 |
INTRODUCTION |
MIP-1
1 is a member of
the chemotactic cytokine (chemokine)
superfamily, a large group of proteins that causes chemotaxis and activation of various subpopulations of cells in the immune system (1).
The activity of chemokines is believed to be mediated primarily through
two types of interactions, the first of which involves chemokines
binding to glycosaminoglycans (GAGs), polyanionic polysaccharides found
on the endothelial surface and the extracellular matrix. This
immobilization of chemokines by GAGs is likely to play a key role in
the formation of chemokine gradients, which are sensed by leukocytes
and trigger localization of leukocyte subpopulations to the site of
infection or injury (2, 3). The other major interaction involves the
tight binding of chemokines to their seven-transmembrane helix,
G-protein-coupled receptors on the surface of leukocytes. This
interaction causes cellular activation and mediates chemotaxis.
The chemokine superfamily has as many as 50 members (1, 4) and is
divided into subfamilies on the basis of the pattern of conserved
cysteine residues at their N termini. One major subfamily, the CC
subfamily, has two contiguous cysteine residues and includes such
members as MIP-1
, MIP-1
, RANTES, and MCP-1. Another major subfamily, the CXC subfamily, has an amino acid separating the conserved cysteines and includes such members IL-8, SDF-1, and PF-4.
Two minor subfamilies also exist, the CX3C and C
subfamilies, which include fractalkine and lymphotactin, respectively.
The G-protein-coupled receptor that interacts with MIP-1
is called
the CC chemokine receptor 5 (CCR5), and this receptor also binds two
other CC chemokines, namely MIP-1
and RANTES. CCR5 has been shown to
be used by strains of HIV-1 known as the "R5 strains," as a
coreceptor, making contacts with the HIV-1 surface that allow viral
entry into the cell. Consequently, it has been shown that the CCR5
ligands MIP-1
, MIP-1
, and RANTES are effective inhibitors of
these strains of HIV-1 infection due to their ability to block viral
access to CCR5, and in some cases, to cause the internalization and
down-regulation of CCR5 upon chemokine binding (5, 6).
The interrelationship between GAG binding and receptor function in
chemokines is not yet clear. We and others have shown that interaction
with GAGs is not required for a chemokine to retain receptor function
in vitro; chemokines retain function both on cells treated
enzymatically to remove GAGs and on cells genetically modified to be
unable to express surface GAGs (7-10). It has been reported, however,
that the presence of GAGs on receptor-bearing cells may lead to an
increased receptor sensitivity due to higher effective chemokine
concentrations (7, 9). Mutagenesis to remove GAG binding residues in
CCR5-binding chemokines affects receptor binding moderately or
minimally, suggesting that the binding site for GAGs may be separate
from that for the receptor (8, 11-13). This may not be the case with
other chemokine receptors, because alteration of GAG binding residues
in CCR1-binding chemokines significantly affects function on the CCR1
receptor (11, 12, 14). Several groups have shown that exogenous soluble
GAGs compete with receptors for chemokine binding (8, 15, 16), although one group reports that a RANTES·GAG complex can bind the
cognate chemokine receptor (17).
Mutagenesis and modeling studies have shown that the interaction of
chemokines with GAGs is mediated by basic residues on the protein
surface (8,10-13,15,18-21). This is in accord with other
proteoglycan-binding proteins, which use positively charged residues in
ionic interactions with the polyanionic GAGs (22). Indeed, chemokines
tend to bind more tightly to more highly sulfated GAGs (15). However,
great variation exists within the chemokine superfamily regarding
residues that are implicated in GAG binding (described in previous
references and summarized in Ref. 12). CXC chemokines such as IL-8
often use basic residues located in their C-terminal
-helix (18,
23), although SDF-1 utilizes basic residues in its first
-strand
(10). CC chemokines tend to use basic residues in the 40s region,
although it has been reported that MCP-1 utilizes C-terminal
-helix
residues (20) and mutation in MIP-1
of Arg18 as well as
40s residues affects GAG binding (14, 19). The single most important
residue for interaction by MIP-1
with heparin is Arg46
(8, 13), and it has been shown that simultaneous elimination of the
three positively charged residues in the 40s region of MIP-1
(K45A/R46A/K48A) completely abrogates heparin binding ability (8).
Similarly, basic residues in this region have been identified as
critical for GAG binding to RANTES (11, 12).
A structural understanding of GAG binding by chemokines is complicated
by the fact that many chemokines have been shown to form tight dimers,
particularly MIP-1
, MIP-1
, and RANTES (24-27), each preferring
acidic conditions and also tending to form higher order aggregates near
neutral pH (26). The physiological role of the chemokine dimer is not
known, because monomeric variants of MIP-1
and other chemokines
effectively bind their cognate receptor in vitro (28, 29).
Because the dimer does not appear to be involved in receptor function,
it is possible that the chemokine dimer may be a critical mediator of
GAG binding (8, 30).
Although the structure of a chemokine in complex with a GAG has not yet
been reported, the NMR spectra of PF-4, IL-8, and RANTES in the
presence of saccharides have allowed some analysis of the binding sites
of those proteins (12, 18, 23). In principle, NMR studies of a
chemokine·GAG complex should provide a clear indication of the GAG
binding site on the protein, because residues whose resonances shift in
the presence of GAGs are likely involved in GAG binding in some manner.
In addition, analysis of the NMR spectra of the titration of a
particular GAG with a chemokine should provide the dissociation
constant for the interaction. The paucity of such data for chemokines
is likely related to the oligomerization that GAGs induce upon binding
chemokines (30), leading to aggregates too large to be analyzed by NMR.
We have found MIP-1
to be amenable to structural study with heparin
disaccharides. In the present study, NMR was used to analyze MIP-1
in the presence of eleven different disaccharides and allowed for the
determination of the dissociation constant for the five found to bind
the chemokine. Similarly, the GAG binding properties of a monomeric
variant of MIP-1
known as MIP(9) were investigated. Combined, these
studies report the binding surfaces for the monomeric and dimeric forms of the protein. Finally, using a mutant of MIP-1
that displays less
of a tendency to aggregate than the wild type protein, we have obtained
what we believe to be the first NMR spectra of an anti-HIV CC chemokine
in the presence of a GAG near neutral pH.
