was irradiated by an
argon-ion laser during the light part of the CIDNP
experiment. The photoexcited dye reacts reversibly
with surface-exposed amino acid side chains of histidine, tyrosine, and tryptophan, thereby generating
protein-dye radical pairs. Nuclear spin-polarization
was obtained from backreactions of the radical pairs.
To enable proton assignment and independent
verification of the apparent consequences of the
CIDNP experiments, TOCSY (total correlated spectroscopy) and NOESY (nuclear Overhauser effect
spectroscopy) spectra of hevein, pseudohevein, and
domB were recorded in H2O as well as in D2O at
temperatures of 293 or 300 K with mixing times from
100 to 200 ms at 500 or 600 MHz on a Bruker 500 or
600 AMX-NMR spectrometer of the Bijvoet Center
(Department for NMR Spectroscopy). In the case of
WGA and UDA, only NOESY spectra in D2O at 300 K
were recorded.
Computational Calculations
MD simulations for hevein, pseudohevein, and
domB were carried out by using the CVFF (consistent valence force field).42,43 In the case of hevein and
pseudohevein we also used the GROMOS (Groningen molecular simulations)44,45 force field for the
molecular dynamics calculations at a simulation
temperature of 300 K.
The starting structures of the lectins were either
taken from the Brookhaven Protein Data Bank or
generated by using knowledge-based homology modeling technique. The generation of the H atoms,
automatic assignment of partial charges for each
atom of the molecule, was accomplished by using the
INSIGHTII software. The center of mass of the lectin
was placed in the center of a 30 3 30 3 30 Å large
water box. A simple point charge model for the water
was used, allowing the atoms of the water molecules
to vibrate. Periodic boundary conditions were applied by using a double cutoff limit of 10 and 12 Å for
the water box. Each molecular ensemble was submitted to a molecular dynamics simulation by using the
CVFF force field at a temperature of 300 K, with an
equilibration time of 20 ps, a production period of
100 ps, an integration step of 1 fs and a dielectric
constant of 1. Each 250 steps of integration a conformation was stored. From the 400 individual conformations obtained within the production time the 10
275
CARBOHYDRATE RECOGNITION BY LECTINS
TABLE VII. Surface Accessibilities [Å2] of CIDNP-Reactive Amino Acid Groups of 10 domB (WGA1) Structures
Representing Relative Local Energy Minima According to MD Simulations*
I
II
III
IV
V
VI
VII
VIII
IX
X
x
107.3
86.3
32.8
115.3
64.5
28.1
117.5
75.6
46.2
129.0
100.8
83.8
133.3
128.9
81.2
122.4
106.3
89.0
120.8
92.8
70.1
119.8
79.4
56.1
127.2
86.8
71.2
a. Start structure: ProMod based on hevein structure. Dot density: 1, sphere radius: 1.5 Å
Tyr64
Tyr66
Tyr73
141.8
77.8
75.9
141.8
68.4
52.8
111.7
79.0
70.4
126.8
78.0
57.7
132.9
66.0
46.5
117.7
71.9
37.9
86.1
92.4
26.3
93.7
67.5
29.7
b. Start structure: ProMod based on all similar structures. Dot density: 1, sphere radius: 1.5 Å
Tyr64
Tyr66
Tyr73
107.2
96.9
73.3
111.8
110.7
79.7
104.4
104.5
90.3
113.8
106.5
72.1
133.3
104.5
95.1
119.2
102.8
110.8
125.7
107.1
103.3
146.3
100.2
100.8
c. Start structure: X-ray data of domB from WGA1. Dot density: 1, sphere radius: 1.5 Å
Tyr64
Tyr66
Tyr73
134.4
83.9
68.2
138.1
88.9
67.7
122.6
82.5
71.8
122.8
85.2
68.0
131.5
88.3
85.7
124.6
89.0
77.5
127.0
92.1
80.0
124.5
85.5
66.7
*MD simulations were carried out in the CVFF (consistence valence force field).42,43
with the lowest potential energy were automatically
selected and processed for energy minimization of
the complete ensemble by using the conjugate gradient method. Generation of possible conformations for
domB was achieved by using different approaches: 1)
singling out the domB from the X-ray structure of
the complete lectin (PDB-entry 7WGA); and 2) knowledge-based homology modeling techniques by using
the ProMod tools46 based on a) 1HEV as 3D template
and b) 1WGC, 2WGC, 1WGT, and 1HEV as 3D
template. The MD calculations were performed on
an IBM-SP2 parallel machine by using four processors. The handling of the molecules and the analysis
of the MD results were accomplished with various
modules of the INSIGHTII software on a Silicon
Graphics Indigo2 workstation.
