From the Institut de Biologie Structurale,
CNRS-CEA-UJF, Laboratoire de Biophysique Moléculaire, 41 Rue
Horowitz, 38027 Grenoble Cedex 01, ¶ Institut Pasteur,
Unité de Chimie Organique, 28 Rue du Dr. Roux, Paris,
75015 and
Centre de Recherches sur les Macromolécules
Végétales, CNRS (affiliated with Université Joseph
Fourier), 38041 Grenoble Cedex 09, France
Received for publication, September 5, 2000, and in revised form, November 14, 2000
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The binding of chemokines to
glycosaminoglycans is thought to play a crucial role in chemokine
functions. It has recently been shown that stromal cell-derived
factor-1 Chemokines are small structurally related chemo-attractant
cytokines, characterized by conserved cysteine residues. Almost 40 chemokines have been identified to date which, based on the position of
the first N-terminal cysteines, fall into four sub-families. Two of
them have been well characterized, the CC group, which includes
regulated on activation, normal T-cell expressed, and secreted,
monocyte chemoattractant protein-1, and
MIP-11 (macrophage
inflammatory peptides-1), and the CXC group, the prototype of which is
interleukin-8. The C chemokine (lymphotactine) and the
CX3C chemokine (fractalkine) sub-families
have been identified more recently (1-4). These proteins signal
through G-protein-coupled seven transmembrane domain receptors (5) and
are primarily involved in immunosurveillance, activation, and
recruitment of specific cell populations during disease (1, 6-8).
Most, if not all, chemokines bind to heparan sulfate (9, 10), a
glycosaminoglycan (GAG) found ubiquitously at the cell surface (11-13)
and in the extracellular matrix (14). This binding is thought to be
functionally important, and current models indicate that heparan
sulfate (HS) either enhances the local concentration of chemokines in
the vicinity of the G-protein-coupled receptor (15) or provides a
haptotactic gradient of the protein along cell surfaces. For example,
leukocyte migration along the endothelium surface, and migration into
the tissues at the site of inflammation, is believed to depend on the
local presentation of chemokines by such cell surface-expressed GAGs
(16-18).
Stromal cell-derived factor-1 (SDF-1) is a member of the CXC chemokine
family of pro-inflammatory mediators and is a potent chemo-attractant
for a variety of cells, including monocytes and T-cells (19, 20).
However, SDF-1 has many other functions that extend well beyond
leukocyte migration, and in many respects, both SDF-1 and its receptor
CXCR4 have several unusual features. For example, mice that lack either
the SDF-1 or the CXCR4 gene die in utero,
with a number of defects including severe developmental abnormalities,
indicating that this chemokine clearly has important roles beyond
chemotaxis (21). Whereas the production of most chemokines is induced
by cytokines or mitogenic stimuli, SDF-1 is constitutively expressed in
a large variety of tissues (22-24). CXCR4, also widely distributed,
raises the question of the specificity of this system. HS-protein
interaction has selectivity on itself (25) and, although the functional
aspects of the SDF/HS interaction have not been reported to date, HS
could be of critical importance in orchestrating the cellular response
to this chemokine, as has already been reported for other ligands
involved in the developmental process (26). In addition to these
physiological functions, SDF-1 is a potent inhibitor of the cellular
entry of CXCR4-dependent human immunodeficiency virus, a
process that relies on the occupation and internalization of CXCR4 (20,
27). In view of the general importance of HS in the activity of
chemokines, the chemokine-HS interaction represents an attractive
target for therapeutic intervention (28). In addition, an obvious
application of the knowledge obtained on the SDF-1-HS complex is the
engineering of drugs blocking human immunodeficiency virus CXCR4
coreceptor. This study was thus undertaken to characterize further the
SDF-1 Equipment and Reagents--
An upgraded BIAcore system, pioneer
F1 sensor chip, amine coupling kit, and HBS (10 mM HEPES,
150 mM NaCl, 3 mM EDTA, 0.005% surfactant P20,
pH 7.4) were from Biacore AB. The Pioneer peptide synthesizer and
chemicals were from PE Biosystems. Amino acid analysis was performed on
a 6300 Beckman amino acid analyser. 125I-Labeled SDF-1
The selectively desulfated heparins were a gift from Prof. J. Gallagher, and the anti-SDF-1 SDF-1 Preparation of Heparin Oligosaccharides--
Porcine mucosal
heparin (1 g) was digested with heparinase I (8 milliunits/ml) in 15 ml
of 0.1 mg/ml bovine serum albumin, 2 mM CaCl2,
50 mM NaCl, and 5 mM Tris buffer, pH 7.5, for
54 h at 25 °C. The enzymatic reaction was stopped by heating
the digest at 100 °C for 5 min. The digestion products were then
size-separated using a Bio-Gel P-10 column (4.4 × 150 cm),
equilibrated with 0.25 M NaCl and run at 1 ml/min (31).
Eluted material, detected by absorbance at 232 nm, consisted of a
graded series of size-uniform oligosaccharides resolved from
disaccharide (dp2) to octadecasaccharide (dp18). To ensure homogeneity,
only the top fractions of each peak were pooled, and each isolated
fraction was re-chromatographed on a gel filtration column to eliminate
further possible contamination. Samples were dialyzed against distilled
water and quantified by colorimetric assay, using the Bitter and Muir
method (32).
Preparation of Heparan Sulfate 3H-Labeled
Oligosaccharides--
HS from Chinese hamster ovary cells were
prepared essentially as described (33). Briefly, confluent cells were
cultured for 48 h in Ham's F-12 medium, without glucose and
supplemented with 10 µCi/ml of [3H]glucosamine. The
labeled material was purified by DEAE-Sephacel chromatography, and the
proteoglycan peak was sequentially digested with chondroitinase ABC (1 unit/ml, 3 h at 37 °C) and papain (1 mg/ml, 16 h at
65 °C). The digest, consisting of intact HS chains, was re-purified
on a small DEAE-Sephacel column. Eluted material was desalted and
freeze-dried. Low pH nitrous acid treatment of a small aliquot of this
material, analyzed by Bio-Gel P-10 chromatography, gave a pattern of
depolymerization typical of HS (34). HS was then digested with
heparitinase (20 milliunits/ml) for 4 h at 30 °C, and
heparitinase resistant oligosaccharides were purified on a 1 × 150 cm Bio-Gel P-10 column.