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EXPERIMENTAL PROCEDURES |
Materials--
Heparin disaccharides IV-A and II-S were obtained
from Calbiochem-Novabiochem Corp. (La Jolla, CA). The following heparin disaccharides were purchased from Sigma-Aldrich Co. (St. Louis, MO):
I-P, I-S, III-S, IV-S, II-H, and III-H. The heparin disaccharides II-A
and III-A and the chondroitin disaccharide
Di-disD were purchased from V-Labs Inc. (Covington, LA). All disaccharides were used
without further purification, and their structures as defined by the
commercial suppliers are shown in Fig. 4. Some anomerization is
possible about the saccharide linkage, which will be investigated in a
later study of GAG selectivity. 2H2O and
13C6-glucose were purchased from Isotec Inc.
(Miamisburg, OH), and 15NH4Cl was obtained from
Martek Biosciences Corp. (Columbia, MD).
Stock solutions (10-30 mM) of disaccharides were prepared
by dissolving in either H2O or 2H2O
and kept frozen until use. The concentrations of the disaccharide stock
solutions were determined based on the amount specified by the
commercial suppliers. Each respective commercial source was contacted
as to the magnitude of possible error in the quantity of disaccharide
purchased: the error in the disaccharides obtained from
Calbiochem-Novabiochem Corp. was ± 1.0%; the amount provided by
Sigma-Aldrich Co. may exceed the specified quantity by a maximum of
20% (several non-binding disaccharides have the possibility of
exceeding the amount specified by as much as 30%, but because these
disaccharides did not bind the proteins of this study, this level of
error does not pertain to our numerical results); and the amount
provided by V-Labs Inc. may exceed that specified by no more than
5%.
Protein Preparation--
The mutations of MIP-1
into the
non-aggregating variant D27A/D65A/E67Q were carried out by standard
methods using the gene for human MIP-1
in a variant of the Novagen
pET32LIC vector as previously described (27). 15N-Labeled
WT-MIP-1
, 15N- and
15N/13C-labeled MIP-1
D27A/D65A/E67Q, and
15N- and 15N/13C-labeled MIP(9)
were expressed and purified following previously reported methods (28).
Protein concentrations were quantified using the absorbance of the
protein at 280 nm (
= 12,000 cm
1
M
1).
NMR Spectroscopy--
All NMR spectra were acquired at 25 °C
on Varian Inova 500 or 600 MHz spectrometers. Chemical shifts were
referenced relative to 2,2-dimethyl-2-silapentane-5-sulfonic acid (31).
Unless otherwise noted, NMR samples were prepared by dissolving
lyophilized protein in a standard buffer of 20 mM sodium
phosphate (pH 2.5) containing 10% 2H2O and
0.02% sodium azide. Data were processed using the program NmrPipe (32)
and analyzed using either PIPP (33) or NMRView (34).
For disaccharide titration experiments, 1H-15N
HSQC spectra were acquired at 500 MHz with 512* points in the
1H (t2) dimension and 128* points in
the 15N dimension (t1), where
n* represents n complex points. The
1H and 15N spectral widths in these experiments
were 6000.6 and 1500 Hz, respectively. These data were processed with
72°-shifted squared sine-bell apodization in both
t1 and t2 followed by
zero-filling to a final digital resolution of 3 Hz/point
(F1) and 6 Hz/point (F2).
For sequential assignment of MIP-1
D27A/D65A/E67Q at pH 2.5 and 6.0, and MIP(9) at pH 2.5, CBCA(CO)NH (35) and HNCA (36) spectra were
acquired. Samples of MIP-1
D27A/D65A/E67Q and MIP(9) at pH 2.5 used
for assignment were 1 mM in concentration and were prepared
in the standard buffer. To prepare the MIP-1
D27A/D65A/E67Q sample
at pH 6.0 for assignment, lyophilized protein was first dissolved in
the standard buffer at pH 2.5 to a final protein concentration of 0.3 mM, NaCl was added to 300 mM, and the pH was
increased by addition of 2 M NaOH. To confirm the identity of several overlapped residues that shifted during titration of MIP-1
D27A/D65A/E67Q and MIP(9) with disaccharides, HNCA spectra were acquired at pH 2.5 in the presence of 1 mM I-P and
compared with the spectra in the absence of the disaccharide. To
confirm the identity of several overlapped residues that shifted upon titration of WT-MIP-1
with disaccharide, an HNHA (37) spectrum was
acquired on a 0.75 mM sample of 15N-labeled
WT-MIP-1
at pH 2.5 in the presence of 1 mM heparin I-P
and compared with an HNHA spectrum acquired in the absence of the disaccharide.
Titrations of WT-MIP-1
and MIP-1
Mutants with
Disaccharides--
At pH 2.5, WT-MIP-1
and MIP(9) were titrated
with heparin disaccharides I-P, I-S, II-S, III-S, IV-S, II-H, III-H,
II-A, III-A, and IV-A and chondroitin disaccharide
Di-disD. MIP-1
D27A/D65A/E67Q was titrated with I-P
and I-S at pH 2.5 (titrations at pH 6.0 are described below). Using
stock solutions (10-30 mM), disaccharides were added to
samples of 0.23-0.26 mM 15N-labeled protein
and 1H-15N HSQC spectra were acquired for each
titration point. To ascertain binding, an initial series of spectra
were acquired for each protein with every disaccharide at varying
intervals between 0 and 4.0 molar equivalents of disaccharide (dilution
effects were taken into account during data analysis). Titrations were
continued on only those disaccharides that caused resonance shifting in the initial series of spectra until no further chemical shift changes
were observed.
As a control for nonspecific binding, 1H-15N
HSQC spectra were acquired for WT-MIP-1
and MIP(9) upon titration
with potassium sulfate (from a stock solution dissolved in the standard
sample buffer) in the amounts of 0, 2, 4, 20, and 40 molar equivalents, respectively. The change in pH of the protein sample upon addition of
disaccharides or potassium sulfate was negligible.
Backbone amide HSQC cross-peaks of the MIP-1
proteins were monitored
for each titration point of every experiment with the various
disaccharides. The observed chemical shift change
(
obs) for each residue was evaluated as the weighted
average of the 1H (
HN) and
15N (
N) chemical shift changes as given
in Equation 1 (38) as follows.
|
(Eq. 1)
|
Because the chemical shifts observed in the presence of
disaccharide are the population-weighted average of the chemical shifts
of free and bound MIP-1
, 
obs is related to the
ratio of bound MIP-1
to total MIP-1
(Mb/Mt) by Equation 2,
|
(Eq. 2)
|
where 
max is the chemical shift difference
between the fully bound and free forms of MIP-1
,
Mt is the total concentration of MIP-1
, and
Mb is the concentration of bound MIP-1
.
