Solution Structure and Dynamics of Myeloid Progenitor Inhibitory Factor-1 (MPIF-1), A Novel Monomeric CC Chemokine*

Krishna RajarathnamDagger §, Yuling Li, Thomas Rohrer, and Reiner Gentz

From the Dagger  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



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta -strands and an overlying alpha -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

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 CKbeta 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 CKbeta 8 and CKbeta 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.

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.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-beta -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.

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,


C<SUB>r</SUB>=C<SUB>0</SUB> <UP>exp</UP>(MH&dgr;)+C<SUP>2</SUP><SUB>0</SUB>K<SUB>a</SUB><UP>exp</UP>(2MH&dgr;)+E (Eq. 1)

where delta  is (r2 - r02), H = (1 - upsilon rho ) (omega 2/2RT), Cr and C0 are the concentrations at radius r and r0, respectively, M is the molecular weight of the monomer, upsilon  is the partial specific volume, rho  is the solvent density, omega  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 chi 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).

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-1alpha and MPIF-1 were expressed in E. coli, interleukin-8 was synthesized from solid-phase chemical synthesis, and HCC-2, MIP-1beta , MCP-3, and RANTES were purchased from R & D Systems.

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, Calpha , and Cbeta 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. phi  Restraints were obtained from a HNHA experiment (23) and stereospecific assignment of the beta  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 Calpha H to Cbeta H protons in NOESY spectra.

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 phi  and 29 chi 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.

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).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


                              
View this table:
[in this window]
[in a new window]
 
Table I
Sedimentation equilibrium ultracentrifugation studies of MPIF-1 and full-length MPIF-1

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.


                              
View this table:
[in this window]
[in a new window]
 
Table II
Structural statistics and r.m.s. differences for 30 calculated MPIF-1 structures



View larger version (32K):
[in this window]
[in a new window]
 
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).

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 (phi  and psi ) 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 chi 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 chi 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.



View larger version (56K):
[in this window]
[in a new window]
 
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 phi  (C), psi  (D), and chi 1 (E), and the fractional solvent accessible area (F) are also shown.

Solution Structure of MPIF-1-- The structure of MPIF-1 consists of an extended loop at the N terminus followed by three beta  strands and a C-terminal alpha  helix (Fig. 1B). The first 10 residues preceding the CC motif show no or only sequential NOEs, have low order parameters for phi  and psi , 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 beta  strand (residues 27-31) is connected by a type III turn to the second beta  strand (residues 39-44), which is in turn connected by a type I turn to the third beta  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 beta  strands, and Cys12 forms a disulfide bond with Cys50 in the third beta  strand. MPIF-1 has two additional cysteines, Cys22 in the 310 helix and Cys62 in the alpha -helix. We have created a data base of cysteine chemical shifts and have observed that the Cbeta 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 alpha -helix and beta -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.

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 - tau c (model 1), S2 - tau c - tau e (model 2), S2 - tau c - Rex (model 3), S2 - tau c - tau 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 tau 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 tau 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. tau c was calculated to be 4.6 ± 0.2 ns on the basis of the remaining 34 residues.



View larger version (21K):
[in this window]
[in a new window]
 
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.



View larger version (38K):
[in this window]
[in a new window]
 
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-1beta (C), and RANTES (D). Residues 11 to 66 of MPIF-1, 11 to 66 of MIP-1beta , 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.

The generalized order parameters (S2), chemical exchange (Rex), and local correlation time (tau 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.

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 alpha -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-1alpha and HCC-2, with comparable affinity to MIP-1beta , and less tightly than MCP-3, RANTES, and IL-8. HCC-2 and MCP-3 are monomeric, IL-8 is dimeric, whereas MIP-1alpha , MIP-1beta , 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).


                              
View this table:
[in this window]
[in a new window]
 
Table III
Heparin binding profiles of MPIF-1 and other chemokines



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta -strands and an overlying alpha -helix. Several CC chemokine structures including those of MIP-1beta , 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-1beta (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.



