Structural basis, stoichiometry, and thermodynamics of binding of the chemokines KC and MIP2 to the glycosaminoglycan heparin

Abstract

Keratinocyte-derived chemokine (KC or mCXCL1) and macrophage inflammatory protein 2 (MIP2 or mCXCL2) play nonredundant roles in trafficking blood neutrophils to sites of infection and injury. The functional responses of KC and MIP2 are intimately coupled to their interactions with glycosaminoglycans (GAGs). GAG interactions orchestrate chemokine concentration gradients and modulate receptor activity, which together regulate neutrophil trafficking. Here, using NMR, molecular dynamics (MD) simulations, and isothermal titration calorimetry (ITC), we characterized the molecular basis of KC and MIP2 binding to the GAG heparin. Both chemokines reversibly exist as monomers and dimers, and the NMR analysis indicates that the dimer binds heparin with higher affinity. The ITC experiments indicate a stoichiometry of two GAGs per KC or MIP2 dimer and that the enthalpic and entropic contributions vary significantly between the two chemokine-heparin complexes. NMR-based structural models of heparin-KC and heparin-MIP2 complexes reveal that different combinations of residues from the N-loop, 40s turn, β₃-strand, and C-terminal helix form a binding surface within a monomer and that both conserved residues and residues unique to a particular chemokine mediate the binding interactions. MD simulations indicate significant residue-specific differences in their contribution to binding and affinity for a given chemokine and between chemokines. On the basis of our observations that KC and MIP2 bind to GAG via distinct molecular interactions, we propose that the differences in these GAG interactions lead to differences in neutrophil recruitment and play nonoverlapping roles in resolution of inflammation.

Keywords: chemokine; glycosaminoglycan; heparin; isothermal titration calorimetry (ITC); molecular dynamics; nuclear magnetic resonance (NMR).

Figure 1.

The dimer is the high-affinity GAG ligand. A and B, sections of the ¹H-¹⁵N HSQC spectra showing the overlay of KC (A) and MIP2 (B) in the free (black) and heparin-bound (red) forms. Dimer and monomer peaks are indicated as D and M. The monomer peaks disappear in both spectra on heparin dp26 binding, indicating that the dimer is the high-affinity GAG ligand. The spectra were collected using 20 μm KC and 15 μm MIP2 samples in 50 mm sodium phosphate, pH 6.0, at 25 °C.

Figure 2.

Thermodynamics of chemokine–heparin interactions. A–D, isotherms corresponding to the binding of heparin dp8 to WT MIP2 (A), WT KC (B), MIP2 dimer (C), and KC dimer (D). The titrations and the integrated data obtained after subtracting the heat of dilution are shown in the upper and lower panels, respectively. The titrations were performed in 50 mm sodium phosphate, pH 6.0, at 25 °C.

Figure 3.

Binding of MIP2 to the heparin dp8. A, sections of the ¹H-¹⁵N HSQC spectra showing overlay of MIP2 in the free (black) and dp8-bound (red) forms. Arrows indicate direction of the peak movement. B, histogram of heparin dp8 binding-induced chemical shift changes as a function of MIP2 residue number. Basic residues are in blue, and nonbasic residues are in red. The spectra were collected using a 100 μm MIP2 sample in 50 mm sodium phosphate, pH 6.0, at 35 °C.

Figure 4.

Binding of KC to the heparin dp14. A, sections of the ¹H-¹⁵N HSQC spectra showing the overlay of KC in the free (black) and dp14-bound (red) forms. Arrows indicate direction of the peak movement. B, histogram of heparin dp14 binding-induced chemical shift changes as a function of KC residue number. Basic residues are in blue, and nonbasic residues are in red. The spectra were collected using a 100 μm KC sample in 50 mm sodium phosphate, pH 6.0, at 35 °C.

Figure 5.

Sequences of mouse and human neutrophil activating chemokines. Heparin-binding basic residues of KC and MIP2 are highlighted in red, shaded in gray, and numbered. Arg-17 and Lys-69 of MIP2 are highlighted in blue. Conserved basic residues of human neutrophil activating chemokines implicated in heparin binding are labeled B1–B8, highlighted in red, and shaded in gray. Similar to mouse chemokines, chemokine-specific residues involved in heparin binding are in blue.

Figure 6.

MIP2-heparin binding models. A–D, structural depiction of model I. In A and B, the MIP2 dimer is shown in ribbon representation, and GAG is shown as sticks. A shows the binding of heparin to one of the monomers of the MIP2 dimer. B shows how the individual basic residues engage the heparin acidic groups. C highlights the GAG-binding residues (blue) in a surface presentation. In D, GAG-binding residues are labeled on the electrostatic surface.

Figure 7.

KC-heparin binding models. A–D, structural depictions of model I. In A and B, the KC dimer is shown in ribbon representation, and GAG is shown as sticks. A shows the binding of heparin to one monomer of the KC dimer. B shows a close-up of how the individual basic residues engage the heparin acidic groups. C highlights the GAG-binding residues (blue) in a surface presentation. In D, GAG-binding residues are labeled on the electrostatic surface.

Figure 8.

H-bonding properties from MD simulations. A and C, occupancy of direct H-bond interactions between MIP2 and heparin (A) and between KC and heparin (C). B and D, occupancy of water-mediated H-bond interactions between MIP2 and heparin (B) and KC and heparin (D).

Figure 9.

Binding energetics from MD simulations. A and B, single-residue energy decomposition values for MIP2–heparin (A) and KC–heparin (B) complexes. The highest to lowest energy are shown from red to blue. The error bar represents the standard deviation.

Figure 10.

Heparin-bound chemokine cannot bind the receptor. NMR data for MIP2 and KC are shown in the left and right panels, respectively. A and F show the overlay of a selected region of ¹H-¹⁵N HSQC spectra showing free chemokine (black), CXCR2 N domain–bound form (green), heparin-bound form (blue), and on titrating CXCR2 N domain to the heparin-bound chemokine (red). Individual spectra of free MIP2 and KC (B and G), CXCR2 N domain–bound MIP2 and KC (C and H), heparin-bound MIP2 and KC (D and I), and on titrating the CXCR2 N domain to the heparin-bound (E and J) are also shown.