Assuming a 1:1 binding stoichiometry of disaccharide for MIP-1
monomer, the ratio Mb/Mt
is given by Equation 3 (39),
|
(Eq. 3)
|
where Dt is the total concentration of
disaccharide and K
is the
apparent dissociation constant of the MIP-1
·disaccharide complex.
For each titration, the ratio Dt/Mt and the

obs for those residues that undergo
disaccharide-induced shifts greater than the average taken over all the
residues were simultaneously fit to Equations 2 and 3 using
Kaleidagraph 3.5 (Synergy Software, Reading, PA) to give an apparent
dissociation constant K
for each residue.
To prepare samples of MIP-1
D27A/D65A/E67Q at pH 6.0 in the presence
of disaccharide, lyophilized protein was dissolved in the standard
sample buffer at pH 2.5, heparin disaccharide I-S was added to 1 mM, and the pH was raised to 6.0. Some precipitation occurred, but the NMR signal was observable and an
1H-15N HSQC spectrum was acquired.
 |
RESULTS |
Titration of WT-MIP-1
with Disaccharides--
As WT-MIP-1
has been sequentially assigned (24), the 1H-15N
HSQC NMR spectrum of the protein was used as a starting point to
monitor spectral changes upon titration of the protein with ten heparin
disaccharides (I-P, I-S, II-S, III-S, IV-S, II-H, III-H, II-A, III-A,
and IV-A) and one chondroitin disaccharide (
Di-disD);
Fig. 4 shows the structure of these disaccharides. Fig.
1A shows the superposition of
the series of spectra acquired for several points in the titration of
WT-MIP-1
with heparin disaccharide I-P. Using chemical shift changes
of the backbone amide 1H and 15N resonances as
probes for disaccharide binding, WT-MIP-1
was found to interact with
seven disaccharides: I-P, I-S, II-S, III-S,
Di-disD,
IIA, and IIIA. However, these latter two disaccharides showed so little
peak movement that a K
determination was impossible, and they were therefore considered to be
non-binding disaccharides for the purposes of discussion. In these
experiments, the resonances for all residues were monitored excluding
the N-terminal Ala1, six prolines not having backbone amide
protons, and Leu20 and Val59 that were not
observable at the protein concentrations used. The superposition of the
spectra clearly indicates that a number of backbone resonances undergo
considerable shifting upon addition of I-P (Fig. 1A). Most
of the same residues were found to undergo shifting upon addition of
I-S, II-S, III-S, and
Di-disD (data not shown). These
spectra all show the clear presence of the dimer throughout the
titration (27). Chemical shift changes of WT-MIP-1
for all
disaccharides tested were found to fall into the fast exchange limit on
the NMR time scale. Therefore, the position of each cross-peak
corresponds to the population-weighted average of the bound and free
chemical shifts of WT-MIP-1
. The backbone amide of Arg46
consistently displays some of the greatest peak movement during the
titrations, having a 
max of about 70 Hz upon
titration with I-P, reflecting a lower limit for the exchange rate of
MIP-1
with the disaccharide of ~450 s
1 (k
2

max) (38). Because each titration shows
smooth, linear peak movement (40) and because the disaccharide unit is
likely to be too small to induce cooperativity between the two binding sites, each subunit of the MIP-1
dimer appears to contain an equivalent, non-cooperative GAG binding site.

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Fig. 1.
The two-dimensional
1H-15N HSQC NMR spectra of
WT-MIP-1 , MIP(9), and MIP-1
D27A/D65A/E67Q, which show their titration with heparin
disaccharide I-P. The superposition of spectra for:
A, the 0.24 mM WT- MIP-1 sample at additions
of 0, 0.25, 0.5, 1.0, 3.0, and 6.0 molar equivalents of I-P;
B, the 0.26 mM MIP(9) sample at additions of 0, 0.23, 0.46, 0.92, 2.79, and 7.53 molar equivalents of I-P; and
C, the 0.22 mM MIP-1 D27A/D65A/E67Q sample at
additions of 0, 0.23, 0.46, 0.92, 1.85, and 7.41 molar equivalents of
I-P. Arrows depict the direction of titration from
black to red. Disaccharide structures are shown
in Fig. 4.
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|
To more clearly delineate the residues affected by the binding of the
various disaccharides, the 1H- and 15N-weighted
average chemical shift difference between the fully bound and free
forms of MIP-1
, 
max, was plotted for each residue in WT-MIP-1
. Fig. 2 (A and
B) gives the plots obtained for disaccharides I-P and I-S,
respectively. In these plots, the average 
max taken over all monitored backbone resonances is shown as a solid
line, and the average 
max plus one standard
deviation is shown as a dotted line. Inspection of these
plots reveals that for all binding disaccharides, cross-peaks
corresponding to residues in three separate regions in the primary
sequence of WT-MIP-1
undergo significant disaccharide-induced
shifting as evidenced by 
max greater than the
average. The first region corresponds to the N terminus of the protein,
including residues Met3, Gly4, and, for all
disaccharides except III-S, Ser5 (for III-S, the shifting
experienced by Ser5 was notable, but not above the average

max). The second region having substantial chemical
shift changes upon addition of disaccharide includes residues
Arg18, Lys19, Arg22,
Val25, Val26, and Tyr28. Because
the intensity of the resonance for Leu20 was too weak to
monitor in these experiments and residue 21 is a proline, it is unknown
whether either of these residues are affected by disaccharide addition.
Finally, the third region encompasses the 40s region, including
residues Gln43, Thr44, Lys45,
Arg46, Ser47, Gln49,
Val50, Cys51, and, for disaccharides I-P and
II-S, residues Phe42 and Lys48. Although each
of the binding disaccharides was determined to affect almost identical
residues of WT-MIP-1
indicating an overlapping binding surface,
differences were noted in the magnitude of peak shifting caused by the
various disaccharides with I-S > I-P >
Di-disD > II-S
III-S.

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Fig. 2.
Plots of the 1H- and
15N-weighted average chemical shift difference between
fully bound and free MIP-1 ,
 max, for residues in WT-
MIP-1 , MIP(9), and MIP-1
D27A/D65A/E67Q in the presence of heparin disaccharides I-P and
I-S. The solid line depicts the average
 max taken over all residues, and the dashed
line represents the average plus one standard deviation.
A and B show WT-MIP-1 with heparin
disaccharides I-P and I-S, respectively. C and D
show MIP(9) with heparin disaccharides I-P and I-S, respectively.