Heparin-Sepharose Chromatography--
Each chemokine (5 µl of
2 × 10 Biotinylation, Heparin Immobilization, and Kinetic
Analysis--
Size-defined heparin (9 kDa) was biotinylated at the
reducing end as described and immobilized on a Biacore sensorchip (29). For this purpose, two flow cells of a F1 sensorchip were activated with
50 µl of a mixture of 0.2 M
1-ethyl-3-(dimethylaminopropyl) carbodi-imine/N-hydroxy-succinimide, 0.05 M
1-ethyl-3-(dimethylaminopropyl) carbodi-imine/N-hydroxy-succinimide
before injection of 50 µl of streptavidin (0.2 mg/ml in 10 mM acetate buffer, pH 4.2). Remaining activated groups were
blocked with 50 µl of 1 M ethanolamine, pH 8.5. Typically, this procedure permitted coupling of ~2000-2500 resonance
units of streptavidin. Biotinylated heparin (5 µg/ml) in HBS
containing 0.3 M NaCl was then injected over the surface to
obtain an immobilization level of 50 resonance units. Flow cells were
then conditioned with several injections of 1 M NaCl. For
binding assays, six different SDF-1
tions in HBS were injected at 50 µl/min onto the heparin surface for 3 min, after which the complexes
formed were washed withrunning buffer. The sensorchip surface was
regenerated with a 2-minpulse of
1 M NaCl in HBS, and the binding curves were analyzed as
described (29).
SDF-1 Enzyme-linked Immunosorbent Assay--
Monoclonal antibodies
(K15C and 4H4) were coated on a 96-well plate (5 µg/ml) overnight at
4 °C in phosphate-buffered saline. The coated mAbs were used to
capture biotinylated SDF-1 Computational Methods--
The coordinates of the crystal
structure of the N33A variant of the SDF-1
The coordinates of heparin were taken from the NMR-derived structures
(41) deposited in the Protein Data Bank (code 1HPN). Both structures,
differing by the ring shape of the L-iduronic acid residues
that can be either 2S0 or
1C4, were considered in the present study. They
are referred to in this paper as 2S0 heparin
and 1C4 heparin. Atom types and partial charges
were defined according to the PIM energy parameters for carbohydrates
(42) to be used within the Tripos force field (37). The torsional
angles of the glycosidic linkages between atom C-1 of residue
i and atom C-4 of residue j were defined as
Docking of Heparin in the Binding Site of SDF-1
The geometry of each of the complexes was optimized by several cycles
of energy minimization. The hydrogen atoms and pendent groups were
first optimized. Finally the whole heparin moiety together with the
side chain of the amino acids in the positively charged cleft were
fully optimized. All energy calculations were performed with the Tripos
force field (37) together with energy parameters specially derived for
carbohydrates (44) and sulfated derivatives (42). To evaluate the
energy of interaction, the complex was also optimized with the heparin
at a distance of 50 Å from the protein, therefore with no
intermolecular interaction.
The Dimeric Association of SDF-1 Experimental Identification of SDF-1 Oligosaccharide Length and Sulfate Dependence of the
SDF-1 The N-terminal Lys Residue of SDF-1 Modeling of the Interactions between Heparin and SDF-1
In the optimized complex, corresponding to the lowest energy binding
mode (Fig. 6), the heparin fragment,
consisting of 14 monomers, appeared to have the appropriate size for
binding the dimer. All the monosaccharides but two interacted directly
with the protein surface. The positively charged amino acids involved in salt bridges with the sulfate and carboxyl groups of heparin were
Lys-1, Lys-24, Lys-27, and Arg-41 of both monomers and Lys-8 of monomer
B and Lys-43 of monomer A. Table III
lists details of the electrostatic interactions. Contacts were
not identical for chains A and B of the dimer, since the
directionality of the polysaccharide chain prevented it from adapting
fully to the pseudo-symmetry axis of the protein. The side chains of
two neutral amino acids, Asn-46 and Gln-48, were also involved in
hydrogen bonding with the heparin molecule. A detailed study of the
interaction indicated that Lys-24, Lys-27, and to a lesser extent Lys-1
and Arg-41 had the major role in binding the polysaccharide fragment
since all of them established at least five salt bridges or hydrogen
bonds with the sugar residues. As displayed in Fig. 6, the heparin
conformation fitted perfectly with the shape of the crevice, whereas
the two extremities established contact with the two N-terminal regions of the SDF-1 Molecular modeling, mutant SDF-1 An additional finding of the present study is the involvement of the
N-terminal lysine residue (Lys-1) in heparin binding. The N-terminal
region of the chemokine (peptide 1-9) is critical for its biological
activity (54). Although it is disordered in solution, and adopts
multiple conformations, a recent study suggests a link between
structuring of the N-terminal SDF-1 peptide and its ability to activate
its receptor (55). Whether or not the interaction of Lys-1 with heparin
stabilizes the N-terminal structure of the chemokine is not known, but
this could represent a way by which heparin acts on SDF-1. N-terminal
truncations showed that SDF-1 One of our aims was also to obtain some information on the heparin
structure required for binding to SDF-1 (SDF-1
), a CXC chemokine with potent anti-human
immunodeficiency virus activity, binds to heparan sulfate through a
typical consensus sequence for heparin recognition (BBXB,
where B is a basic residue KHLK, amino acids 24-27). Calculation of
the accessible surface, together with the electrostatic potential of
the SDF-1
dimer, revealed that other amino acids (Arg-41 and Lys-43)
are found in the same surface area and contribute to the creation of a
positively charged crevice, located at the dimer interface. GRID
calculations confirmed that this binding site will be the most
energetically favored area for the interaction with sulfate groups.
Site-directed mutagenesis and surface plasmon resonance-based binding
assays were used to investigate the structural basis for SDF-1
binding to heparin. Among the residues clustered in this basic surface
area, Lys-24 and Lys-27 have dominant roles and are essential for
interaction with heparin. Amino acids Arg-41 and Lys-43 participate in
the binding but are not strictly required for the interaction to take place. Direct binding assays and competition analysis with monoclonal antibodies also permitted us to show that the N-terminal residue (Lys-1), an amino acid critical for receptor activation, is involved in
complex formation. Binding studies with selectively desulfated heparin,
heparin oligosaccharides, and heparitinase-resistant heparan sulfate
fragments showed that a minimum size of 12-14 monosaccharide units is
required for efficient binding and that 2-O- and
N-sulfate groups have a dominant role in the interaction. Finally, the heparin-binding site was identified on the crystal structure of SDF-1
, and a docking study was undertaken. During the
energy minimization process, heparin lost its perfect ribbon shape and
fitted the protein surface perfectly. In the model, Lys-1, Lys-24,
Lys-27, and Arg-41 were found to have the major role in binding a
polysaccharide fragment consisting of 13 monosaccharide units.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-HS complex. It has been previously demonstrated that SDF-1
binds selectively to HS and heparin (a chemically related GAG) and that
the combined substitution of Lys24, His25, and
Lys27 by serine residues produces a chemokine unable to
bind HS (29). The present study investigates the effect of single
mutations in this domain and the relative importance of several
additional residues of the protein for HS/heparin recognition. From
these results, the heparin-binding site was located on the crystal
structure of SDF-1
, and a docking study was undertaken. The
structural features of heparin required for efficient binding were also
investigated, and these data, together with those obtained from the
directed mutagenesis analysis, were combined with a molecular modeling approach to propose a three-dimensional model of the interaction between SDF-1
and heparin.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
was from New England Nuclear. The Hitrap heparin column was from
Amersham Pharmacia Biotech, and biotin-LC-hydrazide was from Pierce.
