From the Department of Human Biological Chemistry and
Genetics and Sealy Center for Structural Biology, University of
Texas Medical Branch, Galveston, Texas 77555 and the ¶ Department
of Protein Expression, Human Genome Sciences, Inc.,
Rockville, Maryland 20850
Received for publication, June 13, 2000, and in revised form, October 11, 2000
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ABSTRACT |
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MPIF-1, a CC chemokine, is a specific inhibitor
of myeloid progenitor cells and is the most potent activator of
monocytes. The solution structure of myeloid progenitor inhibitor
factor-1 (MPIF-1) has been determined by NMR spectroscopy. The
structure reveals that MPIF-1 is a monomer with a well defined core
except for termini residues and adopts the chemokine fold of three
Chemokines (chemotactic cytokines) mediate diverse biological
processes, including leukocyte trafficking, hematopoiesis, and angiogenesis, and play a fundamental role in host defense against infection (1-3). About 40 chemokines have been identified so far. All
are 70 to 120 amino acids in length, and are characterized by four
conserved cysteine residues. Chemokines are broadly classified into CC
and CXC families on the basis of whether the first
two cysteines are adjacent (CC) or separated by an amino acid
(CXC). The CXC chemokines can be further divided
into two subgroups, "ELR" and "non-ELR." All ELR CXC
chemokines activate neutrophils, whereas non-ELR CXC
chemokines activate different subsets of lymphocytes. CC chemokines
activate monocytes, macrophages, eosinophils, basophils, T cells but
not neutrophils. In addition, a single member of a C family, which
contains only two cysteines, and a single member of a
CX3C family have also been identified.
Chemokines bind and activate 7-transmembrane G-protein-coupled
receptors on leukocytes, and also bind cell surface highly sulfated
proteoglycans. Proteoglycan binding could play a role in forming a
concentration gradient for leukocyte recruitment (4, 5), and also in
receptor activation by sequestering the ligand on the leukocyte cell
surface (6, 7). Various studies have shown that a monomer is sufficient for binding to 7-transmembrane receptors (8-12), and that dimer formation could play a role in binding to proteoglycans (13).
Myeloid progenitor inhibitory factor-1
(MPIF-1)1 (also known as
CK In this study, we have determined the solution structure and
characterized the backbone dynamics of MPIF-1 by NMR spectroscopy. We
have studied the proteoglycan binding of MPIF-1 and full-length MPIF-1
proprotein using heparin affinity chromatography, and the self-association propensities under a variety of solution conditions by
sedimentation ultracentrifugation measurements. The implications of the
structure, dynamics, heparin binding, and the self-association properties are discussed in terms of the protein's functions. The data
from this study will form the structural basis for therapeutics for
preventing chemotherapy-associated myelotoxicity.
Protein Expression and Purification--
The MPIF-1 gene
sequence was chemically synthesized with codons optimized for
expression in Escherichia coli. The gene was then subcloned
into the expression vector pHE4, which contains a strong synthetic
promoter with two lac operators, an efficient ribosomal-binding site and a synthetic transcriptional terminator downstream of the inserted gene. The expression plasmid was transformed into the E. coli K12-derived strain SG13009. The following
protocol was followed for preparing 15N- and
13C-labeled MPIF-1. The seed culture was prepared by
inoculating 1:4000 dilution of frozen seed stock into 125 ml of LB
media with kanamycin. After induction with 5 mM
isopropyl-1-thio- Sedimentation Equilibrium--
Analytical ultracentrifugation
experiments were performed using a Beckman model XL-A ultracentrifuge
at 20 °C at rotor speeds 23,000, 28,000, and 40,000 rpm. Experiments
were carried out at two different starting concentrations (0.6 and 1.2 mgs/ml) in different buffers and ionic strengths to study their effect
on dimerization (Table I). Absorbance was measured at 280 nm, and the
data was collected as an average of five successive radial scans using
a 0.003-cm step size. The data was fitted to the following equation,
where Heparin Affinity Chromatography--
A 0.8-ml heparin-POROS
PE-20 column (4.6 × 50 mm) was equilibrated in a buffer
containing 50 mM Tris-HCl, pH 7, 100 mM NaCl using Waters Alliance HPLC. Chemokine protein was loaded onto the