E and F show MIP-1 D27A/D65A/E67Q with heparin
disaccharides I-P and I-S, respectively.
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|
The concentration-dependent disaccharide-induced chemical
shift changes of individual backbone amides of WT-MIP-1
were plotted as a function of the ratio of the total concentrations of disaccharide to WT-MIP-1
(Dt/Mt)
for each disaccharide and fit using Equations 2 and 3, yielding
apparent dissociation constants, K
, for
each residue. For a given disaccharide, the curves obtained for the
individual residues were similar. Fig. 3
(A and B) shows the fitted data for select
residues in WT-MIP-1
for the titrations with disaccharides I-P and
I-S, respectively. For each disaccharide, an average macroscopic
K
was calculated over residues having a

max greater than the average 
max
and are reported in Table I and Fig.
4. The K
for any residue whose cross-peak in the HSQC experienced overlap with
another resonance was not used in the calculation of the average
K
reported. Also eliminated from the
average K
determination were residues 3 through 5 within the dimer interface of WT-MIP-1
and the resonances
for a very few amino acids (such as Arg22) that reported a
K
significantly deviating from those
revealed by the core set of binding residues. However, all these
residues are considered in the discussion of the binding surface
(under "Discussion"). The average K
determined was disaccharide-dependent, with I-S and I-P
having higher affinity than
Di-disD, which in turn bound
more tightly than II-S and III-S. Some precipitation occurred upon
addition of I-S, which may cause error in its average
K
determination. An additional source of
error in K
is the concentration of
disaccharide used (see "Experimental Procedures" for possible
errors in amounts purchased). In the worst case, this error would cause
the K
to increase by 1.47-fold for
disaccharides I-P and I-S. Although significant, this level of error
does not affect our comparison between dimeric wild type protein and
monomeric MIP(9), because the same stock solutions of disaccharide were
used in these titrations.

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Fig. 3.
Plot of the weighted average of
1H and 15N chemical shifts of select amide
groups of WT-MIP-1 and MIP(9) as a function of
the molar ratio of disaccharide to protein. The weighted average
of 1H and 15N chemical shifts
 obs is given by Equation 1, and the data are fit to
Equations 2 and 3 simultaneously, yielding apparent dissociation
constants (K ) for each residue.
Representative data from each region of interaction are shown.
A and B show WT-MIP-1 with heparin
disaccharides I-P and I-S, respectively. C and D
show MIP(9) with heparin disaccharides I-P and I-S, respectively.
Although not used in the calculation of the average macroscopic
K reported, the curves for
Gly4 in WT-MIP-1 are shown in A and
B to give a sample of the quality of the data obtained for
this region.
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Table I
The average macroscopic K for the MIP-1 proteins
The average macroscopic K is
determined by averaging the K found
for residues whose backbone amide resonances shift greater than the
average chemical shift change, as described under "Experimental
Procedures" and "Results." The error shown in these values is one
standard deviation of the average K
calculated. These error values do not include possible error due to
slightly incorrect quantities provided by the commercial suppliers (see
"Experimental Procedures" and "Results").
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Fig. 4.
The average apparent macroscopic dissociation
constants, K , for
WT-MIP-1 and MIP(9) for disaccharides I-P,
I-S, II-S, III-S, and
Di-disD. A, WT-MIP-1
is shown as filled circles ( ), and MIP(9) is shown as
open circles ( ). The error shown represents one standard
deviation in the average K calculated.
Data for disaccharide I-P is shown in black, I-S in
pink, II-S in blue, III-S in green,
and Di-disD in purple, with the molecular
structure of the disaccharide adjacent to the data. B,
molecular structure of non-binding disaccharides IV-S, II-H, III-H,
IIA, IIIA, and IV-A.
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|
As a control for the effect of nonspecific binding of sulfate to
WT-MIP-1
, the protein was titrated with potassium sulfate to a final
concentration of 10 mM. Small
concentration-dependent changes in the chemical shifts of
the backbone resonances of several residues were observed during the
titration (data not shown). Although these resonances generally
corresponded to those found to be involved in heparin binding, these
shifts were significantly smaller in magnitude than those observed
during the titration of WT-MIP-1
with the binding disaccharides and
had not reached saturation even at 10 mM sulfate (40 molar
equivalents). The small magnitude of peak shifting and the lack of
saturation at 10 mM sulfate suggest that the level of
nonspecific sulfate binding by wild type MIP-1
is about two orders
of magnitude weaker than the binding seen for heparin disaccharides I-S
and I-P.
Titration of MIP(9) with Disaccharides--
MIP(9) is a truncated
mutant of MIP-1
that begins at the ninth residue (Thr9)
and is known to be a folded monomer in solution (27) that retains the
ability to bind the CCR5 receptor (28). Using CBCA(CO)NH and
HNCA data, the assignments for the 1H-15N HSQC
spectrum of MIP(9) were completed (data not shown). Spectral changes in
the 1H-15N HSQC spectrum of MIP(9) were then
used to monitor the titration of MIP(9) with the same eleven
disaccharides used with WT-MIP-1
. Fig. 1B shows the
superposition of the series of spectra acquired for several points in
the titration of MIP(9) with heparin disaccharide I-P. Throughout the
titration experiments, no indication of MIP(9) dimerization was
observed as judged by the relative lack of resonance shifting for
residues involved in the dimer interface of MIP-1
(27). As for
WT-MIP-1
, MIP(9) was found to be in a fast exchange regime with the
disaccharides (as evidenced by single resonances with chemical shifts
that are the population-weighted averages of the free and bound forms
of the protein) and to interact with the same set of disaccharides. A
small percentage of unfolded protein is indicated by several peaks in
the unstructured region of the spectrum (1H ~ 8.1-8.4 ppm). These peaks experience a decrease in the signal-to-noise ratio as the titration progresses and, for some resonances, an increase
in the line width. Although this may indicate an intermediate timescale
exchange process, no disaccharide-induced changes in chemical shift are
seen for these peaks, so it appears unlikely that there is any specific
interaction between the unstructured residues and the disaccharides. A
possible explanation for the behavior of these peaks is a
time-dependent aggregation or possibly a nonspecific
interaction with the disaccharide, resulting in the aggregation of the
unfolded MIP(9) and thereby the disappearance of the peaks.