Streptavidin, extravidin peroxidase conjugate, 3,3',5,5'-tetramethylbenzidine, chondroitinase ABC, and papain were
from Sigma. Heparinase and heparitinase were from Grampian Enzymes, and
Bio-Gel P-10 was obtained from Bio-Rad. [3H]Glucosamine
and DEAE-Sephacel were from Amersham Pharmacia Biotech. Synthetic
oligosaccharides (disaccharide to dodecasaccharide) were from Sanofi
Recherche. These consist of alternating
- and
-linked
3-O-methyl-2,6-di-O-sulfo-D-glucose
units, mimicking the regular region of heparin (30). Their general
structure is shown here for the hexasaccharide.
monoclonal antibodies K15C and 4H4
were from Dr. F. Arenzana-Seisdedos.
View larger version (7K):
[in a new window]
Structure 1.
and SDF-1
Mutant Synthesis--
Wild type SDF-1
and SDF-1
mutants were synthesized by the Merrifield solid-phase
method on a fully automated peptide synthesizer using
fluorenylmethyloxycarbonyl (Fmoc) chemistry, as described (29).
Selective biotinylation at the C-terminal position was achieved by
incorporating an extra lysine residue, bearing a
1-(4,4-dimethyl-2,6-dioscocyclohex-1-ylidene)- ethyl
protective group, on the side chain. Coupling of biotin after
1-(4,4-dimethyl-2,6-dioscocyclohex-1-ylidene)ethyl hydrazine de-protection was performed on the peptide resin. Concentrations of
each chemokine or derivative was determined by amino acid analysis after hydrolysis for 20 h in 6 N HCl, 0.2% phenol in
the presence of a known amount of norleucine as internal standard. The
molecular weights of the synthesized peptides, as measured by ion spray mass spectrometry did not differ from the expected molecular weights. Final purity of wild type or mutant SDF was analyzed by high pressure liquid chromatography and was found to be, on average, greater than
90%. Finally, receptor binding analysis of wild type and mutant SDF
was performed with CXCR4+ CEM cells as described
(29).
4 M solution) was
injected on a 1-ml Hitrap heparin column and submitted to gradient
elution from 0.15 to 1 M NaCl in 20 mM
Na2HPO4/NaH2PO4, pH
7.4, over 20 min at a flow rate of 0.5 ml/min. Detection was carried
out at 280 nm.
concentra-
/Heparin Filter Binding Assay and Competition
Analysis--
To analyze the binding of oligosaccharide to protein in
solution, a modified version of the filter binding assay of Maccarana and Lindahl (35) was used. Briefly, SDF-1
(0.25 µg) and
biotinylated heparin (0.1 µg/ml) were coincubated for 2 h at
room temperature in 200 µl of Tris-buffered saline (TBS). Competition
assays were performed by adding unlabeled oligosaccharides to the
mixture. SDF-1
plus any bound biotinylated heparin were then trapped
on the surface of a nitrocellulose membrane by drawing the incubation mixture through the membrane with a vacuum-assisted dot-blot apparatus. The nitrocellulose filters were washed twice with 200 µl of TBS and
blocked with 5% dry milk in TBS, 0.05% Tween 20. The biotinylated heparin bound to SDF-1
was revealed by incubating the membrane with
extravidin peroxidase (0.5 µg/ml) and ECL detection reagents. The
membrane was exposed to film, and chemiluminescent signals were
quantified by densitometry. Competitors included size-defined, heparin-derived, or synthetic oligosaccharides and selectively de-sulfated heparin. For direct binding assay, SDF-1
(1.5 µg) was
incubated with 3H-labeled HS oligosaccharides (10,000 cpm),
and bound complexes were trapped with a nitrocellulose filter as
described above. The protein-bound HS was dissociated from the filter
with 2 M NaCl and measured in a
-scintillation counter.
(1 µg/ml) that had been preincubated
with either octasaccharide (dp8) or tetradecasaccharide (dp14). Bound
biotinylated SDF-1
was then revealed with extravidin peroxidase (0.7 µg/ml) and 3,3',5,5'-tetramethylbenzidine. The reaction was stopped
with 25 µl of 4 M H2SO4, and
absorbance at 450 nm was measured.
dimer (36) were taken
from the Protein Data Bank (code 1A15). The amino acids missing on one
of the monomers (Lys-1 to Tyr-7 on the N-terminal moiety and Ala-65 to
Asn-67 on the C-terminal moiety) were built by homology with the other monomer. The missing side chains of two amino acids, i.e.
Val-3 and Arg-8, were modeled. The geometry of the modeled amino acids was optimized using the Tripos force field (37) in the Sybyl molecular
modeling package.2 Hydrogen
atoms were added and optimized, and partial atomic charges were derived
using the Pullman procedure. Connolly surfaces of both monomer and
dimer were calculated using the MOLCAD program (39) from the Sybyl
package. The GRID program (40) was used to predict the most favorable
anchoring position for a sulfated oligosaccharide. The probe used in
the calculation was the charged oxygen of a sulfate or phosphate group.
The grid spacing was set to 1 Å.
=
(O-5iC-1iO-1iC-4j) and
=
(C-1iO-1iC-4jC-5j). The
signs of the torsion angles were in agreement with the IUPAC-IUPAB
conventions (43).
--
The
heparin chain was extended to 7 repeating units (i.e. 14 monosaccharides) to obtain a length longer than the dimension of the
SDF-1
dimer positively charged cleft. The heparin chain was located
exactly parallel to the cleft. Since the heparin dimer included a
pseudo axis of symmetry perpendicular to the basic cleft, it was not
necessary to consider the two possible directions (i.e.
location of the nonreducing end) for the heparin chain. Different
starting orientations were generated by rotating the chain along its
long axis by 4 steps of 90°, while keeping it at a van der Waals
contact distance from the protein surface. Due to the 2-fold symmetry
of the heparin ribbons, this was equivalent to combining a 90°
rotation and translation of half a repeating unit. This procedure was
applied to both 2S0 heparin and
1C4 heparin, thereby generating 8 start docking modes.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Creates a Basic Cleft Suitable
for Heparin Binding--
As with most chemokines, SDF-1
is
monomeric in solution (45) but a dimeric association is observed upon
crystallization (36). The possibility that this dimer could result from
a packing artifact in the crystalline state is unlikely because first,
the same dimer has been observed in another crystal form of SDF-1
(46), and second, the observed dimer interface involves the first
-strand of the chemokine as found in all CXC chemokines analyzed to
date. The Connolly surfaces of both the SDF-1
monomer and dimer have
been calculated here. When looking for a possible site of binding for
the negatively charged heparin fragment, the most positively charged
area in the monomer takes the shape of a crest, formed mainly by amino
acids Lys-27, Arg-41, Lys-24, and Lys-43 (Fig.