column, washed in the same buffer, and eluted with a linear gradient of
0.1 M to 0.75 M NaCl over 50 column volumes.
The NaCl concentration for elution of each chemokine was calculated by the conductivity of eluted peak. MIP-1 NMR Spectroscopy--
All spectra were collected at 35 °C on
a Varian Unity Plus 600 or a INOVA 500-MHz spectrometer, both equipped
with field gradient accessories. The protein concentration was 2 mM in 20 mM sodium acetate, 1 mM
sodium azide, pH 5.2, in 90% H2O, 10%
2H2O(v/v) or 99.99%
2H2O. Chemical shifts are referenced to
2,2-dimethyl-2-silapentanesulfonic acid using the method of
Wishart et al. (18). Assignment of the main chain NH, N,
C Hydrogen-bond Restraints--
The potential candidates for
hydrogen bonding were initially identified on the basis of observing
slow-exchanging amide protons from a series of two-dimensional
1H-15N HSQC spectra recorded within 24 h
of dissolving the protein in 2H2O. For each
hydrogen bond, two distance restraints were used (rNH-O,
1.8-2.3 Å and rN-O, 2.4-3.3 Å). The hydrogen bonding
restraints were used only after an initial set of structures had been
calculated. Only the amide protons which satisfied distance and angular
restraints with hydrogen-bond acceptors were used in the structure calculations.
Data Processing and Structure Calculations--
All NMR spectra
were processed using the nmrPipe suite of programs (25). Structures
were calculated by the hybrid distance geometry-dynamical simulated
annealing method using the program XPLOR (26). A total of 711 nonredundant NOE distance restraints (319 intra-residue, 179 sequential, 82 medium range, and 131 long range NOEs) were used. In
addition, 82 dihedral (53 Dynamics--
15N-T1, T2,
and {1H}-15N NOE experiments were recorded
at 35 °C on a uniformly labeled sample of 15N MPIF-1
using gradient version of the pulse sequences (27). {1H}-15N NOEs were measured by recording
HSQC spectra with and without proton saturation. The spectra without
NOE were recorded with delays of 5 s and spectra with NOE with
2 s delay and 3 s of proton saturation to give the same delay
of 5 s between transients. The spectra were processed using
nmrPipe, and T1 and T2 values were obtained by nonlinear least-square fits of the cross peaks to a
two-parameter exponential decay. Uncertainties in the T1
and T2 values were taken as the S.D. of the fit. NOE values
were obtained from the ratio of the peak intensities recorded with and
without proton saturation. Uncertainties in the NOE values were
estimated from the base line of the spectra according to Farrow
et al. (27).
Sedimentation Equilibrium--
Chemokines are known to
self-associate, a process that is dependent on solution conditions such
as pH and ionic strength. The association properties of the MPIF-1 and
the full-length MPIF-1 were studied using ultracentrifugation methods
at different pH and ionic strength conditions. The summary of results
is shown in Table I. The data indicate
that both proteins are monomeric under the experimental conditions and
show no tendency to self-associate. The monomeric state of MPIF-1 is
consistent with both the NMR structure and the calculation of the
correlation time from 15N dynamics.
Structural Statistics of MPIF-1--
The statistics of the 30 final simulated annealing structures are shown in Table
II, and the superimposition of the
individual structures on the average structure is shown in Fig.
1A. The structure of the
protein is well defined except for the terminal residues 1-10 and
67-77. The quality of the structures was tested using the programs
PROCHECK (28) and VADAR (VADAR-structural analysis of protein
structures available for various criteria such as the stereochemistry, hydrogen bonds, the region of occupancy in the Ramachandran plot, van der Waals contacts, buried charged residues, number of buried residues, and packing defects). All 30 structures met the above mentioned criteria, which are expected of a
high resolution structure.
All the structures and the energy-minimized average structure displayed
good covalent geometry (Table II) and minimal NMR constraint
violations. None of the 30 simulated annealing structures had NOE
violations greater than 0.2 Å and dihedral angle violations greater
than 2°. The r.m.s. distribution for residues 11 and 66 between all
30 structures and the average structure is 0.57 Å for the backbone
atoms and 1.09 Å for the heavy atoms (Fig.