View larger version (31K):
[in this window]
[in a new window]
 
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-1alpha , 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 phi , psi  > 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-1alpha ) 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 alpha -helix, and first beta -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 alpha -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-1alpha shows similar functional properties as MPIF-1, the helix in MIP-1alpha 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 alpha -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.



View larger version (49K):
[in this window]
[in a new window]
 
Fig. 6.   Surface charge distribution of MPIF-1 (A), MPIF-1 after rotating 180° (B), IL-8 (C), and MIP-1alpha (D) created using the program MOLMOL (52). The orientation is essentially the same as shown in Fig. 4. Positive and negatively charged regions are shown in blue and red, respectively.

Both MPIF-1 and the full-length MPIF-1 suppress progenitor cell proliferation. Both CC and CXC chemokines, such as MIP-1alpha , IL-8, GRO-beta , platelet factor-4, IP-10, and MCP-1 have been shown to suppress proliferation of progenitor cell whereas related chemokines such as GRO-alpha , neutrophil activating protein-2, MIP-1beta , 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-1alpha 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.


    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.


    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


    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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


1. Baggiolini, M., Dewald, B., and Moser, B. (1997) Annu. Rev. Immunol. 15, 675-705[CrossRef][Medline] [Order article via Infotrieve]
2. Rollins, B. J. (1997) Blood 90, 909-928[Free Full Text]
3. Luster, A. D. (1998) N. Engl. J. Med. 338, 436-445[Free Full Text]
4. Rot, A. (1992) Immunol. Today 13, 291-294[CrossRef][Medline] [Order article via Infotrieve]
5. 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]
6. Ali, S., Palmer, A. C. V., Banerjee, B., Fritchley, S. J., and Kirby, J. A. (2000) J. Biol. Chem. 275, 11721-11727[Abstract/Free Full Text]
7. 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]
8. Rajarathnam, K., Sykes, B. D., Kay, C. M., Dewald, B., Geiser, T., Baggiolini, M., and Clark-Lewis, I. (1994) Science 264, 90-92[Medline] [Order article via Infotrieve]
9. Rajarathnam, K., Kay, C. M., Dewald, B., Wolf, M., Baggiolini, M., Clark-Lewis, I., and Sykes, B. D. (1997) J. Biol. Chem. 272, 1725-1729[Abstract/Free Full Text]
10. Laurence, J. S., Blanpain, C., Burgner, J. W., Parmentier, M., and LiWang, P. J. (2000) Biochemistry 39, 3401-3409[CrossRef][Medline] [Order article via Infotrieve]
11. Paavola, C. D., Hemmerich, S., Grunberger, D., Polsky, I., Bloom, A., Freedman, R., Mulkins, M., Bhakta, S., McCarley, D., Wiesent, L., Wong, B., Jarnagin, K., and Handel, T. M. (1998) J. Biol. Chem. 273, 33157-33165[Abstract/Free Full Text]