Fig. 2 (C and D) shows the plots obtained of

max for the residues of MIP(9) for heparin
disaccharides I-P and I-S, respectively. These plots reveal that, for
the interacting disaccharides I-P, I-S, II-S, III-S, and
Di-disD, residues in two distinct regions of MIP(9)
experience sizeable chemical shift changes. The residues Phe13, Tyr15, Ala17,
Arg18, Lys19, and Arg22 compose the
first region of substantial shifting. Residues in and surrounding the
40s region, including Lys45, Arg46,
Ser47, Lys48, Val50,
Cys51, and in most cases Gln43 and/or
Thr44, comprise the second region. Although many of the
residues found to be involved in MIP(9) binding to disaccharides are
the same as those for WT-MIP-1
, some differences were observed. The
first obvious difference comes from the fact that MIP(9) lacks the
first eight amino acids of WT-MIP-1
and, hence, the first region of WT-MIP-1
interaction
(Met3-Gly4-Ser5) can not be seen in
MIP(9). The second more subtle difference involves residues surrounding
Arg18, Lys19, and Arg22, all of
which appear to be involved in disaccharide binding for both
WT-MIP-1
and MIP(9). In WT-MIP-1
, residues just C-terminal to
these were seen to shift upon disaccharide addition (Val25,
Val26, and Tyr28), whereas in MIP(9) the
residues immediately N-terminal (Phe13, Tyr15,
and Ala17) to this region are affected. Finally, in the 40s
region Gln49 in WT-MIP-1
was found to have one of the
largest peak movements, whereas Lys48 shifted less than or
just equal to the average. In contrast, in MIP(9) Gln49 was
found to shift very little and Lys48 was consistently the
residue with the greatest chemical shift change.
The concentration-dependent disaccharide-induced chemical
shift changes of individual backbone amides of MIP(9) were plotted as a
function of the ratio
Dt/Mt for each
disaccharide, and the data were fit to find
K
as described above. Fig. 3
(C and D) shows the fitted data for select
residues in MIP(9) for the titrations with disaccharides I-P and I-S,
respectively. For a given disaccharide, an average macroscopic
K
was calculated and is reported in
Table I and Fig. 4. The K
of any residue
whose resonance in the HSQC experienced overlap, and residues
Arg22 and Tyr62, which consistently exhibited
an anomalously high K
, were not used in
the calculation of the average K
reported. However, these residues are included in the discussion of the binding surface (under "Discussion"). The average
K
varied with the disaccharide tested,
with I-S and I-P binding tighter than
Di-disD, which
bound more strongly than II-S and III-S. MIP(9) was determined to
interact less tightly than WT-MIP-1
for every disaccharide (Table I
and Fig. 4). The effect of nonspecific binding of sulfate to MIP(9) was
examined by titration with potassium sulfate (up to 10 mM).
The small magnitude of chemical shift changes (data not shown) suggests
that the level of nonspecific sulfate binding by MIP(9) is quite low at
the concentrations comparable to those used for the disaccharide titrations.
Titration of MIP-1
D27A/D65A/E67Q with
Disaccharides--
To obtain a MIP-1
mutant that was soluble to NMR
concentrations at pH 6.0, we found it necessary to simultaneously
neutralize three acidic residues of MIP-1
resulting in the mutant
MIP-1
D27A/D65A/E67Q. Although this mutant was significantly more
soluble at pH 6.0 than WT-MIP-1
, 100-200 mM NaCl was
still required to produce NMR samples of 0.1-0.2 mM
protein in the absence of heparin. Using CBCA(CO)NH and HNCA
data collected both at pH 2.5 and pH 6.0, the assignments for the
1H-15N HSQC spectrum of MIP-1
D27A/D65A/E67Q
at each pH were obtained (data not shown). At pH 2.5, MIP-1
D27A/D65A/E67Q retained the spectral attributes of WT-MIP-1
at the
same pH, with little variation in the peak positions other than for
peaks corresponding to those of the mutated residues, indicating that
the mutations had not perturbed the overall structure of the protein
(Fig. 1, A and C). Similarly, at pH 6.0 this
mutant exhibited narrow lines in the HSQC spectrum, and peaks
associated with the N-terminal dimer interface were present indicating
that the protein was folded and its structure at pH 6.0 was consistent
with that determined for the wild type protein at pH 2.5 (Fig.
5A).

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Fig. 5.
The spectra of MIP-1
D27A/D65A/E67Q. A, superposition of the
1H-15N HSQC spectra at pH 2.5 (blue)
and pH 6.0 (black) of MIP-1 D27A/D65A/E67Q. The sample at
pH 2.5 consisted of 0.22 mM protein in 20 mM
sodium phosphate buffer containing 10% 2H2O
and 0.02% sodium azide. The sample at pH 6.0 was 0.2 mM
protein in 20 mM sodium phosphate buffer containing 150 mM NaCl, 10% 2H2O, and 0.02%
sodium azide. B, comparison of
1H-15N HSQC spectra at pH 6.0 of MIP-1
D27A/D65A/E67Q with heparin disaccharide I-S (red, 0.99 mM disaccharide) and without I-S (black). The
sample in the presence of I-S contained no NaCl, whereas the sample in
the absence of I-S contained 150 mM NaCl. Residues labeled
indicate several of those that are affected by disaccharide binding at
both pH 2.5 and pH 6.0.
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For comparison to the titration data collected at pH 2.5 for
WT-MIP-1
and MIP(9), spectral changes in the
1H-15N HSQC spectrum of MIP-1
D27A/D65A/E67Q
at pH 2.5 were used to follow its titration with heparin disaccharides
I-P and I-S (Figs. 1C, 2E, and 2F). As
expected, inspection of the spectra showed that essentially all
resonances in MIP-1
D27A/D65A/E67Q that shift considerably upon
addition of either I-P or I-S correspond directly to those in
WT-MIP-1
that experience disaccharide-induced chemical shift
changes. As was observed for WT-MIP-1
, the chemical shift changes
were in the fast exchange limit on the NMR timescale. As described
above, the average macroscopic K
was
calculated for the binding of MIP-1
D27A/D65A/E67Q with
disaccharides I-P and I-S (Table I).
At pH 6.0, titrations of MIP-1
D27A/D65A/E67Q were again conducted
with I-P and I-S. As mentioned previously, at least 100 mM
NaCl was required to achieve even fairly dilute NMR samples of MIP-1
D27A/D65A/E67Q. Under these conditions, no disaccharide-induced chemical shift changes for the protein were observed upon addition of
either disaccharide (data not shown), likely due to the NaCl interfering with the interaction. As an alternative approach, heparin
disaccharide I-S was added directly to 0.2 mM MIP-1
D27A/D65A/E67Q at pH 2.5 to a final concentration of 1 mM
disaccharide. The pH was raised to 6.0 with no addition of NaCl, and an
HSQC spectrum was acquired. Although some precipitation of the protein
was noted upon raising the pH, sufficient protein remained in solution
to provide an NMR signal. It is noteworthy that only 1 mM
disaccharide was sufficient to keep the protein largely soluble at pH
6.0, whereas, in the absence of disaccharide, at least 100 mM NaCl was required.