The surface accessibilities of the respective amino
acid side chains were calculated by using the X-ray
structures of WGA1 and WGA2 (Brookhaven Protein
Data Bank) or MD structures of hevein and pseudohevein that have been ascertained independently by
two-dimensional NMR measurements. The calculations were carried out with the help of the Connolly
program in InsightII on a Silicon Graphics Personal
Iris following an established method.47,48 The assessment of the routine ‘‘surface’’ based on the Connolly
program49,50 pinpoints the accessible exterior part of
the relevant portions of the molecule by smoothing
the van der Waals surface with a test sphere that
displays the average radius of the solvent water (1.5
Å) or the average radius of flavin (4 Å). Both average
radii were calculated according to published data.51
The dot density was generally set to a value of 1.
RESULTS AND DISCUSSION
Hevein
The small size of the protein allows unequivocal
assignment of signals to individual protons and
comparison of the NMR-derived structure with calculated surface accessibilities as quality control. The
CIDNP signals of hevein indicate a high accessibility
for Trp21 and for Trp23 in contrast to a low accessibility for Tyr30 (Fig. 1). This agrees well with calculations that assess the surface accessibilities of the
respective residues in several hevein NMR conformations. For example, the surface display of the CIDNPreactive moieties was calculated for eight hevein
energy-minimized conformations we derived from
combined application of our own NMR data (unpublished results) and MD simulations by using the
GROMOS force field (Table I). When this set was
compared to seven published conformational variants of hevein,52 especially the binding region showed
a high degree of conformational conservation. The
calculated surface accessibilities of the CIDNPreactive amino acids in these resolvable spatial
arrangements are compiled in Table II. The average
radius of flavin (4 Å) was chosen in addition to that of
water (1.5 Å) to reflect the fact that the CIDNP effect
is generated by an interaction with the dye, as given
in Tables I and II. In general, the calculated accessibilities of the tyrosine, tryptophan and histidine
residues decrease with the assumed increase of the
sphere radius. At both test sphere radii the same
residues can be identified as having low, medium, or
high accessiblity. For comparison, surface accessibility values for the different hevein conformations
derived from MD calculations in the CVFF are also
given in Table III. A significant difference between
protein conformational aspects and surface accessibility was detected only for His35, which is positioned in a highly flexible part of the protein chain.
By including the accessibility values derived from
different energy minimum conformations into our
276
H.-C. SIEBERT ET AL.
Fig. 6. Laser photo CIDNP difference spectra (aromatic part)
of domB (WGA1) and domB (WGA1)–(GlcNAc)4 complexes. The
small positive CIDNP signals originate from the d1, d2 protons of
the tyrosine residues. a: 2 mmol domB (WGA1) 1 1 mmol
(GlcNAc)4. b: 2 mmol domB (WGA1) 1 1.5 mmol (GlcNAc)4. c:
CIDNP-difference spectrum of the aromatic part of the corresponding one-dimensional spectra, given above. 2 mmol domB (WGA1) 1 2
mmol (GlcNAc)4.
tables, we take into account the inherent protein
flexibility and the resulting fluctuation of the surface
presentation of this side chain. It is evident that
movement of side chains alters their surface exposure within certain limits. When the accessible area
adopts a value above 80 Å2 (with a test sphere radius
of 1.5 Å), the tyrosine, histidine, and tryptophan
residues can legitimately be considered as surfaceexposed and are thereby able to generate a CIDNP
signal of high intensity. When chitobiose is added to
hevein, CIDNP signals are altered. The intensities of
the signals of the protons, whose reactivities to the
photoactivated dye are affected by ligand binding,
decrease (Fig. 1). As can be seen in Figures 1 and 2,
the overlapping h2-proton signals of Trp21 and Trp23
are broadened but their intensity is not weakened.
In Fig. 2c one can see that the intensity of the
h2-proton signal is even enhanced. These results
indicate that the h2-protons are not strongly shielded
by the ligand, which means that distinct parts of the
CARBOHYDRATE RECOGNITION BY LECTINS
277
Fig. 7. Energy minimum conformation of domB of WGA1, emphasizing the surface exposition of
Tyr64 and Tyr66 in the upper part of the figure.