12. Czaplewski, L. G., McKeating, J., Craven, C. J., Higgins, L. D., Appay, V., Brown, A., Dudgeon, T., Howard, L. A., Meyers, T., Owen, J., Palan, S. R., Tan, P., Wilson, G., Woods, N. R., Heyworth, C. M., Lord, B. I., Brotherton, D., Christison, R., Craig, S., Cribbes, S., Edwards, R. M., Evans, S. J., Gilbert, R., Morgan, P., Hunter, M. G., Randle, E., Schofield, N., Varley, P., Fisher, J., and Waltho, J. (1999) J. Biol. Chem. 274, 16077-16084[Abstract/Free Full Text]
13. Koopmann, W., and Krangel, M. S. (1997) J. Biol. Chem. 272, 10103-10109[Abstract/Free Full Text]
14. Patel, V. P., Kreider, B. L., Li, Y., Li, H., Leung, K., Salcedo, T., Nardelli, B., Pippalla, V., Gentz, S., Thotakura, R., Parmelee, D., Gentz, R., and Garotta, G. (1997) J. Exp. Med. 185, 1163-1172[Abstract/Free Full Text]
15. Youn, B.-S., Zhang, S. M., Broxmeyer, H. E., Cooper, S., Antol, K., Fraser, M., Jr., and Kwon, B. S. (1998) Blood 91, 3118-3126[Abstract/Free Full Text]
16. Nardelli, B., Tiffany, H. L., Bong, G. W., Yourey, P. A., Morahan, D. K., Li, Y., Murphy, P. M., and Aldrson, R. F. (1999) J. Immunol. 162, 435-444[Abstract/Free Full Text]
17. Berkhout, T. A., Gohil, J., Gonzalez, P., Nicols, C. L., Moores, K. E., Macphee, C. H., White, J. R., and Groot, P. H. E. (2000) Biochem. Pharmacol. 59, 591-596[CrossRef][Medline] [Order article via Infotrieve]
18. Wishart, D. S., Bigam, C. G., Yao, J., Abildgaard, F., Dyson, H. J., Oldfield, E., Markley, J. L., and Sykes, B. D. (1995) J. Biomol. NMR 6, 135-140[Medline] [Order article via Infotrieve]
19. Muhandiram, D. R., and Kay, L. E. (1994) J. Magn. Reson. 103, 203-216[CrossRef]
20. Zhang, O., Kay, L. E., Olivier, J. P., and Forman-Kay, J. D. (1994) J. Biomol. NMR 4, 845-858[Medline] [Order article via Infotrieve]
21. Kay, L. E., Xu, G. Y., Singer, A. U., Muhandiram, D. R., and Forman-Kay, J. D. (1993) J. Magn. Reson. B 101, 333[CrossRef]
22. Pascal, S. M., Ratliff, N., and Kay, L. E. (1994) J. Magn. Reson. B 103, 197-201[CrossRef]
23. Kuboniwa, H., Tjandra, N., Grzesiek, S., Ren, H., Klee, C. B., and Bax, A. (1995) Nat. Struct. Biol. 2, 768[Medline] [Order article via Infotrieve]
24. Grzesiek, S., Kuboniwa, H., Hinck, A. P., and Bax, A. (1995) J. Am. Chem. Soc. 117, 5312-5315
25. Delaglio, F., Gresiek, S., Vuister, G. W., Zhu, G., Pfeifer, J., and Bax, A. (1995) J. Biomol. NMR 6, 277-293[Medline] [Order article via Infotrieve]
26. Brünger, A. T. (1993) XPLOR Version 3.1. Manual , Yale University, New Haven, CT
27. Farrow, N. A., Muhandiram, R., Singer, A. U., Pascal, S. M., Kay, C. M., Gish, G., Shoelson, S. E., Pawson, T., Forman-Kay, J. D., and Kay, L. E. (1994) Biochemistry 33, 5984-6003[Medline] [Order article via Infotrieve]
28. Laskowski, R. A., MacArthur, M. W., Moss, D. S., and Thornton, J. M. (1993) J. Appl. Crystallogr. 26, 283-291[CrossRef]
29. Hyberts, S. G., Goldberg, M. S., Havel, T. F., and Wagner, G. (1992) Protein Sci. 1, 736-751[Abstract/Free Full Text]
30. Lipari, G., and Szabo, A. (1982a) J. Am. Chem. Soc. 104, 4546-4559
31. Lipari, G., and Szabo, A. (1982b) J. Am. Chem. Soc. 104, 4559-4570
32. Clore, G., Szabo, A., Bax, A., Kay, L. E., Driscoll, P. C., and Gronenborn, A. M. (1990) J. Am. Chem. Soc. 112, 4989-4991