2, A and B). The
precision of the torsion angles is assessed in terms of the order
parameter S (29). The torsion angle order parameters ( Solution Structure of MPIF-1--
The structure of MPIF-1 consists
of an extended loop at the N terminus followed by three Dynamics--
The 15N T1, T2,
and NOE relaxation data could be obtained for 60 out of 73 expected
resonances (Fig. 3, A-C). The
15N T1, T2, and NOE relaxation data
were analyzed to describe the internal dynamics of MPIF-1 using the
model-independent formalism of Lipari-Szabo (30, 31). Relaxation data
for each residue were fitted to different models that include
S2
The generalized order parameters (S2), chemical
exchange (Rex), and local correlation time
( Heparin Binding--
The surface positively charged residues in
chemokines mediate binding to proteoglycans that are sulfated and are
highly negatively charged. MPIF-1 is highly basic (pI >9) and the
sequence analysis showed cluster of positively charged residues in the
40-s loop and the C-terminal Alternative splicing of the MPIF-1 gene results in two forms of
the protein that are 99 and 116 amino acids long, respectively (15).
The N-terminal domain of 32 and 49 amino acids in these two proteins is
exceptional compared with an average of 10 residues in most CC
chemokines. Both forms of the protein were shown to have similar
properties of myeloid progenitor inhibition and monocyte chemotaxis
(15), and further truncation to 76 residues results in substantial
increase in potency (16), an observation that suggests that the longer
versions could be considered as proproteins. Our data for MPIF-1 and
the full-length MPIF-1, corresponding to the 76 and 99 amino acid
versions of the protein, indicate that they are monomers, and the
latter actually binds to heparin with lesser affinity (Table III).
These observations suggest that there is no correlation between the
length of the N-terminal residues, the ability to form dimers, and
proteoglycan binding. One aspect of leukocyte activation is the release
of peptidases and proteases that could act on the chemokines and thus
modulate their activity and function. The differences in potency of the
variants of MPIF-1 may play an important role in vivo
because the recruitment and activation of leukocytes is highly
regulated spatially and temporally.
NMR structure, the rotational correlation time from 15N
dynamics, and ultracentrifugation studies under a variety of solution conditions indicate that MPIF-1 shows no propensity to dimerize and is
a monomer (Table I). MPIF-1 adopts a typical chemokine fold of three
-strands and an overlying
-helix. In addition to the four
cysteines that characterize most chemokines, MPIF-1 has two additional
cysteines that form a disulfide bond. The backbone dynamics indicate
that the disulfide bonds and the adjacent residues that include the functionally important N-terminal and N-terminal loop residues show
significant dynamics. MPIF-1 is a highly basic protein (pI >9), and
the structure reveals distinct positively charged pockets that could be
correlated to proteoglycan binding. MPIF-1 is processed from a longer
proprotein at the N terminus and the latter is also functional though
with reduced potency, and both proteins exist as monomers under a
variety of solution conditions. MPIF-1 is therefore unique because
longer proproteins of all other chemokines oligomerize in solution. The
MPIF-1 structure should serve as a template for future functional
studies that could lead to therapeutics for preventing
chemotherapy-associated myelotoxicity.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
8), a member of CC family, was initially identified in a large
scale sequencing effort and is constitutively expressed in liver, lung,
pancreas, and bone marrow (14). In addition to inhibiting colony
formation of bone marrow cells that give rise to granulocyte and
monocyte lineages, MPIF-1 is a potent activator of monocytes and
eosinophils (14, 15). Alternative splicing results in two forms of the
protein, named CK
8 and CK
8-1, that are 99 and 116 amino acids in
length, respectively (15). Further processing at the N terminus results
in a 76-residue protein, henceforth called MPIF-1, that was
significantly more active (16, 17). Cross-desensitization experiments
in both monocytes and eosinophils indicate that MPIF-1 binds
predominantly to the CCR1 receptor but incomplete desensitization also
suggests that additional receptor(s) may be involved (16). MPIF-1
induces a rapid dose-dependent release of arachidonic acid
from monocytes that is dependent on extracellular calcium and is
blocked by phospholipase A2 inhibitors. Furthermore,
phospholipase A2 activation is shown to be necessary for
filamentous actin formation in monocytes.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside, MPIF-1 was
produced as an insoluble protein in minimal media supplemented with
[15N]ammomium sulfate and [13C]glucose
(Cambridge Isotope Laboratory, Andover, MA) and was extracted and
refolded in 1.75 M guanidine HCl in the presence of 5 mM cysteine. The expressed protein carries an extra Met
(the initiation codon) in the N terminus that for simplicity is
considered as the first residue of MPIF-1. The protein was purified to
homogeneity by successive passages through strong cation (poros HS-50),
anion (poros HQ-50), and cation (poros CM-20) exchange columns and
finally through a size exclusion (Sephacryl S-100) column. The
full-length 99-amino acid MPIF-1 proprotein was cloned, expressed, and
purified as outlined for the MPIF-1 protein.