1a). Amino acids Lys-24,
His-25, and Lys-27, present in the first
-strand of the chemokine,
constitute a typical BBXB heparin-binding motif (47) and
have already been shown to be collectively involved in heparin
recognition. Fig. 1b displays the Connolly surface of the
dimer color-coded for the projection of the electrostatic potential. In
the crystallographic dimer, the two crests formed by residues 24, 27, 41, and 43 are on the sides of the protein-protein interface, each of
them forming the edge of a deep, straight crevice (Fig. 1b).
Arg-41 and Lys-43, present in the second
-strand of the chemokine,
protrude within the BBXB cluster and appear to be ideally
located to participate in the interaction. On the opposite side, His-25
is buried at the dimer interface and is more likely to play a role in
protein-protein association rather than in the interaction with
heparin. It is worth noting that the two N-terminal peptides running
parallel to the crevice in both directions extend the dimension of the cleft. In particular, the side chain of the two Lys-1s are responsible for another positively charged area on the accessible surface of the
protein and could also be involved in heparin binding. The binding
region was further characterized using the GRID program. In this
protocol, the interaction of a probe group with a protein of known
structure is computed at sampled positions throughout and around the
macromolecule, giving an array of energy values. The analysis of the
GRID calculations, performed with a probe consisting of a charged
oxygen from a sulfate or phosphate group, is graphically displayed in
Fig. 1c. Indeed, the most favorable region for binding
sulfate is at the protein-protein interface, in the cleft bordered by
four basic amino acids, i.e. Lys-27 and Arg-41 of each
protein chain. Lys-24 and Lys-43 also represent a favorable binding
site on each chain, therefore extending the binding area along the
crevice. It is worth noting that a single 9-kDa heparin chain can
accommodate up to six SDF-1
molecules, indicating that each SDF-1
occupies, on average, 6 monosaccharide units (29). Such dense packing
of the chemokine along the chain implies protein-protein contact,
further suggesting that SDF-1
has a dimeric association when bound
to heparin. In addition, using the "zero-length cross-linking"
strategy described by Pye and Gallagher (48) dimerization of SDF-1
has been observed in the presence of heparin-derived
oligosaccharides.3
View larger version (39K):
[in a new window]
Fig. 1.
a, ribbon representation of the
SDF-1 monomer. The side chains of the basic residues of interest are
displayed as sticks (WelLab Viewer, MSI). b,
Connolly surface of the SDF-1
dimer color-coded according to the
electrostatic potential (blue for negative regions and
red for positive regions). c, GRID iso-energy
contour (
7 kcal/mol) for the interaction of an oxygen atom from a
sulfated group superimposed on a CPK representation of the SDF-1
dimer. The basic amino acids of interest are colored
green.
Amino Acids Involved in
Heparin Binding--
Based on the charge distribution on the Connolly
surface of the proteins, we produced a set of mutant SDF-1
s, in
which residues 24, 27, 41, or 43 were substituted with a Ser residue,
or in which the first Lys was omitted. To analyze the relative
importance of these amino acids in heparin binding, we used heparin
chromatography and a surface plasmon resonance binding assay that we
have already described for wild type SDF-1 (29). Typical sensorgrams
were obtained for the wild type (Fig.
2a) and mutants SDF-1
(Fig. 2, b-g), and sensorgram evaluations are reported in Table
I. A double mutant K24S/K27S was unable
to bind heparin (Fig. 2b), demonstrating the importance of
these two residues for heparin recognition. Single substitution at
these positions (mutant K24S or K27S, Fig. 2, c and
d) also strongly impaired the binding, indicating that both
Lys-24 and Lys-27 are critically involved in the complex formation.
However, significant binding of these mutants was observed at higher
concentrations (Fig. 2h), indicating that other residues
contributed to form the binding site. Substitution of Lys-43 (Fig.
2f) and Arg-41 (Fig. 2g) produced more subtle changes, mainly reducing the association rate constants (Table I).
Finally, removal of Lys-1, an amino acid outside the charged crevice
(see Fig. 1), also led to a significantly reduced affinity (Fig.
2e) demonstrating the involvement of this amino acid in heparin binding. Chemokines were also applied to a heparin
chromatography column and eluted with a linear salt gradient. The
retention time on the column for each mutant is also reported in Table
I and correlates well with the measured changes in affinity. It is
worth noting that a mutant SDF, in which Lys-24, Lys-27, and His-25 were mutated to Ser residues fold with a structure comparable with the
structure of the wild type chemokine (29). Thus, misfolding of the SDFs
mutated at positions 24 or 27 could not be the cause of the absence of
heparin binding activity. In addition, the similar affinity of SDF-1
and mutant SDFs for CXCR4 (Fig. 3 and see Ref. 45 for
SDF-(2-67)), further support the
correct folding of these peptides and allowed us to conclude that the
residues analyzed here all take part in the heparin-binding
reaction.
View larger version (29K):
[in a new window]
Fig. 2.
Binding of wild type and mutant
SDF-1 to immobilized heparin. Wild type
SDF-1
(a), mutant K24S/K27S (b), K24S
(c), K27S (d), SDF-(2-67) (e),
mutant K43S (f), and R41S (g) were injected over
a heparin-activated surface, at a flow rate or 50 µl/min for 3 min,
after which running buffer was injected, and the response in resonance
units was recorded as a function of time. Each set of sensorgrams was
obtained with SDF-1
molecules at (from top to
bottom) 200, 133, 89, 59, 39.5, and 0 nM. In
some cases, mutant SDF-1
(K24S, square; K27S,
triangle; or K24S/K27S, circle) was injected in
the range 300-2000 nM, and the amount of bound SDF-1
at
the end of the injection is reported (h).
Sensorgrams of Fig. 2 were analyzed as described (29)
. ND, not determined.
View larger version (12K):
[in a new window]
Fig. 3.
Comparative analysis of the CXCR4-binding
properties of wild type and mutant SDF-1 .
Binding of 125I-labeled SDF-1
(0.25 nM) to
CXCR4+ CEM cells (1.5 × 106) was competed
with either cold SDF-1
(open squares), K24S/K27S
(closed squares), R41S (closed circles) or K43S
mutants (closed triangles). Nonspecific binding was
determined in the presence of 1 µM unlabeled
SDF1
.