Fig. 5B shows the superposition at pH 6.0 of the spectrum of
MIP-1
D27A/D65A/E67Q in the presence of I-S with a control spectrum of MIP-1
D27A/D65A/E67Q containing 150 mM NaCl in the
absence of disaccharide. This control spectrum is not ideal because it contains NaCl, which does cause peak shifts, but we were unable to
obtain a control spectrum at pH 6.0 in the absence of NaCl due to
extensive protein precipitation. Although small in magnitude, clear
chemical shift changes are seen between the spectra at pH 6.0 in the
presence and absence of heparin disaccharide. All of these shifted
residues are the same as those that experienced disaccharide-induced
shifts at pH 2.5. These effects would likely be more pronounced if
compared with an ideal (no salt) control in the absence of heparin,
because the salt required for solubility (greater than 100 mM) of the control spectrum causes some shifting of
resonances in the same direction as does heparin at pH 2.5 and at pH 6 (data not shown).
 |
DISCUSSION |
Disaccharide Selectivity of WT- MIP-1
and MIP(9)--
Some of
the most common GAGs are heparin, heparan sulfate (related to heparin
and found on the cell surface), and chondroitin sulfate. At the most
basic level, GAGs are comprised of a repeating disaccharide unit of a
hexuronic acid and an amino sugar. For heparin and heparan sulfate, the
hexuronic acid can be either glucuronic acid or its epimer, iduronic
acid, and the amino sugar is D-glucosamine. The repeating
disaccharide units of GAGs have variable amounts of N- and
O-sulfation and N-acetylation (22). Kuschert
et al. (15) demonstrated that chemokines show varying degrees of selectivity for the different types of GAGs, with the highly
basic RANTES demonstrating the most pronounced selectivity and the
acidic MIP-1
the least of the chemokines tested. In addition, these
studies also demonstrated that the interaction between chemokines and
GAGs was dependent on GAG length and required both N- and O-sulfation for optimal binding. The same group has also
demonstrated that RANTES, MIP-1
, MCP-1, and IL-8 aggregate in the
presence of heparin (30), which complicates structural analysis of
chemokine-GAG interactions. However, we have found that MIP-1
is
amenable to study by NMR with disaccharides. Our studies were limited
to disaccharides available from commercial sources and so included a
set of eleven such disaccharides. Fig. 4 shows the chemical structures
of these sugars; unsaturation of the uronic acid unit in the
disaccharides is the result of heparin lyase digestion used in the
commercial production of these molecules.
Fig. 4 and Table I show the average macroscopic
K
values obtained for the binding of
these disaccharides to MIP-1
and its variants. Our studies indicate that multiple sulfation of the disaccharide unit is an important factor
in the binding of both dimeric WT-MIP-1
and monomeric MIP(9),
because non- and mono-sulfated disaccharides were not seen to
significantly bind either protein. Of the sugars tested, heparin
disaccharides I-P (di-sulfated) and I-S (tri-sulfated) bound the most
tightly to WT-MIP-1
with average
K
values of ~44 ± 12 and
68 ± 15 µM, respectively. The similarity of
dissociation constants for I-P and I-S with WT-MIP-1
indicates that
O-sulfation at the two positions that these sugars have in common (positions 2' of the unsaturated hexuronic acid unit and 6 of
the D-glucosamine unit, Fig. 4) are important in the
interaction of MIP-1
with disaccharides. Because I-S shows no clear
increased affinity, its additional site of N-sulfation at
position 2 in the D-glucosamine unit does not appear to
facilitate tighter I-S binding. Although showing weaker binding than
for WT-MIP-1
, MIP(9) also bound I-P and I-S the most tightly among
the disaccharides with average K
values
of ~94 ± 23 and 139 ± 25 µM, respectively.
It should be noted that errors in the amount of disaccharide purchased
(see "Experimental Procedures") do not affect this conclusion or
the argument regarding sulfate selectivity. For both the wild type
dimer and the monomeric MIP(9), the binding of heparin disaccharides
II-S and III-S was significantly weaker than for I-S or I-P. In
accordance with this observation that O-sulfation at both
positions 2' and 6 is important to binding, each of these
disaccharides, while being di-sulfated overall, has an
N-linked sulfate at the amino moiety of position 2 of the D-glucosamine unit, with the second site of sulfation being
O-linked sulfate at position 6 (II-S) or at
position 2' (III-S). The binding of these sugars also produces
significantly less NMR peak movement, increasing the error in
K
measurements, leading to the larger
error bars in Fig. 4 (also Table I). In support of our
findings regarding the importance of O-sulfation at
positions 2' and 6, Kuschert et al. (15) found that,
although both N- and O-sulfation of GAGs are
required for optimal GAG-chemokine binding, enzymatic removal of
O-sulfation was more deleterious to chemokine binding than
the elimination of N-sulfation.
Chemokines are known to exhibit a range of selectivity for the
different types of GAGs (11, 15). Because a number of chondroitin disaccharides are commercially available, we investigated the chondroitin disaccharide
Di-disD, which has a sulfation
pattern identical to heparin I-P but contains a
D-galactosamine unit instead of a D-glucosamine
unit. Within error, our experiments indicate that the chondroitin
disaccharide
Di-disD binds essentially as well as the
heparin disaccharides I-P and I-S to WT-MIP-1
. Therefore, unlike
studies using much larger heparin- and chondroitin-like GAG fragments
(11, 15), where small differences in selectivity may be additive over
the large number of GAG units, little sequence selectivity between
heparin and chondroitin for WT-MIP-1
could be inferred using the
disaccharides in our binding studies.
Although sulfation appears to influence the binding of disaccharides to
dimeric WT-MIP-1
and monomeric MIP(9) in similar manners, the main
difference in affinity that our studies reveal between these proteins
is a clear trend of WT-MIP-1
binding more tightly than MIP(9).
Although there is some overlap in the error range of the two proteins
in Fig. 4 and Table I, this is eliminated if only the NMR resonances
with the largest magnitude of disaccharide-induced chemical shift
changes (and therefore the least amount of error) are used in the calculations.