(Eq. 1)
is (r2
r02), H = (1
) (
2/2RT), Cr and
C0 are the concentrations at radius r
and r0, respectively, M is the
molecular weight of the monomer,
is the partial specific volume,
is the solvent density,
is the angular velocity of the rotor,
Ka is the association constant of the monomer-dimer equilibrium, and E is the baseline offset. Partial specific
volumes were calculated from the weight average of the partial specific volumes for individual amino acids. Data were fitted to the equation by
nonlinear least squares using the Microcal Origin 4.1 software provided
by Beckman for the XL-A. The quality of the fit was characterized by
2, the sum of the squares of the residuals, and
examination of the residuals for systematic deviation. The data were
fitted to a single species or to a monomer-dimer model. The molecular
weights calculated from the fit indicated that both proteins are
monomers under all solution conditions (Table I).
and MPIF-1 were expressed in
E. coli, interleukin-8 was synthesized from solid-phase
chemical synthesis, and HCC-2, MIP-1
, MCP-3, and RANTES were
purchased from R & D Systems.
, and C
chemical shifts were made on the
basis of HNCACB and CBCA(CO)NH experiments (19). The chemical shifts of
the side chain atoms were assigned on the basis of
15N-edited total correlation spectroscopy (TOCSY) (20) and
HCCH-TOCSY (21) experiments. A high-resolution two-dimensional
1H-1H nuclear Overhauser enhancement
spectroscopy (NOESY), TOCSY, and double-quantum-filtered correlated
spectroscopy experiments were used to assign the aromatic protons.
Inter-proton distances were derived from 15N-edited NOESY
(mixing time 50 ms and 150 ms) and
15N/13C-edited NOESY (mixing time 75 ms)
experiments (22). NOE buildup curve showed that the NOE intensities
were essentially linear for the mixing times used in generating the
distance restraints. NOE cross-peak intensities were classified as
strong, medium, weak, or very weak, corresponding to upper distance
restraints of 2.8, 3.5, 4.0, and 5.0 Å, respectively.
Restraints
were obtained from a HNHA experiment (23) and stereospecific assignment
of the
protons was obtained from 3J coupling constants
derived from a HACAHB experiment (24), and the relative intensities of
the NOEs from the NH and the C
H to C
H protons in NOESY spectra.
and 29
1) and 36 hydrogen
bonding restraints (from 18 hydrogen bonds) were used in the final
structure calculations. The initial structures were generated with NOE
restraints alone and, in subsequent structure calculations, the
dihedral and hydrogen-bond restraints were included. The simulated
annealing calculations were carried out using the standard force-field
parameter set and topology file in XPLOR version 3.1. A total of 50 structures were generated and the top 30 structures were selected on
the basis of the lowest energies.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Sedimentation equilibrium ultracentrifugation studies of MPIF-1 and
full-length MPIF-1
Structural statistics and r.m.s. differences for 30 calculated MPIF-1
structures
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Fig. 1.
A, superposition of the 30 simulated annealing structures about the average structure.