-Heparin Interaction--
Oligosaccharides of defined length
that have been enzymatically produced from heparin (dp2, disaccharide
to dp18, octadecasaccharide), or chemically synthesized heparin mimics
(dp2 to dp12), were investigated for their ability to compete with
biotinylated heparin for binding to SDF-1
. As shown in Fig.
4a, no efficient competition
was observed until a fragment length of 8 monosaccharide units (dp8)
was included as a competitor. However, for heparin-derived
oligosaccharides, the inhibition was very low, and significant
competition was only observed using oligosaccharides of at least dp10.
To investigate further the length dependence, we also produced
3H-labeled HS oligosaccharides from Chinese hamster ovary
cells. SDF-1
was coincubated with 10,000 cpm of size-defined
heparitinase-resistant oligosaccharides, and the nitrocellulose filter
assay was used again to quantitate the bound 3H-labeled
oligosaccharides. The smallest HS fragment able to bind to SDF-1 was an
octasaccharide, albeit at a very low level. A first increase in binding
was observed with dp10 and dp12, and a second increase was observed for
oligosaccharides containing 14 or more monosaccharide units (Fig.
4b). To determine which sulfate groups were important in the
binding with SDF-1
, a competition study, using a range of
specifically desulfated heparins, was carried out (Fig. 4c).
A similar decrease in competition effectiveness was observed with
N- and 2-O-desulfated heparin, indicating that both N-sulfate and 2-O-sulfate groups were
involved in SDF-1
binding. However, since
2,6-O-desulfated material was ineffective as a competitor,
N-sulfate by itself did not allow heparin to bind
efficiently to SDF-1
. 6-O-Desulfated heparin had slightly reduced efficiency compared with native heparin, demonstrating that
this position was less important.
View larger version (17K):
[in a new window]
Fig. 4.
Binding of heparin/heparan sulfate fragments
and selectively desulfated heparin to
SDF-1 . a, biotin-labeled
heparin was coincubated with SDF-1
and size-defined, heparin-derived
(open bars), or chemically synthesized (closed
bars) oligosaccharides (each at 2.5 µg/ml). Samples were
filtered through a nitrocellulose membrane, and retained biotinylated
heparin was quantified by chemiluminescent assay and densitometry.
b, size-defined heparan sulfate oligosaccharide (10,000 cpm)
was mixed with SDF-1
, and then bound material was recovered by
filtration through nitrocellulose membrane and quantified by
scintillation counting. c, SDF-1
and biotin-labeled
heparin were coincubated with a range of concentrations of selectively
desulfated heparin (closed triangles,
de-O-sulfated heparin; open squares,
de-2-O-sulfated heparin; closed circles,
de-N-sulfated, re-N-acetylated heparin;
open triangles, de-6-O-sulfated heparin;
closed squares, heparin) and then filtered through a
nitrocellulose membrane. SDF-1
-bound heparin was quantified as in
a.
Is Involved in Heparin
Complex Formation--
In view of the reduced affinity of SDF-1
lacking the first N-terminal lysine residue, we further investigated
the involvement of this amino acid in the SDF-1
-heparin complex. For
this purpose, we analyzed the ability of heparin-derived
oligosaccharides to prevent the binding of SDF-1
to mAb K15C, a mAb
that recognizes the two first amino acids of SDF-1
(29).
Biotinylated SDF-1
was coincubated with a range of concentrations of
either dp14 or dp8, and the complexes were captured with mAb K15C. The
amount of captured biotinylated SDF-1
was quantified with extravidin peroxidase. The results showed that dp14 bound to SDF-1
prevents the
binding to mAb K15C (Fig. 5), suggesting
that, as a result of its involvement in the complex with the
oligosaccharide, Lys-1 became inaccessible to the mAb. It is worth
noting that dp8, an oligosaccharide too short to bridge the two Lys-1s
in the SDF-1
dimer (see below), failed to compete with the
interaction between mAb K15C and the chemokine. As a control we also
show here that the binding of mAb 4H4 (which recognizes the C-terminal
part of SDF-1
) was only slightly inhibited by dp14.
View larger version (10K):
[in a new window]
Fig. 5.
Biotinylated SDF-1
(1 µg/ml) was preincubated with a range
of concentrations of size-defined, heparin-derived
oligosaccharides (octasaccharide, dp8 or tetradecasaccharide, dp14),
and captured with coated monoclonal antibodies (K15C, closed
bars; 4H4, open bars). The amount of
captured biotinylated SDF-1
was quantified with extravidin
peroxidase and 3,3',5,5'-tetramethylbenzidine development.
--
To
define further the SDF-1
-heparin complex, we combined the above
experimental data with a molecular modeling approach. For this purpose,
a heparin chain was docked in the positively charged cleft formed at
the SDF-1
dimer interface. The starting geometry of heparin was the
elongated conformation with a 2-fold axis that has been demonstrated to
exist in solution (41). A combination of translation and rotation along
the polysaccharide chain was investigated, together with the two
possible conformations of the iduronic acid ring. This resulted in the
investigation of eight different docking modes. In all cases, the
polysaccharide extended above the cleft, and 13 to 14 monosaccharides
were needed to cover the distance between the two Lys-1 residues. Due
to the large number of variables already included in the docking study, it was not possible to consider polysaccharides containing various amounts of 1C4 and 2SO
conformations, the case that is more likely to occur in solution. However, this variation in ring shape does not alter the general ribbon
shape of the polysaccharide. The geometry of the eight binding modes
was optimized, and the resulting potential energies are listed in Table
II. All the complexes displayed a very
favorable energy of interaction, mainly due to the electrostatic
contact between the heparin negative charges and the positively charged amino acids. In the two energetically preferred binding modes, the
heparin ribbon-like chain sat "flat" on the cleft and the L-iduronic acid residues displayed a
1C4 ring shape. The calculated energy
difference between the two best modes was not significant since
changing the orientation of one sulfate group could result in the
creation or destruction of a salt bridge. However, the two best modes
displayed a potential energy significantly lower than the others, and
it could be hypothesized that this particular binding mode, with the
negative charges interacting with the edges of the crevice, was the
preferred one.
Potential energies for the optimized complexes following the 8 modes of binding
Etot is partitioned into
contributions from the protein and the ligand
(
Eprot and
Eheparin) and
the interaction energy
Einter.
dimer. During the energy minimization process, heparin lost the perfect ribbon shape with a 2-fold axis that had been demonstrated to exist in solution (41). The final shape was still
extended but less regular and matched perfectly the protein surface.
Analysis of the conformation of the polysaccharide indicated that all
of the L-iduronic acid remained in its starting
1C4 ring shape, but with small variations in
the puckering parameters. Further variations were observed at the
glycosidic linkages. The values of the
and
torsion angles after
optimization differed by up to 60° from the starting conformation.
