RESUMO
Glycosaminoglycans (GAGs) are essential functional components of the extracellular matrix (ECM). Artificial GAGs like sulfated hyaluronan (sHA) exhibit pro-osteogenic properties and boost healing processes. Hence, they are of high interest for supporting bone regeneration and wound healing. Although sulfated GAGs (sGAGs) appear intracellularly, the knowledge about intracellular effects and putative interaction partners is scarce. Here we used an affinity-purification mass spectrometry-based (AP-MS) approach to identify novel and particularly intracellular sGAG-interacting proteins in human bone marrow stromal cells (hBMSC). Overall, 477 proteins were found interacting with at least one of four distinct sGAGs. Enrichment analysis for protein localization showed that mainly intracellular and cell-associated interacting proteins were identified. The interaction of sGAG with α2-macroglobulin receptor-associated protein (LRPAP1), exportin-1 (XPO1), and serine protease HTRA1 (HTRA1) was confirmed in reverse assays. Consecutive pathway and cluster analysis led to the identification of biological processes, namely processes involving binding and processing of nucleic acids, LRP1-dependent endocytosis, and exosome formation. Respecting the preferentially intracellular localization of sGAG in vesicle-like structures, also the interaction data indicate sGAG-specific modulation of vesicle-based transport processes. By identifying many sGAG-specific interacting proteins, our data provide a resource for upcoming studies aimed at molecular mechanisms and understanding of sGAG cellular effects.
Assuntos
Glicosaminoglicanos/metabolismo , Serina Peptidase 1 de Requerimento de Alta Temperatura A/metabolismo , Carioferinas/metabolismo , Proteína Associada a Proteínas Relacionadas a Receptor de LDL/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Células Cultivadas , Cromatografia Líquida , Glicosaminoglicanos/química , Serina Peptidase 1 de Requerimento de Alta Temperatura A/química , Serina Peptidase 1 de Requerimento de Alta Temperatura A/isolamento & purificação , Humanos , Carioferinas/química , Carioferinas/isolamento & purificação , Proteína Associada a Proteínas Relacionadas a Receptor de LDL/química , Proteína Associada a Proteínas Relacionadas a Receptor de LDL/isolamento & purificação , Células-Tronco Mesenquimais/química , Células-Tronco Mesenquimais/metabolismo , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/isolamento & purificação , Espectrometria de Massas em Tandem , Proteína Exportina 1RESUMO
Glycosaminoglycans (GAG) as long, unbranched polysaccharides are major components of the extracellular matrix. Many studies provided additional evidence of a specific binding between mediators and sulfated GAG, at which the sulfation code-which means the number and positions of sulfate groups along the polysaccharide chain-plays an important role. GAG from natural sources are very inhomogeneous regarding their sulfation patterns and molecular weight. Additionally, there is a high risk of contamination. This results in a growing interest in the careful characterization of native GAG and the synthesis of artificial GAG. Additionally, chemically oversulfated GAG analogues show many favorable properties. However, the structural characterization of these carbohydrates by mass spectrometry remains challenging. One significant problem is the sulfate loss during the ionization, which increases with the number of sulfate residues. We used the sulfated pentasaccharide fondaparinux as model substance to optimize sample preparation and measurement conditions, compared different established desalination methods and already existing protocols for sulfated oligosaccharides, and investigated their impact on the quality of the mass spectra. After optimization of the measurement conditions, we could establish a gentle and fast protocol for the mass spectrometry characterization of (fully) sulfated, artificial GAG-like oligosaccharides with minimized sulfate loss in the positive and negative ion mode. Here, the negative ion mode was more sensitive in comparison with the positive one, and fondaparinux species with sulfate loss were not detectable under the optimized conditions in the positive ion mode.
