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. 2013 Aug 30;288(35):25143-25153.
doi: 10.1074/jbc.M113.492579. Epub 2013 Jul 17.

Molecular basis of glycosaminoglycan heparin binding to the chemokine CXCL1 dimer

Krishna Mohan Poluri  1 , Prem Raj B Joseph  1 , Kirti V Sawant  2 , Krishna Rajarathnam  3
Affiliations

Molecular basis of glycosaminoglycan heparin binding to the chemokine CXCL1 dimer

Krishna Mohan Poluri et al. J Biol Chem. .

Abstract

Glycosaminoglycan (GAG)-bound and soluble chemokine gradients in the vasculature and extracellular matrix mediate neutrophil recruitment to the site of microbial infection and sterile injury in the host tissue. However, the molecular principles by which chemokine-GAG interactions orchestrate these gradients are poorly understood. This, in part, can be directly attributed to the complex interrelationship between the chemokine monomer-dimer equilibrium and binding geometry and affinities that are also intimately linked to GAG length. To address some of this missing knowledge, we have characterized the structural basis of heparin binding to the murine CXCL1 dimer. CXCL1 is a neutrophil-activating chemokine and exists as both monomers and dimers (Kd = 36 μm). To avoid interference from monomer-GAG interactions, we designed a trapped dimer (dCXCL1) by introducing a disulfide bridge across the dimer interface. We characterized the binding of GAG heparin octasaccharide to dCXCL1 using solution NMR spectroscopy. Our studies show that octasaccharide binds orthogonally to the interhelical axis and spans the dimer interface and that heparin binding enhances the structural integrity of the C-terminal helical residues and stability of the dimer. We generated a quadruple mutant (H20A/K22A/K62A/K66A) on the basis of the binding data and observed that this mutant failed to bind heparin octasaccharide, validating our structural model. We propose that the stability enhancement of dimers upon GAG binding regulates in vivo neutrophil trafficking by increasing the lifetime of "active" chemokines, and that this structural knowledge could be exploited for designing inhibitors that disrupt chemokine-GAG interactions and neutrophil homing to the target tissue.

Keywords: Chemokines; Glycobiology; Glycosaminoglycan; Heparin; NMR; Neutrophil.