However, when compared with the previously calculated energy maps of
each linkage (41, 49), all 13 glycosidic linkages remained in the
conformational region that contained the global minimum. When compared
with MM3 energy maps (49), the energy cost of the variation ranged
between 0 and 4 kcal/mol depending on the glycosidic linkage. The
deformation involving the higher energy was restricted to the center of
the binding site, where interaction with the protein was strongest.
View larger version (48K):
[in a new window]
Fig. 6.
Orthogonal representations of the model for
the interaction of heparin with SDF-1
dimer. The protein is represented as a ribbon.
The heparin polysaccharide, together with the basic amino acids
involved in the interaction, are represented as sticks.
Drawings prepared with MOLSCRIPT (57) and RASTER-3D (38).
Details of the electrostatic interactions established between the side
chains of the basic amino acids and the acidic groups of heparin in the
optimized complex
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, and structurally defined
heparin oligosaccharides were used to characterize the SDF-1
-heparin complex. Based on the heparin-binding consensus sequence proposed by
Cardin and Weintraub (47), a first group of basic amino acids (KHLK,
residues 24-27) has been shown to be collectively involved in heparin
binding. The present study shows that within this cluster both Lys-24
and Lys-27 are essential, since the substitution of either of these two
residues strongly impairs the heparin binding activity of SDF-1
. We
also identified three other amino acids that play a secondary role in
heparin binding. These new residues (Lys-1, Arg-41, and Lys-43) are
located in a different peptide sequence, but interestingly, two of them
(Arg-41 and Lys-43) fold together with Lys-24 and Lys-27 to form a
single basic domain. Furthermore, electrostatic calculations show that
Lys-24/Arg-41/Lys-27/Lys-43 are remarkably aligned to form a cationic
crest on the SDF-1
surface. Widely separated residues in a primary
sequence, organized into a single domain to form a functional
heparin-binding site, have been reported in other chemokines, including
MIP-1
(50) and MIP-1
(51). It is not well understood why of the
four SDF-1
amino acids that constitute the basic surface
(i.e. Lys-24/Arg-41/Lys-27/Lys-43) only two of them (Lys-24
and Lys-27) are essential for binding, whereas the other two are only
slightly involved. Such differences in the importance of spatially
colocalized basic residues for heparin binding have been reported for
MIP-1
, in which, of the six basic residues that are clustered into a
basic domain, one was found to be critical (Arg-46), two played a
secondary role (Arg-18 and Lys-45), and the remaining three (Lys-19,
Arg-22, and Lys-48) appeared not to participate in the interaction
(51). It is worth noting that in our model of the SDF-1
-heparin
complex, Lys-24 and Lys-27, the two critical residues, create more salt bridges with heparin than Arg-41 and Lys-43, but more structural data
will be required to understand these differences. Interestingly, the
heparin-binding amino acids are located close to the protein-protein interface in the SDF-1
dimer. Analysis with the GRID software showed
that the most energetically favorable domain for binding sulfated
groups is located at the protein-protein interface, and thus the
functional binding site appears to be created by the dimerization of
the protein. Similarly, it has been demonstrated that monomeric
interleukin-8 has affinity too weak for heparin to bind the GAG at
physiological ionic strength, whereas the dimeric form of this
chemokine mediates binding to heparin or to HS (52). Our study strongly
suggests that indeed, when bound to heparin, SDF-1
behaves as a
dimer. In particular, while the stoichiometry of the binding indicated
that each SDF-1
occupies an average of six monosaccharide units on
heparin (29), competition assay and direct binding analysis with HS-
derived oligosaccharides clearly indicate that 12-14 monosaccharides
are required to interact efficiently with the chemokine. These data are
also consistent with our model in which 13 monosaccharide units
interact with the dimeric form of the chemokine. As SDF-1
has been
reported to be monomeric in solution (45), it is likely that the
chemokine is dimerized by heparin. It has recently been shown that
CXCR4 undergoes dimerization after being bound by SDF-1
(53), and it
is possible that this process could be promoted by HS at the cell
surface. SDF-1
-induced dimerization of CXCR4 has been proposed to be
an important step in triggering the biological response via activation
of the JAK/signal transducers and activators of transcription pathway
(53). Thus, in addition to providing a chemokine gradient along cell
surfaces, HS could help in the constitution of a signaling complex
through the dimerization process. However, SDF-1
and a mutant SDF
(called SDF-1
3/6) that does not bind to heparin are almost
indistinguishable with regard to CXCR4 binding, chemotaxis, and
intracellular calcium mobilization (29). It is thus possible that
different activities may be differentially dependent on HS binding.
, lacking the first residue
(SDF-1
-(2-67)), still had an affinity for the receptor but
was unable to trigger CXCR4 signaling (45). Of interest is the fact
that CD26, a leukocyte-activating antigen, which possesses dipeptidyl
peptidase IV activity, cleaves the first two residues (KP) of SDF-1 and
thus inactivates the chemokine. It is tempting to imagine that heparin
protects the chemokine from such cleavage, and this will be
investigated in a future study. The conformation of the N-terminal
region proposed in our model is, of course, hypothetical since both NMR
and crystallographic studies indicate that this peptide is flexible.
However, the proposed conformation not only permits rationalization of
the biochemical data but also agrees with a recent crystal structure of
SDF-1 in which the N-terminal tail can be located and is shown to
extend from the globular region, parallel to the
-trands (46).
Finally, our model also suggests that Asn-46 and Gln-48 are involved in hydrogen bonding with heparin and, as such, could contribute to the
interaction. Although this has not been experimentally analyzed here,
it is worth noting that such bonds have been found in other chemokine-GAG complexes, including PF4, in which Thr-25 and Asn-47 are
implicated in the interaction (56).
. Our data show that a
dodeca- to tetradecasaccharide was required to efficiently interact
with the chemokine, and in our model this corresponds exactly to the
length necessary to span the heparin-binding site on the SDF-1
dimer, including the two N-terminal lysine residues. Removal of
2-O- or N-sulfate groups in heparin impedes the
binding to SDF, indicating that these particular groups contribute to the binding, whereas removal of 6-O-sulfate has less effect.
In contrast, the binding of heparin to interleukin-8 required all types
of sulfate substituents and a minimal sequence of 18-20 monosaccharide
units (52). The exact organization of various sulfated saccharide units
optimal for SDF-1
binding remains to be defined. We are currently
analyzing HS-derived oligosaccharides with high affinity for SDF-1
with the aim of obtaining sequence information. This information
together with the present study should help us design some HS mimics
able to modulate some functions of the chemokine.