Assuntos
Heparina/análise , Heparina/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Sulfatos/análise , Sulfatos/química , Oligossacarídeos/químicaRESUMO
Supporting the wound healing process by sending the appropriate cytokine signals can shorten healing time and overcome chronic inflammation syndromes. Even though adhesion peptides consisting of Arg-Gly-Asp (RGD) are commonly used to enhance cell-surface interactions, peptide-mediated cytokine delivery has not been widely exploited so far. Cytokines interact with high affinity with their cognitive receptors but also with sulfated glycosaminoglycans (GAGs), both of which form a base for incorporation of cytokines into functional biomaterials. Here, we report on a mussel-derived surface coating as a prospective cytokine delivery system using covalently bound heparin mimetics, receptor-derived chemokine-binding peptides, and heparin-binding peptides (HBP). The latter enabled non-covalent immobilization of heparin on the surface followed by chemokine binding and release, whereas the former allowed direct non-covalent chemokine immobilization. The peptide displayed excellent binding to custom-made polystyrene 96-well plates, enabling convenient testing of several compounds. Released chemokine successfully induced migration in Jurkat cells, especially for the non-covalent heparin immobilization approach using HBPs as evaluated in a transwell assay. In comparison, heparin-mimetic coatings, comprised of sulfated peptides and GAG derivatives, proved less efficient with respect to amount of immobilized chemokine and migratory response. Thus, our study provides a roadmap for further rational optimization and translation into clinics.
Assuntos
Materiais Revestidos Biocompatíveis/química , Citocinas/farmacologia , Peptídeos/química , Cicatrização/efeitos dos fármacos , Animais , Bivalves/química , Movimento Celular/efeitos dos fármacos , Quimiocina CXCL12/farmacologia , Química Click , Di-Hidroxifenilalanina/química , Sistemas de Liberação de Medicamentos , Heparina/química , Humanos , Células Jurkat , Poliestirenos/química , Ligação Proteica/efeitos dos fármacos , Propriedades de SuperfícieRESUMO
Pathological healing characterized by abnormal angiogenesis presents a serious burden to patients' quality of life requiring innovative treatment strategies. Glycosaminoglycans (GAG) are important regulators of angiogenic processes. This experimental and computational study revealed how sulfated GAG derivatives (sGAG) influence the interplay of vascular endothelial growth factor (VEGF)165 and its heparin-binding domain (HBD) with the signaling receptor VEGFR-2 up to atomic detail. There was profound evidence for a HBD-GAG-HBD stacking configuration. Here, the sGAG act as a "molecular glue" leading to recognition modes in which sGAG interact with two VEGF165-HBDs. A 3D angiogenesis model demonstrated the dual regulatory role of high-sulfated derivatives on the biological activity of endothelial cells. While GAG alone promote sprouting, they downregulate VEGF165-mediated signaling and, thereby, elicit VEGF165-independent and -dependent effects. These findings provide novel insights into the modulatory potential of sGAG derivatives on angiogenic processes and point towards their prospective application in treating abnormal angiogenesis.
Assuntos
Glicosaminoglicanos/metabolismo , Ácido Hialurônico/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Sítios de Ligação , Sulfatos de Condroitina/farmacologia , Simulação por Computador , Glicosaminoglicanos/química , Células Endoteliais da Veia Umbilical Humana , Humanos , Proteínas Imobilizadas/metabolismo , Modelos Moleculares , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Neovascularização Fisiológica , Fosforilação , Domínios Proteicos , Esferoides Celulares , Relação Estrutura-Atividade , Ressonância de Plasmônio de Superfície , Fator A de Crescimento do Endotélio Vascular/química , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Binding of sulfated glycosaminoglycans (GAG) to a wide spectrum of extracellular regulatory proteins is crucial for physiological processes such as cell growth, migration, tissue homeostasis and repair. Thus, GAG derivatives exhibit great relevance in the development of innovative biomaterials for tissue regeneration therapies. We present a synthetic strategy for the preparation of libraries of defined sulfated oligohyaluronans as model GAG systematically varied in length, sulfation pattern and anomeric substitution in order to elucidate the effects of these parameters on GAG recognition by regulatory proteins. Through an experimental and computational approach using fluorescence polarization, ITC, docking and molecular dynamics simulations we investigate the binding of these functionalized GAG derivatives to ten representative regulatory proteins including IL-8, IL-10, BMP-2, sclerostin, TIMP-3, CXCL-12, TGF-ß, FGF-1, FGF-2, and AT-III, and we establish structure-activity relationships for GAG recognition. Binding is mainly driven by enthalpy with only minor entropic contributions. In several cases binding is determined by GAG length, and in all cases by the position and number of sulfates. Affinities strongly depend on the anomeric modification of the GAG. Highest binding affinities are effected by anomeric functionalization with large fluorophores and by GAG dimerization. Our experimental and theoretical results suggest that the diversity of GAG binding sites and modes is responsible for the observed high affinities and other binding features. The presented new insights into GAG-protein recognition will be of relevance to guide the design of GAG derivatives with customized functions for the engineering of new biomaterials.