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Figures

FIGURE 1.
FIGURE 1.
Sedimentation equilibrium analysis of wild-type CXCL1 in 50 mm phosphate and 50 mm NaCl (pH 6. 0) (A). The concentrations calculated in fringe displacement units (mg/ml) are plotted against the radius. Residuals of the corresponding least square fit are random, indicating the goodness of the fit. B, 1H-15N HSQC spectrum of WT-CXCL1 (120 μm) at pH 6.0 at 25 °C. The spectra show approximately twice the number of peaks than what is expected for a single species, indicating the presence of both the CXCL1 monomer and dimer.
FIGURE 2.
FIGURE 2.
Sequence alignment of two murine and seven human CXC neutrophil-activating chemokines (A). The murine chemokines are represented with their common names (mKC for CXCL1 and mMIP2 for CXCL2). The functional ELR sequence motif is shown in green. The conserved cysteine residues and their respective disulfide linkages are shown in red. The positively charged residues His-20, Lys-22, Lys-62, and Lys-66 (numbered with respect to mKC) are shown in blue. Residues Lys-28 in mKC, Arg-26 in CXCL8, and Asn-27 in CXCL1 represent the 2-fold symmetry and act as an anchor point for introducing the intermolecular disulfide bond (asterisk). B, schematic showing the design strategy for connecting two monomeric CXCL1 units using the K28C mutation. C, SDS-PAGE analysis of murine CXCL1 variants. The formation of intermolecular disulfide bond is evident for the CXCL1 dimer (dCXCL1) from the band at 16 kDa. M, protein marker; R, reduced; NR, non-reduced. D, HNCACB strips of all the cysteine residues. The Cβ shifts indicate that all form disulfides. E, the chemotactic activity of the WT and trapped dimer at 10 and 100 nm concentrations was measured using a Boyden chamber-type assay. The data were collected in quadruplicate, and the results are expressed as mean ± S.D. and are representative of three independent experiments.
FIGURE 3.
FIGURE 3.
1H-15N HSQC spectrum of dCXCL1 at 35 °C (A). Backbone N-H resonance assignments are shown for all the resolved peaks in the spectrum. The peaks enclosed in the rectangles correspond to the Asn/Gln side chain NH2 resonances. B, two-dimensional strip plots from the 15N-edited three-dimensional NOESY spectrum. Representative two-dimensional strips are shown for Lys-27, Val-29, Val-64, Gln-65, and Lys-66 amides. The characteristic interstrand intermolecular NOEs between the β1 and β′1 strand residues are highlighted as prime (′), and the proton type is shown in italics.
FIGURE 4.
FIGURE 4.
Structural features of the murine CXCL1 dimer. A, tertiary fold of a single monomeric subunit of the dCXCL1 dimer. The structural elements, including disulfide linkages and functionally important N loop and ELR motif regions, are highlighted. B, the individual monomers in the dimer are shown in cyan and pink. The Cys-28-Cys-28′ intermolecular disulfide bond is shown in yellow, and other disulfides are not shown for clarity. C, electrostatic surface representation of dCXCL1. All structures were generated using PYMOL. Blue, positively charged residues; red, negatively charged residues; white, hydrophobic residues).
FIGURE 5.
FIGURE 5.
Structural comparison of the monomeric subunit of dimeric murine CXCL1/mKC with other ELR-CXC chemokines. A, murine CXCL2/MIP2 (PDB code 3N52). B, human CXCL1/melanoma growth-stimulatory activity (PDB code 1MGS). C, human CXCL8/IL-8 (PDB code 3IL8).
FIGURE 6.
FIGURE 6.
A section of the 1H-15N HSQC spectrum of the free dCXCL1 (blue) and in the presence of heparin octasaccharide (red) (A). The arrow indicates the direction of binding-induced chemical shift changes. B, representative binding profile for calculating the binding constant of dCXCL1-octasaccharide (Octa) interactions. C, CSP map of dCXCL1-octasaccharide interactions. The horizontal line at 0.1 ppm represents the cutoff for a residue to be considered perturbed. The sequence-specific secondary structural elements are shown on the top of the CSP map with arrows for β sheets and cylinders for helices. D, molecular plot of the dCXCL1 residues that are significantly perturbed on octasaccharide binding. The perturbed residues are shown in red.
FIGURE 7.
FIGURE 7.
The 10 lowest energy-minimized structures of the dCXCL1-heparin octasaccharide complex calculated using HADDOCK 2.1. (A). B, magnification highlighting the electrostatic interactions between the positively charged residues (blue) of dCXCL1 and the negatively charged sulfate groups (yellow) of heparin octasaccharide (backbone in green). C, schematic of the electrostatic model of the dCXCL1-glycosaminoglycan complex. GAG is shown in red. Positively charged residues that are perturbed upon GAG binding are shown in blue. Other residues that are perturbed are shown in yellow. D, electrostatic surface representation of the quadruple mutant dCXCL1-M4. Blue, positively charged residues; red, negatively charged residues; white, hydrophobic residues. E, overlay of a section of the 1H-15N HSQC spectrum of dCXCL1-M4. Blue, dCXCL1-M4; red, dCXCL1-M4 + heparin octasaccharide (ratio ∼1:11).
FIGURE 8.
FIGURE 8.
1H-15N heteronuclear steady-state NOE relaxation measurements of dCXCL1 (blue) and dCXCL1-octasaccharide complex (red). B, 1H-15N HSQC spectra of free dCXCL1 (blue) and the dCXCL1-octasaccharide complex (red) after initiating hydrogen exchange with 100% D2O at pH 6.0 and 25 °C (experiment time, 90 min).
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