The GAG Binding Surface of the Wild Type MIP-1
Dimer--
The
GAG binding surface on WT-MIP-1
was characterized by
1H-15N HSQC NMR spectroscopy using
15N-labeled protein and a total of eleven different heparin
and chondroitin disaccharides. The changes in these spectra upon
disaccharide titration could result from the direct interaction of the
amide group with the GAG, the participation of the side chain of an amino acid (resulting in a shift of its backbone amide resonance), or
be due to a general shifting of the structure in that region of the
protein. Spheres in Fig.
6A represent the backbone
amide nitrogen atoms of all the residues in the dimer of WT-MIP-1
that are demonstrated by the present titration studies to have
above-average disaccharide-induced resonance movement. As is evident
from Fig. 6A, the distal surfaces of the protein are
involved in GAG binding, whereas the intervening regions of the dimer,
including much of the dimer interface, and the C-terminal
-helices
are unaffected. Fig. 6B shows a single monomeric subunit of
the wild type protein with the same residues highlighted. The binding
surface contains the basic residues Arg18,
Lys19, Arg22, Lys45,
Arg46, and Lys48 that surround a cleft that
includes residues Val25, Val26,
Tyr28, Gln43, Thr44,
Ser47, Gln49, Val50, and
Cys51. In addition, although not included in Fig.
6A or 6B, the side chain of Asn23
(found directly in the binding cleft) is likely to be part of the
binding surface, because its side-chain amide resonances were significantly shifted in the presence of disaccharide.

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Fig. 6.
The disaccharide binding site of
MIP-1 . Magenta spheres in
A and B represent the backbone amide nitrogen
atoms of residues of WT-MIP-1 shown by NMR to be affected by
disaccharide binding on the dimeric and monomeric units, respectively.
The structure used for WT-MIP-1 was determined by NMR at pH 2.5 (24). C, a close up of the proposed binding surface of
WT-MIP-1 . Magenta spheres symbolize the backbone amide
nitrogen atoms of residues originating from the monomeric unit shown,
and those in blue correspond to residues Met3,
Gly4, and Ser5 of the other monomer in the
dimer. D, the NMR structure determined for the monomeric
mutant MIP-1 F13A (42) onto which the GAG binding surface for the
monomeric MIP(9) is mapped in magenta spheres. Residues
truncated in MIP(9) are shown in light blue at the N
terminus. Shown as green spheres are the backbone amide
nitrogen atoms of residues 13, 15, and 17, which are affected by
addition of disaccharide to monomeric MIP(9) but are not affected in
dimeric WT-MIP-1 .
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Basic residues at positions corresponding to Arg18,
Lys45, Arg46, and Lys48 in MIP-1
are conserved in both MIP-1
and RANTES, the latter three forming a
BBXB motif (B representing basic residues), which has been
shown to be a common heparin binding motif for a number of proteins
(22). For MIP-1
, the involvement of Arg18,
Lys45, and Arg46 in heparin binding is
consistent with previous studies (8, 13) that demonstrated that
mutagenesis of any of these residues in MIP-1
can diminish heparin
binding ability, although each mutation alone does not have a large
effect. Studies have reported similar results for MIP-1
(14, 19),
whereas only the conserved residues in the 40s region (44, 45, 47) are
important for RANTES (11, 12). Additionally, the backbone amide
resonances of Lys19, not conserved in either RANTES or
MIP-1
, and Arg22, not conserved in MIP-1
, were found
to undergo considerable disaccharide-induced chemical shift changes in
the present studies, but mutation of both of these residues in MIP-1
(13) and modification of the residue corresponding to Arg22
in RANTES (11) appeared to show no effect on heparin binding.
To understand the involvement of Met3, Gly4,
and Ser5, the binding of GAGs to MIP-1
must be
approached from the perspective of the MIP-1
dimer. Fig.
6B depicts the residues in the monomeric unit of WT-MIP-1
that are affected by disaccharide binding, showing that residues 3 through 5 are located on the opposite end of the subunit from the GAG
binding site. This suggests the cluster
Met3-Gly4-Ser5 is not involved with
GAG binding to its own monomer. On the other hand, in the context of a
dimer these residues are located such that they are in close proximity
to the conserved basic residues on the other monomer unit as shown in
blue in Fig. 6C, and it appears these residues form part of
the GAG binding cleft.
The GAG Binding Surface of the MIP(9) Monomer--
Figs.
2C, 2D, and 4, and Table I show the results of
titrating the monomeric MIP(9) with the various GAG disaccharides. In each case, MIP(9) was found to bind a particular GAG more weakly than
the wild type protein, although for the case of heparin disaccharide II-S the errors in the average K
values
overlapped for the two proteins. This observation is in contrast to a
recent report that the IL-8 monomer binds GAGs more tightly than its
dimer (41). The binding surface described by the NMR studies for MIP(9)
is largely the same region of the protein as that found for the wild
type dimer. Fig. 6D shows the NMR-derived structure of the
monomeric MIP-1
variant F13A (the only available structure of a
monomeric MIP-1
variant) (42), with spheres at the backbone amide
nitrogen atoms of the residues that shift more than average in MIP(9)
during the GAG titrations. A notable difference in the peak movements
for MIP(9) compared with the wild type protein is the significant
movement of residues Phe13, Tyr15, and
Ala17 in MIP(9), whereas in the wild type protein these
resonances are little affected by GAGs (Fig. 2, A and
B). Interestingly, these residues (shown in green
in Fig. 6D) are at the back of the GAG binding site (using
approximately the same orientation shown in Fig. 6C), on the
same side of the site as the dimer residues Met3,
Gly4, and Ser5, which form the back of the
pocket in the wild type protein.
Since MIP(9) is a monomer, it lacks the full binding pocket provided by
the dimer. This not only potentially provides a rationale for the trend
toward lower GAG affinity for the monomer as compared with the dimer,
but it also appears that residues Phe13, Tyr15,
and Ala17 may be recruited in MIP(9) to help form the
pocket or are structurally affected due to the elimination of the dimer
residues. Furthermore, often the single largest peak movement in
monomeric MIP(9) is displayed by residue Lys48, which
contrasts with the wild type dimer where this residue is only perturbed
somewhat by GAG binding. The location of Lys48 in the wild
type dimer is within 5 Å of the dimer residues 3-5, so in the MIP(9)
monomer the lack of these residues may cause an increased role for
Lys48 in binding GAGs.