Trp residues are covered differently. Such an observation can be helpful in case of computer-assisted
docking studies because a defined reorientation of
the two Trp residues in case of ligand binding has
been detected. Our results thus reveal a carbohydrate-dependent effect on the CIDNP spectrum,
which in the absence of a major conformational
change by ligand binding can be attributed to ligandshielding of certain amino acid side chains.
Involvement of Ser19, Trp21, Trp23, and Tyr30 in
carbohydrate binding has been proven independently by two-dimensional NMR experiments53 (unpublished results). The availability of NMR-derived
coordinates of the hevein–chitobiose complex, kindly
provided in detail by Dr. Jimenez-Barbero, allows
comparison of surface accessibility values of this
complex in solution to the corresponding values of
hevein in the absence of the ligand, as given in Table
IV. These results corroborate the evidence from the
CIDNP experiments that the ligand reduces the extent
of interaction between these residues and the dye.
CIDNP experiments at two different pH values
were carried out in order to test whether the surface
accessibility is affected by this parameter. An apparent pH dependence of the hevein conformation was
indicated by the change in intensity of the signals of
Trp21, Trp23, Tyr30, and His35 (Figs. 1, 2). En-
hanced intensity at increasing pH values can be
attributed to an increased accessibility of Tyr30 due
to conformational changes at elevated pH values.
This interpretation is in agreement with results of a
recent study, in which the pH dependence of the
conformation in a distinct part of hevein, that is, the
part between amino acid residues 28 and 35, has
been described.52
Pseudohevein
For pseudohevein, where the highly exposed and
flexible Trp21 is a tyrosine moiety, we observed a
strong Tyr signal in the CIDNP spectrum (Fig. 3a).
The CIDNP signal of the conserved Trp23 is maintained. The different degrees of accessibility as illustrated by the CIDNP data could be confirmed by
molecular dynamics calculations, compiled in Tables
V and VIa,b. During the simulation period, 10 intermediate coordinate frames were collected and energetically minimized. The initial structure was derived from an energy-minimized coordinate frame of
hevein in which the six variant amino acids were
replaced by the appropriate residues of pseudohevein. The resulting coordinate frame was energetically minimized. Possible differences between the
conformations of hevein and pseudohevein were
analyzed by MD simulations. The MD calculations of
278
H.-C. SIEBERT ET AL.
Fig. 8. Laser photo CIDNP difference spectra (aromatic part) of UDA and UDA-(GlcNAc)4
complexes. a: Ligand-free UDA. b: 1 mmol UDA 1 1 mmol (GlcNAc)4.
pseudohevein indicate that Tyr21 has a significantly
higher accessibility than Tyr30, independent of the
applied force field, either GROMOS (Table V) or
CVFF (Table VIa,b). However, comparisons between
the accessibility values given in Tables V, VIa,b show
remarkable differences. Taking the strong CIDNP
signals from tyrosine into account, the values of
Table VIb obtained with the ProMod procedure yield
the closest agreement to the experimental data (Fig.
3a,b). A low-energy minimum conformation of a
complete pseudohevein molecule, depicted in Figure
4, illustrates the high degree of surface exposure of
Tyr21 and Trp23. The surface exposure of Tyr30 is
increased relative to hevein (Table VIb). The CIDNP
spectrum of the pseudohevein–(GlcNAc)4 complex
(Fig. 3b) suggests an involvement of residues 21, 23,
and 30 in carbohydrate binding, which is consistent
with the results obtained for hevein. To illustrate the
quantitative relationship between signal intensity
and ligand concentration, the degree of suppression
of the nonoverlapped signal of proton Trp23e3 in
dependence of the ligand concentration is shown in
Figure 3c. Moreover, the pH dependence of conformational features of pseudohevein is similar to that of
hevein (not shown). It is remarkable that pseudohevein and hevein do not lose their binding abilities, not
even at pH 1, despite the presence of conformational changes. Having monitored the CIDNP behav-
ior of these two small lectins, we proceeded to use a
single domain of the more complex agglutinin WGA,
obtained from genetic engineering, in the next series
of measurements, extending the experimental basis
to more complex GlcNAc-binding lectins of this
family.