B, the same as in panel A except the N-terminal
(1-10) and the C-terminal (67) residues have been omitted for
clarity. C, a schematic representation of MPIF-1 in the same
orientation as shown in panel B created using the program
MOLMOL (52).
and
) for most of the residues in the structured region is >0.95,
an observation that indicates that the backbone of the structures is
well defined (Fig. 2, C and D). The order
parameter for
1 is shown in Fig. 2E. Fig. 2F
shows the solvent accessible area, and a low value implies that the
side chain is buried and inaccessible to the bulk solvent. These
residues tend to be hydrophobic and in general are highly structured as
evidenced by high order parameter for
1 > 0.9. One exception
of a large hydrophobe that shows a low S is
Ile13. It is solvent exposed and structure-function studies
have shown an essential role for this residue in receptor binding in
several CXC and CC chemokines. It is observed that all of
the torsion angles in all 30 structures fall in the favored region of
the Ramachandran plot. Seventy-two percent of the residues fall in the
core (most favored) region, 23% in the allowed region, and 1% in the
generous region.
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Fig. 2.
Atomic r.m.s. distribution of the 30 simulated annealing structures about the average structure best fitted
for residues 11-66 for the backbone atoms (A) and all
heavy atoms (B). Angular order parameter
(S) for (C),
(D), and
1
(E), and the fractional solvent accessible area
(F) are also shown.
strands and
a C-terminal
helix (Fig. 1B). The first 10 residues
preceding the CC motif show no or only sequential NOEs, have low order
parameters for
and
, and therefore lack defined structure. These
are followed by a N-terminal loop that contains a series of turns
(residues 13-20) that leads into a 310 helix (residues
21-24). The first
strand (residues 27-31) is connected by a type
III turn to the second
strand (residues 39-44), which is in turn
connected by a type I turn to the third
strand (residues 48-52).
The third strand leads into the helix (residues 56-66) via a type III
turn. Residues 67-77 are largely unstructured, a feature that is
consistent with the lack of long range NOEs, a few medium NOEs, and low
order parameters. Hydroxyl protons of Thr31 and
Thr44, located at the end of the 1st strand and 2nd strand,
respectively, are H-bonded to the backbone carbonyls across the strand
and therefore play a structural role. Of the six cysteines, four are
characteristic of all CC chemokines: Cys11 forms a
disulfide with Cys35, which is part of the turn linking
first and second
strands, and Cys12 forms a disulfide
bond with Cys50 in the third
strand. MPIF-1 has two
additional cysteines, Cys22 in the 310 helix
and Cys62 in the
-helix. We have created a data base of
cysteine chemical shifts and have observed that the C
shifts are uniquely different in the free and the disulfide bonded form
(53). The shifts of Cys22 and Cys62 indicate
that they are involved in disulfide bond formation. The structure
reveals that the cysteines are proximal to form a disulfide bond, and
this is also evident from the NMR properties such as the small coupling
constant, slow exchanging amide proton, and NOE pattern for
Cys62 that are characteristic of a helical residue. The
Cys12-Cys51 disulfide bond adopts a left-handed
twist in the majority of the structures whereas the other two disulfide
bonds are unstructured. The core of the structure is well defined by a
number of long-range hydrophobic contacts (Ile20,
Leu25, Tyr28, Phe29,
Val40, Phe42, Phe50,
Ala52, Val59, Met63, and
Leu66) between residues of the
-helix and
-strands.
Besides the disulfide bonds, long range NOEs between Thr31,
Gly39, Tyr15, Cys11,
Cys12, and Cys51 orient the N-terminal loop and
the N-terminal residues with respect to the core structure, which could
be essential for their function.
c (model 1),
S2
c
e (model 2),
S2
c
Rex
(model 3), S2
c
e
Rex (model 4), and a two-time scale model (model
5) that allow internal motions to occur at two distinct time scales
(32). The appropriate model was chosen for each residue, by evaluating
the quality of the fit. The optimal
c was initially
calculated on a per residue basis by minimizing the experimental and
the calculated T1, T2, and NOE values using the
isotropic spectral density function. For calculating the overall correlation time
c, 26 residues with either
15N-{1H} NOE values <0.65 and/or with
T1/T2 ratio outside 1 S.D. (Fig. 4D) from the mean were
excluded.
c was calculated to be 4.6 ± 0.2 ns on the
basis of the remaining 34 residues.
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Fig. 3.
