Format

Send to

Choose Destination
J Biol Chem. 2016 Feb 5;291(6):3100-13. doi: 10.1074/jbc.M115.681759. Epub 2015 Dec 16.

Identification of the Glycosaminoglycan Binding Site of Interleukin-10 by NMR Spectroscopy.

Author information

1
From the Institute of Medical Physics and Biophysics, University of Leipzig, Härtelstrasse 16/18, 04107 Leipzig, Germany and.
2
From the Institute of Medical Physics and Biophysics, University of Leipzig, Härtelstrasse 16/18, 04107 Leipzig, Germany and the Institute of Pharmacy, Medicinal Chemistry, Freie Universität Berlin, Königin-Luise-Strasse 2+4, 14195 Berlin, Germany.
3
From the Institute of Medical Physics and Biophysics, University of Leipzig, Härtelstrasse 16/18, 04107 Leipzig, Germany and daniel.huster@medizin.uni-leipzig.de.

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.

KEYWORDS:

carbohydrate function; extracellular matrix; glycosaminoglycan; heparin; interleukin-10; nuclear magnetic resonance (NMR); paramagnetic NMR; pseudocontact shifts

PMID:
26677224
PMCID:
PMC4742770
DOI:
10.1074/jbc.M115.681759
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for HighWire Icon for PubMed Central
Loading ...
Support Center