33. Lodi, P. J., Garrett, D. S., Kuszewski, J., Tsang, M. L., Weatherbee, J. A., Leonard, W. J., Gronenborn, A. M., and Clore, G. M. (1994) Science 263, 1762-1767[Medline] [Order article via Infotrieve]
34. Skelton, N. J., Aspiras, F., Ogez, J., and Schall, T. J. (1995) Biochemistry 34, 5329-5342[Medline] [Order article via Infotrieve]
35. Handel, T. M., and Domaille, P. J. (1996) Biochemistry 35, 6569-6584[CrossRef][Medline] [Order article via Infotrieve]
36. Kim, K. S., Rajarathnam, K., Clark-Lewis, I., and Sykes, B. D. (1996) FEBS Lett. 395, 277-282[CrossRef][Medline] [Order article via Infotrieve]
37. Crump, M. P., Rajarathnam, K., Kim, K. S., Clark-Lewis, I., and Sykes, B. D. (1998) J. Biol. Chem. 273, 22471-22479[Abstract/Free Full Text]
38. Sticht, H., Escher, S. E., Schweimer, K., Forssmann, W.-G., Rosch, P., and Adermann, K. (1999) Biochemistry 38, 5995-6002[CrossRef][Medline] [Order article via Infotrieve]
39. Rajarathnam, K., Dewald, B., Baggiolini, M., Sykes, B. D., and Clark-Lewis, I. (1999) Biochemistry 38, 7653-7658[CrossRef][Medline] [Order article via Infotrieve]
40. Crump, M. P., Spyracopoulos, L., Lavigne, P., Kim, K. S., Clark-Lewis, I., and Sykes, B. D. (1999) Protein Sci. 8, 2041-2054[Abstract]
41. Ye, J., Mayer, K. L., and Stone, M. J. (1999) J. Biomol. NMR 15, 115-124[CrossRef][Medline] [Order article via Infotrieve]
42. LiWang, A. C., Cao, J. J., Zheng, H., Lu, Z., Peiper, S. C., and LiWang, P. J. (1999) Biochemistry 38, 442-453[CrossRef][Medline] [Order article via Infotrieve]
43. Clark-Lewis, I., Kim, K. S., Rajarathnam, K., Gong, J.-H., Dewald, B., Moser, B., Baggiolini, M., and Sykes, B. D. (1995) J. Leukocyte Biol. 57, 703-711[Abstract]
44. Gong, J.-H., and Clark-Lewis, I. (1995) J. Exp. Med. 181, 631-640[Abstract]
45. Moser, B., Dewald, B., Barella, L., Schumacher, C., Baggiolini, M., and Clark-Lewis, I. (1993) J. Biol. Chem. 268, 7125-7128[Abstract/Free Full Text]
46. Pakianathan, D. R., Kuta, E. G., Artis, D. R., Skelton, N. J., and He'bert, C. A. (1997) Biochemistry 36, 9642-9648[CrossRef][Medline] [Order article via Infotrieve]
47. Jarnagin, K., Grunberger, D., Mulkins, M., Wong, B., Hemmerich, S., Paavola, C., Bloom, A., Bhakta, S., Diehl, F., Freedman, R., McCarley, D., Polsky, I., Ping-Tsou, A., Kosaka, A., and Handel, T. M. (1999) Biochemistry 38, 16167-16177[CrossRef][Medline] [Order article via Infotrieve]
48. Kuschert, G. S. V., Hoogewerf, A. J., Proudfoot, A. E. I., Chung, C., Cooke, R. M., Hubbard, R. E., Wells, T. N. C., and Sanderson, P. N. (1998) Biochemistry 37, 11193[CrossRef][Medline] [Order article via Infotrieve]
49. Chakravarty, L., Rogers, L., Quach, T., Breckenridge, S., and Kolattukudy, P. E. (1998) J. Biol. Chem. 273, 29641-29647[Abstract/Free Full Text]
50. Koopmann, W., Ediriwickrema, C., and Krangel, M. S. (1999) J. Immunol. 163, 2120-2127[Abstract/Free Full Text]
51. 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[Abstract/Free Full Text]
52. Koradi, R., Billeter, M., and Wüthrich, K. (1996) J. Mol. Graph. 14, 29-42
53. Sharma, D., and Rajarathnam, K. (2000) J. Biomol. NMR 18, 165-171[CrossRef][Medline] [Order article via Infotrieve]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.