15N dynamics of MPIF-1. The
15N T1, T2, NOE, and
T1/T2 ratio, are shown in panels A, B,
C, and D, respectively. Dynamics parameters calculated
from fitting the 15N T1, T2, and
NOE data are shown in the remaining panels. Order parameter
(S2); internal correlation time
(Te) and conformational exchange rate are shown in
panels E, F, and G, respectively.
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Fig. 4.
Structural comparison of MPIF-1 and other CC
chemokines. Structure of MPIF-1 (shown in magenta) is
superimposed on CC chemokine structures HCC-2 (A), MCP-3
(B), MIP-1 (C), and RANTES (D).
Residues 11 to 66 of MPIF-1, 11 to 66 of MIP-1
, 6 to 61 of HCC-2, 10 to 65 of RANTES, and 11 to 67 of MCP-3 were used for superimposition.
The remaining terminal residues are not displayed for clarity.
c) as a function of amino acid sequence are shown in Fig.
3, E-G. Generalized order parameters provide a measure of
the amplitude of internal motion, where S2 = 1 means that the given N-H bond vector is rigid and
S2 = 0 indicates that the motion is
unrestricted. Residues 67-77 in the C terminus and residues 1-10
preceding the first cysteine in the N terminus exhibit low order
parameters (S2 <0.7). Both termini are poorly
defined in the NMR structures, and the dynamics data confirm that these
residues are intrinsically mobile and that the lack of structure is not
due to lack of experimental restraints. Excluding the termini, the
average S2 value for residues 11-66 is
0.84 ± 0.06. Other residues that exhibit low order parameters are
Arg18 and Ile20 (S2 = 0.7), which are part of the N-terminal loop. Relaxation data for
residues Tyr15, Cys22, Thr44,
Cys51, Ala52, and Asn77 required an
exchange term (model 3 and 4) (Fig. 3G) and for C-terminal residues 69-73, the two time scale model.
-helix, regions that are known to play
a role in proteoglycan binding. The ability of MPIF-1 and other
chemokines to interact with proteoglycans was assessed by elution of
the chemokines from a heparin-POROS column with a linear NaCl gradient (Table III). MPIF-1 eluted from the
column at 0.30 M NaCl, and the full-length eluted at 0.25 M NaCl. The observation that full-length MPIF-1 actually
eluted at slightly lower NaCl concentration indicates that the
additional N-terminal residues do not play a role in this interaction.
MPIF-1 binds more tightly than MIP-1
and HCC-2, with comparable
affinity to MIP-1
, and less tightly than MCP-3, RANTES, and IL-8.
HCC-2 and MCP-3 are monomeric, IL-8 is dimeric, whereas MIP-1
,
MIP-1
, and RANTES are highly associated at neutral pH (>100 kDa).
For chemokines that are known to self-associate, proteoglycan binding
has been shown to promote oligomerization (7).
Heparin binding profiles of MPIF-1 and other chemokines
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-strands and an overlying
-helix. Several CC chemokine structures
including those of MIP-1
, RANTES, MCP-1, MCP-3, eotaxin, and HCC-1
have been elucidated (33-38). The secondary and tertiary structural
elements of MPIF-1 are similar to those observed in the other CC
chemokines although the sequence identity between MPIF-1 and these CC
chemokines varies from ~25 to 60% (Fig.
5). Superposition of the backbone of the
structured region (residues 11 to 66) of MPIF-1 and other CC chemokines
shows a r.m.s.d. from 1.5 to 2.0 Å. The structure of MPIF-1 is
compared with two monomeric CC chemokines, HCC-2 (A) and
MCP-3 (B), and two CC chemokines that oligomerize at neutral
pH, MIP-1
(C) and RANTES (D) in Fig. 4. The
lowest r.m.s.d. is observed for the structured regions (the strands and
the helix), and higher r.m.s.d. are observed for the N-terminal
residues, N-terminal loop, and the 30-s turn. However, the latter
regions show the largest sequence differences, and are also the regions
that are relatively less defined and functionally important. A
signature of most chemokines is the conserved four cysteines that form
disulfide bonds. The disulfide bonds have been shown to be essential
for structure and function. 15N dynamics data indicate that
some of the cysteines and the residues in the vicinity of all the
disulfide bonds show conformational exchange, suggesting that these
regions of the protein are mobile. The
Cys11-Cys35 disulfide bond shows the largest
segmental motion and it has been shown for example in IL-8, that subtle
perturbations to the disulfide bond result in significant loss of
function with no changes in structure (39). Interestingly, the dynamic
regions of the protein, the N-terminal region, the disulfide bond, and the 30-s turn, are the critical regions in ligand binding and receptor
activation (40-42). One characteristic difference between MPIF-1 and
other CC chemokines is a Gln at position 58 and a Trp in others. MPIF-1
structure reveal that the steric bulk of the indole side chain would be
in the way of the disulfide bond.