![]() |
ACKNOWLEDGEMENTS |
---|
We are indebted to Dr. F. Arenzana-Seisdedos (Institut Pasteur) for the kind help and the gift of mAbs K15C and 4H4, to Dr. M. Petitou (Sanofi-Recherche) for the kind gift of synthetic oligosaccharides, and to Prof. J. Gallagher for the gift of desulfated heparins.
![]() |
FOOTNOTES |
---|
* This work was supported in part by grants from the Agence National de la Recherche Contre le SIDA and by the CNRS.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by a postdoctoral fellowship from the Agence National de la Recherche Contre le SIDA.
** To whom correspondence should be addressed: IBS/LBM, 41 Rue Horowitz, 38027 Grenoble Cedex 01, France. Tel.: 33 476 88 95 69; Fax: 33 476 88 54 94; E-mail: Hugues.Lortat-Jacob@ibs.fr.
Published, JBC Papers in Press, November 21, 2000, DOI 10.1074/jbc.M008110200
2 SYBYL Tripos Associates, Suite 303, 1699 S. Hanley Rd., St. Louis, MO 63144.
3 R. Vivès, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
MIP-1, macrophage
inflammatory peptides-1;
GAG, glycosaminoglycan;
HS, heparan sulfate;
SDF-1, stromal cell-derived factor-1
;
TBS, Tris-buffered saline;
mAb, monoclonal antibody;
dp, degree of polymerization.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Rossi, D., and Zlotnik, A. (2000) Annu. Rev. Immunol. 18, 217-242[CrossRef][Medline] [Order article via Infotrieve] |
2. | Bazan, J. F., Bacon, K. B., Hardiman, G., Wang, W., Soo, K., Rossi, D., Greaves, D. R., Zlotnik, A., and Schall, T. J. (1997) Nature 385, 640-644[CrossRef][Medline] [Order article via Infotrieve] |
3. | Kelner, M. P., Kennedy, J., Bacon, K. B., Kleyensteuber, S., Largaespada, D. A., Jenjins, N. A., Copeland, N. G., Bazan, J. F., Moore, K. W., Schall, T. J., and Zlotnik, A. (1994) Science 266, 1395-1399[Medline] [Order article via Infotrieve] |
4. |
Rollins, B. J.
(1997)
Blood
90,
909-928 |
5. | Wells, T. N. C., Power, C. A., and Proudfoot, A. E. I. (1998) Trends Pharmacol. Sci. 19, 376-380[CrossRef][Medline] [Order article via Infotrieve] |
6. | Luster, A. D. (1998) N. Engl. J. Med. 7, 436-445 |
7. | Gale, L. M., and McColl, S. R. (1999) BioEssays 21, 17-28[CrossRef][Medline] [Order article via Infotrieve] |
8. | Melchers, F., Rolink, A. G., and Schaniel, C. (1999) Cell 99, 351-354[Medline] [Order article via Infotrieve] |
9. | Witt, D. P., and Lander, A. D. (1994) Curr. Biol. 4, 394-400[Medline] [Order article via Infotrieve] |
10. | Kuschert, G. S. V., Coulin, F., Power, C. A., Proudfoot, A. E. I., Hubbard, R. E., Hoogewerf, A. J., and Wells, T. N. C. (1999) Biochemistry 38, 12959-12968[CrossRef][Medline] [Order article via Infotrieve] |
11. | David, G., and Bernfield, M. (1998) Matrix Biol. 17, 461-463[CrossRef][Medline] [Order article via Infotrieve] |
12. |
Zimmermann, P.,
and David, G.
(1999)
FASEB J.
13,
91-100 |
13. | Tumova, S., Woods, A., and Couchman, J. R. (2000) Int. J. Biochem. Cell Biol. 23, 269-288 |
14. | Iozzo, R. V. (1998) Annu. Rev. Biochem. 67, 609-652[CrossRef][Medline] [Order article via Infotrieve] |
15. | Hoogewerf, A. J., Kuschert, G. S. V., Proudfoot, A. E. I., Borlat, F., Clark-Lewis, I., Power, C. A., and Wells, T. N. C. (1997) Biochemistry 36, 13570-13578[CrossRef][Medline] [Order article via Infotrieve] |
16. | Tanaka, Y., Kimata, K., Wake, A., Mine, S., Morimoto, I., Yamakawa, N., Habuchi, H., Ashikari, S., Yamamoto, H., Sakurai, K., Yoshida, K., Suzuki, S., and Eto, S. (1996) J. Exp. Med. 184, 1987-1997[Abstract] |
17. | Tanaka, Y., Fujii, K., Hubscher, S., Aso, M., Takazawa, A., Saito, K., Ota, T., and Eto, S. (1998) Arthritis & Rheum. 41, 1365-1377[CrossRef][Medline] [Order article via Infotrieve] |
18. | Weber, K. S., von Hundelshausen, P., Clark-Lewis, I., Weber, P. C., and Weber, C. (1999) Eur. J. Immunol. 29, 700-712[CrossRef][Medline] [Order article via Infotrieve] |
19. | Bleul, C. C., Fuhlbrigge, R. C., Casasnovas, J. M., Aiuti, A., and Springer, T. A. (1996) J. Exp. Med. 184, 1101-1109[Abstract] |
20. | Oberlin, E., Amara, A., Bachelerie, F., Bessia, C., Virelizier, J. L., Arenzana-Seisdedos, F., Schwartz, O., Heard, J. M., Clark-Lewis, I., Legler, D. F., Loetscher, M., Baggiolini, M., and Moser, B. (1996) Nature 382, 833-835[CrossRef][Medline] [Order article via Infotrieve] |
21. | Tachibana, K., Hirota, S., Iizasa, H., Yoshida, H., Kawabata, K., Kataoka, Y., Kitamura, Y., Matsushima, K., Yoshida, N., Nishikawa, S., Kishimoto, T., and Nagasawa, T. (1998) Nature 393, 591-594[CrossRef][Medline] [Order article via Infotrieve] |
22. |
Coulomb-L'Hermin, A.,
Amara, A.,
Schiff, C.,
Durand-Gasselin, I.,
Foussat, A.,
Delaunay, T.,
Chaouat, G.,
Capron, F.,
Ledee, N.,
Galanaud, P.,
Arenzana-Seisdedos, F.,
and Emilie, D.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
8585-8590 |
23. |
Pablos, J. L.,
Amara, A.,
Bouloc, A.,
Santiago, B.,
Caruz, A.,
Galindo, M.,
Delaunay, T.,
Virelizier, J. L.,
and Arenzana-Seisdedos, F.
(1999)
Am. J. Pathol.
155,
1577-1586 |
24. | Agace, W. W., Amara, A., Roberts, A. I., Pablos, J. L., Thelen, S., Uguccioni, M., Li, X. Y., Marsal, J., Arenzana-Seisdedos, F., Delaunay, T., Ebert, E. C., Moser, B., and Parker, C. M. (2000) Curr. Biol. 10, 325-328[CrossRef][Medline] [Order article via Infotrieve] |
25. |
Lindahl, U.,
Kusche-Gulberg, M.,
and Kjellén, L.