Domain B of Wheat Germ Agglutinin
To answer the question whether the so far documented ligand-dependent signal suppression of
CIDNP-reactive amino acids will also be observed in
a single heveinlike domain of WGA1, we investigated the recombinant domain B (domB). Interpretation of the CIDNP signals of the complete WGA
molecule can be more easily analyzed, when spectra
of the single domain are available. The CIDNP
spectra of ligand-free domB display overlapping
signals for Tyr66e1,2 and Tyr73e1,2 overlap (Fig. 5).
Tyr64e1,2 generates the most pronounced CIDNP
signal which points to a very high accessibility (Fig.
5). This observation deserved special attention in the
molecular modeling studies of domB. One initial
structure was derived from a low-energy minimum
coordinate frame of hevein. The second start structure was obtained by comparison with all other
similar structures by using the ProMod procedure.46
The third start structure was taken from the X-ray
data of WGA1 in the Brookhaven protein data bank.
CARBOHYDRATE RECOGNITION BY LECTINS
Fig. 9. Laser photo CIDNP difference spectra (aromatic part) of WGA2 and WGA2-(GlcNAc)4
complexes. a: Ligand-free WGA2. b: 0.5 mmol WGA2 1 0.5 mmol (GlcNAc)4. c: 0.5 mmol WGA2 1
0.75 mmol (GlcNAc)4. d: 0.5 mmol WGA2 1 1 mmol (GlcNAc)4. e: Signal intensity of Tyr in
dependence of the ligand concentration.
279
280
H.-C. SIEBERT ET AL.
Urtica dioica Agglutinin
Figure 9.
(Continued.)
All MD simulations demonstrated that Tyr64 has
the highest accessibility (Table VIIa,b,c). As observed for hevein and pseudohevein, the CIDNP
signals of domB can be suppressed by addition of
N-acetylglucosamine oligomers. When the concentration of domB in CIDNP studies was increased (Fig.
6), all three Tyre1,2 signals were separated at a 2:1
molar ratio between domB and (GlcNAc).4 The three
Tyr-dependent CIDNP signals behaved differently
as a function of the ligand (Fig. 5). The CIDNP signal
of Tyr64e1,2 was most strongly affected when
(GlcNAc)4 was added to domB in a molar ratio of 1:2.
By contrast, the CIDNP signals of Tyr66e1,2 and
Tyr73e1,2 decreased after further enhancement of
the ligand concentration and finally were suppressed
more strongly than the Tyr64e1,2 signal. This result
illustrates that it is possible to detect different
degrees of responsiveness of CIDNP-sensitive residues. The ligand-dependent CIDNP reactivity of all
three Tyr residues underscores their role in stabilizing protein–carbohydrate complexes. The relative
orientations of three Tyr residues are modeled in
Figure 7. Based on these results, we conclude that all
three structure-generating procedures lead to comparable conformations of the binding region that are in
agreement with the CIDNP data, although distinct
differences in surface accessibility of Tyr73, for example, occur. These findings are of special interest
because deviations between the X-ray structure and
NMR structures of hevein have been reported.52,54
Having so far evaluated the properties of proteins
with one GlcNAc-binding site, the next two paragraphs will report data on agglutinins with two or
four domains.
UDA consists of two hevein domains. Alignment of
UDA with the other heveinlike lectins shows that
Trp21, Trp23, and Tyr30 as well as His67, Trp69, and
Tyr76 are the corresponding aromatic amino acids in
the binding pocket. Trp16, Trp40, His47, and Tyr84
are other CIDNP-reactive amino acid residues of
UDA, which are located in exposed surface regions
other than the GlcNAc-binding sites.26 CIDNP spectra of hevein–, pseudohevein– or domB–carbohydrate complexes show a reduced signal intensity in
relation to the CIDNP spectra of ligand-free protein
(Figs. 1–3, 5, 6). In contrast to this, the CIDNP
spectrum of UDA with its two overlapping Tyr
signals is not strongly affected after ligand addition
(Fig. 8). This points to a lesser extent of shielding of
the binding site residues of UDA by (GlcNAc)–
oligomers in comparison to that observed for the
hevein, pseudohevein and domB binding sites, which
show reduced signals. Assignment of the signals is in
agreement with published data.55 The occurrence of
the Trp21e3 signal in the CIDNP spectra of UDA–
(GlcNAc)–oligomer complexes raises the question
whether GlcNAc binding is accompanied by a conformational change of and/or around Trp21 (Fig. 8). A
similar observation has been made based on a biochemical experiment in the case of an insect lectin,
whose positive surface charge and susceptibility to
trypsin are altered by lactose binding.56 The presence of a ligand-induced conformational change allows consideration of various models. In one of these
models a significant conformational change of the
carbohydrate receptor has previously been predicted.57,58 In view of the remarkable homology to
hevein, it is notable that the resonances of Trp21 and
Trp23 of UDA are broadened in the presence of the
ligand.55 This homology to hevein is obviously indicated also by the similarities of the CIDNP spectra of
hevein (Fig. 1) and that of UDA (Fig. 8).