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Fig. 5.
The alignment of amino acid sequences of
MPIF-1 and related CC chemokines.
MPIF-1 binds to CCR1 with high affinity and has been shown to be the
most potent activator of monocytes (16, 17). MCP-3, RANTES, MIP-1,
and HCC-2 also bind and activate CCR1. Mutagenesis studies have
indicated that the N-terminal residues preceding the first cysteine and
the N-terminal loop residues (between the second cysteine and the
310 helix) play important roles in receptor binding for
both CXC and CC chemokines (43-46). In the MPIF-1
structure, the N-terminal loop residues are fairly well defined
(S for
,
> 0.95) and adopt a unique
conformation (Fig. 2). The 15N dynamics data, on the other
hand, indicate that this region of the protein is relatively mobile and
shows fluctuations in the subnanosecond and conformational exchange in
the slower millisecond time scale (Fig. 3). The N-terminal loop domain
is adjacent and proximal to all the disulfide bonds. Sequence analysis
reveals that Ile13, Tyr15, Arg18,
and Ile20 of MPIF-1 are conserved or similarly substituted
in other CC chemokines. Mutagenesis of these residues in those
chemokines results in reduced binding to their respective receptors.
Ile13, the first residue after the second cysteine, is
solvent exposed in all CXC and CC chemokines and most likely
plays a direct role in activation of receptors. The importance of the
N-terminal loop residues was observed in RANTES to be receptor
specific: Arg17 was necessary for binding to CCR1,
Phe12 for binding to CCR3, Phe12 and
Ile15 for binding to CCR5 (46). In most chemokine
structures, Tyr15 and Ile20 are buried and
adopt a similar conformation and are packed against other hydrophobic
residues indicating a structural role. These observations suggest that
in MPIF-1, Ile13 and Arg18 play a functional
role and are directly involved in receptor binding, and that
Tyr15 and Ile20 function as a part of the
structural scaffold.
The NMR structure (Fig. 1) and the dynamics data of MPIF-1 (Fig. 2)
indicate that the N-terminal residues preceding the first cysteine are
unstructured. All CXC chemokines that activate neutrophils have the characteristic ELR sequence that is essential for binding and
activation (43). Such a signature sequence is absent for CC chemokines
and sequence analysis does not provide any insight into receptor
specificity. A comparison of the N-terminal residues of MPIF-1 with
other CC chemokines that bind CCR1 (HCC-2, MCP-3, RANTES, and MIP-1)
shows very little or no similarity. HCC-2 shows the highest overall
sequence homology to MPIF-1 (~60%) but none in the N terminus.
Interestingly, it has been shown recently that it is the length of the
N-terminal domain, and not the nature of the side chain, that is
critical for MCP-1 binding to CCR2 (47).
It is well established that the chemokines immobilized on proteoglycans
play a role in leukocyte recruitment by forming a concentration
gradient on the endothelial cell surface. Proteoglycans are also found
on leukocytes, and chemokines bind to these proteoglycans and mediate
the activation of the leukocyte 7-transmembrane receptor. Removal of
leukocyte cell surface GAG by glycosaminidases reduces chemokine
binding, thus modulating the kinetics of binding to the 7-transmembrane
receptor. Sequence analysis indicates a distinct charge distribution
for MPIF-1 compared with other chemokines. Binding of MPIF-1 and other
chemokines to proteoglycans has been measured in terms of the
concentration of NaCl required to elute the bound chemokine from a
heparin-Sepharose column (Table III). The mutagenesis studies in
different chemokines suggest that a combination of positively charged
residues in the N-terminal loop, 40-s loop, C-terminal -helix, and
first
-strand participate in proteoglycan binding (13, 48-51).
