Abstract
The biological function of interleukin-10 (IL-10), a pleiotropic cytokine with an essential role in inflammatory processes, is known to be affected by glycosaminoglycans (GAGs). GAGs are highly negatively charged polysaccharides and integral components of the extracellular matrix with important functions in the biology of many growth factors and cytokines. The molecular mechanism of the IL-10/GAG interaction is unclear. In particular, experimental evidence about IL-10/GAG binding sites is lacking, despite its importance for understanding the biological role of the interaction. Here, we report the experimental determination of a GAG binding site of IL-10. Although no co-crystal structure of the IL-10·GAG complex could be obtained, its structural characterization was possible by NMR spectroscopy. Chemical shift perturbations of IL-10 induced by GAG binding were used to narrow down the location of the binding site and to assess the affinity for different GAG molecules. Subsequent observation of NMR pseudocontact shifts of IL-10 and its heparin ligand, as induced by a protein-attached lanthanide spin label, provided structural restraints for the protein·ligand complex. Using these restraints, pseudocontact shift-based rigid body docking together with molecular dynamics simulations yielded a GAG binding model. The heparin binding site is located at the C-terminal end of helix D and the adjacent DE loop and coincides with a patch of positively charged residues involving arginines 102, 104, 106, and 107 and lysines 117 and 119. This study represents the first experimental characterization of the IL-10·GAG complex structure and provides the starting point for revealing the biological significance of the interaction of IL-10 with GAGs.
Highlights
Introduction of a Lanthanide Ion SpinLabel into IL-10 —In a second NMR approach, structural information on the IL-101⁄7GAG complex was obtained from measurement of pseudocontact shifts (PCSs) using a protein-attached paramagnetic spin label
Calculations were tried with two types of protein PCS data sets, one obtained from mixed IL-10-lanthanide binding tag (LBT) with one single lanthanide site and another one measured on uniform IL-10-LBT
This led to the determination of the crystal structure of mouse IL-10 at a resolution of 2.1 Å, which was deposited in the PDB under accession number 4X51
Summary
Materials—GAG oligosaccharides were purchased from Iduron (Manchester, UK). The proteins equine myoglobin, human carbonic anhydrase 1, and bovine carbonic anhydrase 2 were ordered from Sigma-Aldrich (Taufkirchen, Germany). A uniformly composed IL-10 dimer with two identical subunits, each carrying one LBT, was prepared This “uniform” protein was expressed unlabeled (for ligand PCS measurements) or with 15N labeling (for protein PCS measurements) and purified solely by StrepTactin affinity chromatography and gel filtration. Calculations were tried with two types of protein PCS data sets, one obtained from mixed IL-10-LBT with one single lanthanide site and another one measured on uniform IL-10-LBT. Because the symmetry of the PCS isosurfaces caused two degenerated solutions, including one model in which the ligand was placed away from the protein, a binding restraint was added to the calculation This was set up as a loose distance restraint between both molecules with an upper border of 20 Å using the NOE potential of Xplor-NIH. Rithms were used to keep rigid all bonds involving hydrogen atoms, allowing a 2-fs time step
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