(1998)
J. Biol. Chem.
273,
24979-24982 |
26. | Perrimon, N., and Bernfield, M. (2000) Nature 404, 725-728[CrossRef][Medline] [Order article via Infotrieve] |
27. |
Amara, A.,
Gall, S. L.,
Schwartz, O.,
Salamero, J.,
Montes, M.,
Loetscher, P.,
Baggiolini, M.,
Virelizier, J. L.,
and Arenzana-Seisdedos, F.
(1997)
J. Exp. Med.
186,
139-146 |
28. | McFadden, G., and Kelvin, D. (1997) Biochem. Pharmacol. 54, 1271-1280[CrossRef][Medline] [Order article via Infotrieve] |
29. |
Amara, A.,
Lorthioir, O.,
Valenzuela, A.,
Magerus, A.,
Thelen, M.,
Montes, M.,
Virelizier, J. L.,
Delepierre, M.,
Baleux, F.,
Lortat-Jacob, H.,
and Arenzana-Seisdedos, F.
(1999)
J. Biol. Chem.
274,
23916-23925 |
30. | Petitou, M., Herault, J. P., Bernat, A., Driguez, P. A., Duchaussoy, P., Lormeau, J. C., and Herbert, J. M. (1999) Nature 398, 417-422[CrossRef][Medline] [Order article via Infotrieve] |
31. |
Pye, D. A.,
Vives, R. R.,
Turnbull, J. E.,
Hyde, P.,
and Gallagher, J. T.
(1998)
J. Biol. Chem.
273,
22936-22942 |
32. | Bitter, T., and Muir, H. (1962) Anal. Biochem. 4, 330-334 |
33. | Lortat-Jacob, H., Turnbull, J. E., and Grimaud, J. A. (1995) Biochem. J. 310, 497-505[Medline] [Order article via Infotrieve] |
34. | Turnbull, J. E., and Gallagher, J. T. (1990) Biochem. J. 265, 715-724[Medline] [Order article via Infotrieve] |
35. | Maccarana, M., and Lindahl, U. (1993) Glycobiology 3, 271-277[Abstract] |
36. |
Dealwis, C.,
Fernandez, E. J.,
Thompson, D. A.,
Simon, R. J.,
Siani, M. A.,
and Lolis, E.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
6941-6946 |
37. | Clark, M., Cramer, R. D., III., and van den Opdenbosch, N. (1989) J. Comput. Chem. 10, 982-1012 |
38. | Merrit, E. A., and Murphy, M. E. (1994) Acta Crystallogr. Sect. D Biol. Crystallogr. 50, 869-873[CrossRef][Medline] [Order article via Infotrieve] |
39. | Waldherr-Teschner, M., Goetze, T., Heiden, W., Knoblauch, M., Vollhardt, H., and Brickmann, J. (1992) in Advances in Scientific Visualization (Post, F. H. , and Hin, A. J. S., eds) , pp. 58-67, Springer-Verlag, Heidelberg |
40. | Goodford, P. J. (1985) J. Med. Chem. 28, 849-857[Medline] [Order article via Infotrieve] |
41. | Mulloy, B., Forster, M. J., Jones, C., and Davies, D. B. (1993) Biochem. J. 293, 849-858[Medline] [Order article via Infotrieve] |
42. | Imberty, A., Bettler, E., Karababa, M., Mazeau, K., Petrova, P., and Pérez, S. (1999) in Perspectives in Structural Biology (Vijayan, M. , Yathindra, N. , and Kolaskar, A. S., eds) , pp. 392-409, Indian Academy of Sciences and Universities Press, Hyderabad |
43. | IUPAC-IUB. (1971) Arch. Biochem. Biophys. 145, 405-621 |
44. | Pérez, S., Meyer, C., and Imberty, A. (1995) in Modeling of Biomolecular Structures and Mechanisms (Pullman, A. , Jortner, J. , and Pullman, B., eds) , pp. 425-454, Kluwer Academic Publishers Group, Drodrecht, Netherlands |
45. |
Crump, M. P.,
Gong, J. H.,
Loetscher, P.,
Rajarathnam, K.,
Amara, A.,
Arenzana-Seisdedos, F.,
Virelizier, J. L.,
Baggiolini, M.,
Sykes, B. D.,
and Clark-Lewis, I.
(1997)
EMBO J.
16,
6996-7007 |
46. | Ohnishi, Y., Senda, T., Nandhagopal, N., Sugimoto, K., Shioda, T., Nagai, Y., and Mitsui, Y. (2000) J. Interferon Cytokine Res. 20, 691-700[CrossRef][Medline] [Order article via Infotrieve] |
47. | Cardin, A. D., and Weintraub, H. J. R. (1989) Arteriosclerosis 9, 21-32[Abstract] |
48. |
Pye, D. A.,
and Gallagher, J. T.
(1999)
J. Biol. Chem.
274,
13456-13461 |
49. | Cros, S., Petitou, M., Sizun, P., Perez, S., and Imberty, A. (1997) Bioorg. Med. Chem. 5, 1301-1309[CrossRef][Medline] [Order article via Infotrieve] |
50. |
Koopmann, W.,
and Krangel, M. S.
(1997)
J. Biol. Chem.
272,
10103-10109 |
51. |
Koopmann, W.,
Ediriwickrema, C.,
and Krangel, M. S.
(1999)
J. Immunol.
163,
2120-2127 |
52. |
Spillmann, D.,
Witt, D.,
and Lindahl, U.
(1998)
J. Biol. Chem.
273,
15487-15493 |
53. |
Vila-Coro, A. J.,
Rodriguez-Frade, J. M.,
Martin De Ana, A.,
Moreno-Ortiz, M. C.,
Martinez-A, C.,
and Mellado, M.
(1999)
FASEB J.
13,
1699-1710 |
54. |
Loetscher, P.,
Gong, J. H.,
Dewald, B.,
Baggiolini, M.,
and Clark-Lewis, I.
(1998)
J. Biol. Chem.
273,
22279-22283 |
55. |
Elisseeva, E. L.,
Slupsky, C. M.,
Crump, M. P.,
Clark-Lewis, I.,
and Sykes, B. D.
(2000)
J. Biol. Chem.
275,
26799-26805 |
56. | Mayo, K. H., Ilyina, E., Roongta, V., Dundas, M., Joseph, J., Lai, C. K., Maione, T., and Daly, T. J. (1995) Biochem. J. 312, 357-365[Medline] [Order article via Infotrieve] |
57. | Kraulis, P. (1991) J. Appl. Crystallogr. 24, 946-950[CrossRef] |