Wheat Germ Agglutinin
The WGA monomer consists of four hevein domains (A, B, C, D), each with an architecturally
unique binding site characterized by aromatic amino
acid residues. These residues are tyrosines in the
binding sites of domains A and B (Tyr21, -23, -30; and
Tyr64, -66, and -70, respectively). In WGA2 Tyr66 is
replaced by His. Domains C and D have a somewhat
different character where the corresponding residues are Trp107, Phe109, and Phe116 as well as
Trp150 and Tyr159, respectively. A conserved Ser
residue, which interacts with the carbonyl group of
bound GlcNAc, is present in all four binding sites. A
subsidiary binding region located on an adjacent
domain of the second monomer further contributes
to ligand stabilization in all binding sites except that
of domain A. Thus, as an example of a more complex
structure, WGA can be used to test the conclusions
281
CARBOHYDRATE RECOGNITION BY LECTINS
drawn from the simple one- or two-domain lectins.
As illustrated in Figure 9a, several surface-exposed
aromatic residues (His, Trp, Tyr) of WGA2 produce
signals in the difference CIDNP spectrum. Upon
ligand binding a decrease of the intensity of the Tyr
signal is observed as a function of the oligosaccharide chain length (N,N8-diacetylchitobiose, N,N8,N9triacetylchitotriose or N,N8,N9,N--tetraacetylchitotetraose) (not shown) and ligand concentration (Fig.
9a–d).
The gradual decrease of the peak areas as a
function of increases in saccharide concentration is
shown in Fig. 9b–d. When the measured intensities
are plotted against the ligand concentration, the
pronounced shielding of the ligand is readily apparent (Fig. 9e). Remarkably, it is evocative of the
correlation of these two parameters determined in
the case of pseudohevein (Fig. 3c). The Trp/His
signals are also strongly reduced after stepwise
addition of N,N8,N9,N--tetraacetylchitotetraose to
WGA2 as demonstrated in Fig. 9b–d. In order to
identify which aromatic amino acid side chains can
contribute most to the intensity of the CIDNP signals, we have calculated the surface accessibilities of
the tyrosine, tryptophan and histidine residues from
the available crystal structures (Brookhaven protein
data bank), as shown in Table VIII. It is apparent
from these values that Tyr21 and Tyr64 possess the
highest accessibility and presumably contribute most
strongly to the tyrosine CIDNP signal intensity in
the difference spectrum.
The surface accessible Tyr residues generate only
one Tyr CIDNP signal at 6.8 ppm which decreases at
a slower rate with increasing ligand-concentration
(Fig. 9a–d) than the CIDNP signals of the Trp/His
residues do. This is in accordance with the fact that
four tyrosine moieties are present in the two weak
binding sites (3 in site A and one in site D) and that
these sites may not be fully occupied at low ligand
concentration. As mentioned above, histidine is present only in the B binding site of WGA2, and its
accessibility is rather low (Table VIII). A highly
accessible Trp107 appeared to be present in the
binding site in the C-domain of WGA. Although
GlcNAc binding at the C-domain site was not observed in the crystal due to molecular packing
interactions, binding of NeuNAc was obtained in
another crystal form in which it participates in a
crosslinking binding mode. The evidence from our
CIDNP spectra suggests that GlcNAc–oligomers occupy the C-domain site due to the high accessibility
of Trp107 and the strong ligand-dependent responsiveness of the Trp/His signal (Fig. 9). The CIDNP
technique thus provides evidence for occupancy of all
four sites at high ligand concentration. However, the
spectrum is complicated by the fact that aromatic
amino acids that are not located in the binding sites
also contribute to the CIDNP signals (His59, Trp41,
Tyr145). Thus, the resonances of the aromatic amino
TABLE VIII. Surface
Accessibilities [Å2]
of CIDNP-Reactive Amino
Acid Groups
I
II
a. WGA1 (monomer I,
monomer II). Dot density: 1,
sphere radius: 1.5 Å
Tyr21
Tyr23
Tyr30
Trp41
Tyr64
Tyr66
Tyr73
Trp107
Tyr145
Trp150
Tyr159
96.6
94.0
59.8
136.0
111.4
69.5
63.8
125.7
55.6
56.7*
62.0
103.7
96.3
43.3
132.3
102.1
82.4
52.2
121.9
57.9
142.2
73.6
b. WGA2 (monomer I,
monomer II) according to the
X-ray structures taken from
the Protein Data Bank. Dot
density: 1, sphere radius: 1.5 Å
Tyr21
Tyr23
Tyr30
Trp41
His59
Tyr64
His66
Tyr73
Trp107
Tyr145
Trp150
Tyr159
91.2
98.0
46.7
131.4
44.3
97.9
50.1
55.4
142.0
50.2
161.0
58.1
96.2
87.2
61.5
130.5
31.6
101.5
57.9
62.9
136.3
40.8
55.5*
69.8
*The complete structural coordinates of this residue are not given
in the Protein Data Bank.