MPIF-1 has four charged residues, two lysines and two arginines, in the
40-s loop whereas others have three or less, and also a cluster of
positive charges in the C-terminal tail (Table III). The structure of
MPIF-1 reveals that the C-terminal
-helix extends up to residue 66, and the remaining 10 residues that contain a cluster of positive
charges is unstructured. Orientation of MPIF-1 that highlights the
positive surface charge distribution is shown in Fig.
6A, and it is observed that
the opposite face is actually negatively charged (Fig. 6B). Although MIP-1
shows similar functional properties as MPIF-1, the
helix in MIP-1
is actually negatively charged, and binds through the
40-s and the N-terminal loops (Fig. 6D). On the other hand,
the helix is positively charged in IL-8, and is involved in binding to
proteoglycans (Fig. 6C). There is no correlation between the
net positive charge (number of positively charged residues minus the
negatively charged residues), and the strength of binding indicating
that only a few amino acids participate in binding. Furthermore,
chemokines can bind to proteoglycans as dimers or oligomers
(e.g. RANTES) or could oligomerize on binding to
proteoglycans (7). We propose that MPIF-1, on the basis of the
structure, self-association properties, and heparin binding, binds as a
monomer through the 40-s loop and the
-helix, although we cannot
strictly rule out that it could dimerize on binding to proteoglycans.
This model is consistent with the differential functional properties of
the full-length MPIF-1 and MPIF-1 as the former binds heparin less
tightly and is less active.
|
Both MPIF-1 and the full-length MPIF-1 suppress progenitor cell
proliferation. Both CC and CXC chemokines, such as MIP-1, IL-8,
GRO-
, platelet factor-4, IP-10, and MCP-1 have been shown to
suppress proliferation of progenitor cell whereas related chemokines such as GRO-
, neutrophil activating protein-2, MIP-1
, and RANTES are nonsuppressive. The chemokine activity profile clearly suggests that the ability to modulate progenitor cell proliferation is not
related to their binding to their cognate chemokine receptors in
vitro, and the molecular basis for this function is not clear. Oligomerization clearly plays a role as a monomeric form of MIP-1
is
significantly more potent in suppressing stem cell proliferation (12).
MPIF-1 is naturally monomeric, and the knowledge of the structure,
dynamics, and functional properties could lead to better therapeutics
aimed at preventing chemotherapy-associated myelotoxicity for patients
undergoing chemotherapy.
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ACKNOWLEDGEMENTS |
---|
We thank Protein Engineering Network Centers of Excellence, Edmonton, Canada, for access to NMR instrumentation. We thank Dr. Brian Sykes for support and encouragement, Dr. Leo Spyracopoulos for help during NMR data collection, Dr. Jim Lee and Chris Chin for ultracentrifugation studies, Dr. Yuan Xu for figures, Olga Galperina for technical assistance, and Regina Grochowski for editorial assistance.
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FOOTNOTES |
---|
* The purchase of NMR instruments was funded by the Government of Canada's Network of Centers of Excellence Program and the Medical Research Council of Canada. The ultracentrifuge core was supported by National Institutes of Health Grant RR08961.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.
The atomic coordinates and the structure factors (code 1G91) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/). .
§ To whom correspondence should be addressed. Tel.: 409-772-2238; Fax: 409-772-1790; E-mail: krishna@hbcg.utmb.edu.
Published, JBC Papers in Press, November 1, 2000, DOI 10.1074/jbc.M005085200
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ABBREVIATIONS |
---|
The abbreviations used are: MPIF-1, myeloid progenitor inhibitory factor-1; HCC-2, human CC chemokine 2; HSQC, heteronuclear single quantum coherence; IL-8, interleukin-8; MCP, monocyte chemoattractant protein; MIP, macrophage inflammatory protein; NOE, nuclear Overhauser enhancement; NOESY, two-dimensional nuclear Overhauser enhancement spectroscopy; RANTES, regulated upon activation, normal T-cell expressed and secreted; r.m.s.d., root mean square deviation; TOCSY, total correlation spectroscopy.
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