MIP(9) is a truncation mutant and does not contain residues 1-8, but
if our structural interpretation is correct the lower binding affinity
and difference in titration spectra found for MIP(9) are expected to be
observable in other MIP-1
monomeric variants that do contain
residues 3-5. The N-terminal residues of full-length monomers should
not be effective at binding GAGs, because, unlike in the dimer, their
distance from the GAG binding site precludes participation in GAG
binding. Preliminary titration studies have been conducted on a
functional full-length monomeric variant, MIP-1
P8A (28), with
heparin disaccharide I-P. These titration studies of MIP-1
P8A with
I-P indicate that nearly identical residues exhibit
disaccharide-induced chemical shift perturbations as were found for
MIP(9), including residues 13, 15, 17, 18, 19, 45-48, and 62 among
others.2 The relative overall
magnitudes of the perturbations were consistent with those of monomeric
MIP(9) rather than that for the wild type dimer. In addition, the
affinity of P8A for the I-P disaccharide is even weaker than that for
monomeric MIP(9).2 Due to the lack of a dimer in this
variant, there is considerable N-terminal heterogeneity in P8A, but the
major peaks for residues Met3, Gly4, and
Ser5 (which are involved in GAG binding in the dimer) do
not shift in the P8A monomer upon GAG titration, consistent with our
structural model that the chemokine dimer itself causes the involvement
of the residues. However, minor peaks in P8A that have chemical shifts consistent with residues 3 through 5 do shift about an average amount
upon titration with I-P (
max ranging from 0.03 to
0.06). The magnitudes of these perturbations are less than seen with the wild type protein, where the resonance for Gly4
(
max = 0.11) consistently displays some of the
greatest disaccharide-induced shifting observed for the protein. Work
continues on this and other monomeric variants (data not shown).
MIP-1
Binding to Heparin Disaccharide I-S at pH
6.0--
Although several chemokines are naturally monomeric (39, 43),
many chemokines form dimers even at low concentrations (28, 29).
Several CC chemokines, including MIP-1
, MIP-1
, and RANTES, self-associate further to form high molecular weight aggregates in a
pH-dependent manner (25, 26), which precludes study of their GAG binding properties at physiological pH by NMR methods. Due to
the tendency of WT-MIP-1
and MIP(9) to self-associate, the
disaccharide titration studies of these MIP-1
proteins were conducted at pH 2.5, where WT-MIP-1
has been shown to be a stable dimer (24) and MIP(9) a monomer (27). The question is therefore raised
whether the GAG binding surface on MIP-1
determined at pH 2.5 accurately portrays the surface under physiological conditions. To
answer this question, a mutant of MIP-1
was sought that would not
form high molecular weight aggregates at physiological pH. Neutralization of charged residues on the surfaces of MIP-1
, MIP-1
, and RANTES has been reported to decrease their tendency for
self-association at physiological pH (8, 26). For example, we have
shown that the triple mutant MIP-1
K45A/R46A/K48A does not exhibit
pH-dependent aggregation at pH 7.0 (8), but this mutant
could not be used in the present studies because these mutations
adversely affect heparin binding. Studies conducted by Czaplewski
et al. (26) demonstrated that a single mutation of either of
the acidic residues at positions 26 or 66 in MIP-1
and their counter
residues in MIP-1
(Asp27 or Glu67) and
RANTES (Glu26 or Glu66) significantly
disaggregated the chemokines (26). In our hands, significant salt
concentrations were still required to obtain sample concentrations
above 0.1 mM for any single point mutant (data not shown).
To obtain a MIP-1
mutant that was soluble to NMR-amenable
concentrations at pH 6.0, we constructed the mutant MIP-1
D27A/D65A/E67Q.
Fig. 5B shows the spectrum of MIP-1
D27A/D65A/E67Q in the
presence of heparin disaccharide I-S at pH 6.0, with no salt present except 20 mM sodium phosphate, 0.02% sodium azide. As
stated previously, we are unable to obtain a pH 6.0 control spectrum of
MIP-1
D27A/D65A/E67Q in the absence of NaCl, so Fig. 5B
shows the superposition of the spectrum of the MIP-1
D27A/D65A/E67Q·disaccharide I-S complex at pH 6.0 with the spectrum
of MIP-1
D27A/D65A/E67Q at the same pH in the presence of 150 mM NaCl. This superposition clearly shows chemical shift
changes for resonances of the same residues that were seen to shift
upon addition of disaccharide to both this mutant and to WT-MIP-1
at
pH 2.5. Therefore, the surface determined at pH 2.5 is consistent with
the available spectrum at pH 6.0, indicating that studies performed at
pH 2.5 are a valid assessment of the surface under physiological conditions.
Biological Significance of Disaccharide Binding Results--
The
GAG binding surface of MIP-1
forms a clear cleft (Fig.
6C) that involves some of the residues (such as
Arg18 and Arg46) that are also participate in
receptor binding (44). The overlapping nature of the receptor binding
surface and the GAG binding groove is consistent with results that GAGs
can compete with the receptor for chemokine binding (8, 11, 16).
Mutations of some GAG binding residues moderately affect CCR5 binding
by MIP-1
(8). It has been observed in MIP-1
and RANTES that
mutations affecting the GAG binding surface alter the ability of the
mutant to bind CCR1 more than CCR5 (11, 12, 14). This suggests that the GAG binding surface in those proteins has greater overlap with the CCR1
binding surface than the CCR5 binding surface. The role of the MIP-1
dimer in GAG binding is also indicated, because residues 3 through 5 from the other subunit appear to have a role in facilitating higher
affinity GAG binding than a monomer alone could provide. This is
consistent with a model in which chemokine multimers can bind to GAGs
in the formation of a concentration gradient. The high homology between
MIP-1
and MIP-1
suggests similar binding clefts for each
chemokine. The applicability of our results to larger GAGs of in
vivo lengths is not known, although the clear binding site,
selectivity, and reasonable affinity revealed by our data suggest that
the characteristics of both MIP-1
and GAG that we have found to be
important are likely characteristics that are also utilized in
vivo.
Conclusions--
We have used NMR to determine the affinity of
wild type MIP-1
and a monomeric variant MIP(9) for several GAG
disaccharides. This work has delineated a GAG-binding cleft in MIP-1
and shown the importance of GAG O-sulfation at the 2' and 6 positions of the hexuronic acid and the D-glucosamine
units, respectively. In addition, a possible role for the dimer in
forming part of the GAG binding site in the chemokine is revealed.