acid residues are not quenched in the presence of the
specific ligand (Fig. 9).
With regard to the binding of NeuNAc oligosaccharide or 2-deoxy-2,3-didehydro-N-acetylneuraminic
acid (Neu5Ac2en) (data not shown) to WGA, our
method fails to detect any changes in the CIDNP
spectrum relative to the ligand-free lectin. This
result suggests either that NeuNAc covers the binding pocket differently or that the method is not
suitable to analyze weak interactions of a ligand to
the binding site.
Commercially available isolectin mixtures of WGA
exhibit the same changes in the CIDNP signals, as
observed in either the WGA1 or WGA2 spectrum,
when a ligand is added (not shown). This observation
suggests that both isoforms of WGA behave similarly
upon ligand binding.
282
H.-C. SIEBERT ET AL.
CONCLUSIONS
GlcNAc-specific lectins of increasing structural
complexity have been analyzed in CIDNP experiments monitoring the surface exposure of histidine,
tyrosine and tryptophan as sensors for carbohydrateprotein interaction. It could be demonstrated that
carbohydrate binding reduces the intensity of distinct signals due to ligand shielding of several aromatic amino acids in the binding regions. In the case
of UDA the surface accessibility of a Trp residue
increases as a result of addition of GlcNAc–oligomers, suggesting a ligand-induced change of surface
presentation of that residue.
The single domain lectins hevein and pseudohevein and recombinant domB of WGA1 show a similar
pattern of responses, corroborating the notion that
aromatic amino acids are present in all homologous
binding sites. In addition to ligand-dependent differences in signal intensity variations of the pH value
can also lead to conformational changes, which,
however, appear not to affect ligand binding.
Since only three types of aromatic amino acids are
CIDNP-sensitive, the probability of signal overlap in
a CIDNP difference spectrum is lower than in the
conventional one-dimensional 1H-NMR spectrum.
Furthermore, as shown in Fig. 6c, an aromatic amino
acid signal in an one-dimensional 1H-NMR spectrum
can be broadened and/or have reduced intensity in
the presence of a saccharide-ligand. The CIDNP
approach allows correlation of signal intensity and
signal responsiveness as a function of surface exposure and ligand shielding of a specific residue. These
measurable parameters can therefore be helpful in
optimizing molecular models for computer-aided
docking studies. As also revealed in this study, it is
possible to comparatively assess the validity of computer-assisted structural calculations by different
modeling techniques. The significant structural differences between the pentraxins serum amyloid P
component and C-reactive protein despite their remarkably high degree of sequence homology call
attention to the necessity to take any reliable conformational parameter into account for meaningful
modeling studies to avoid being led astray.59
In sum, the CIDNP method provides a valuable
tool to study protein–carbohydrate interaction in
addition to multidimensional NMR-techniques, Xray-analysis and computer-assisted molecular modeling.16,60–65
ACKNOWLEDGMENTS
We are indebted to Dr. N. H. Andersen and Dr. B.
Cao for information on their hevein NMR data, Dr. J.
Jimenez-Barbero for information on his hevein–
ligand complex NMR data, Dr. L. Kroon-Batenburg
for advice to perform calculations of surface accessibility, and P. Edebrink and J. O. Koopmann for
technical assistance. This work was supported by the
Human Capital and Mobility Program of the European Community, and the Dr. M. Scheel-Stiftung für
Krebsforschung.
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