RESUMO
BACKGROUND AND PURPOSE: Gain of function mutations in TRPC6 channels can cause autosomal dominant forms of focal segmental glomerulosclerosis (FSGS). Validated inhibitors of TRPC6 channels that are biologically active on FSGS-related TRPC6 mutants are eagerly sought. EXPERIMENTAL APPROACH: We synthesized new TRPC6-inhibiting modulators from larixol, a resiniferous constituent of Larix decidua, and tested the potency and selectivity in cell lines stably expressing various TRPC channel isoforms. Channel activation was followed by Ca2+ influx analyses and electrophysiological recordings. The most promising compound larixyl carbamate (LC) was tested on native TRPC6 channels and TRPC6 constructs carrying FSGS-related point mutations. KEY RESULTS: LC exhibited an about 30-fold preference for TRPC6 over TRPC3 channels and a fivefold preference for TRPC6 over TRPC7 channels. Six FSGS-related TRPC6 mutants, including the highly active M132T and R175Q variants, were strongly inhibited by 1 µM LC. Surprisingly, no TRPC6-related Ca2+ signals were detectable in primary murine podocytes, or in acutely isolated glomeruli. in these preparations. Quantitative PCR revealed a 20-fold to 50-fold lower abundance of TRPC6 transcripts in rat or mouse podocytes, compared with pulmonary artery smooth muscle cells from the same species. Accordingly, electrophysiological recordings demonstrated that DAG-induced currents in murine podocytes are very small, but sensitive to LC. CONCLUSIONS AND IMPLICATIONS: In spite of their low abundance in native podocytes, native TRPC6 channels are targetable using larixol-derived TRPC6 inhibitors. As observed with wild-type TRPC6 channels, FSGS-related TRPC6 mutants were sensitive to the newly developed inhibitors, paving the way for experimental therapies.
Assuntos
Diterpenos/farmacologia , Canal de Cátion TRPC6/antagonistas & inibidores , Animais , Sinalização do Cálcio/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Glomerulosclerose Segmentar e Focal , Células HEK293 , Humanos , Glomérulos Renais/citologia , Glomérulos Renais/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Miócitos de Músculo Liso/fisiologia , Podócitos/efeitos dos fármacos , Podócitos/metabolismo , Podócitos/fisiologia , Artéria Pulmonar/citologia , Ratos Wistar , Canal de Cátion TRPC6/genética , Canal de Cátion TRPC6/metabolismo , Canal de Cátion TRPC6/fisiologiaRESUMO
Glycosaminoglycans are known to bind biological mediators thereby modulating their biological activity. Sulfated hyaluronans (sHA) were reported to strongly interact with transforming growth factor (TGF)-ß1 leading to impaired bioactivity in fibroblasts. The underlying mechanism is not fully elucidated yet. Examining the interaction of all components of the TGF-ß1:receptor complex with sHA by surface plasmon resonance, we could show that highly sulfated HA (sHA3) blocks binding of TGF-ß1 to its TGF-ß receptor-I (TßR-I) and -II (TßR-II). However, sequential addition of sHA3 to the TßR-II/TGF-ß1 complex led to a significantly stronger recruitment of TßR-I compared to a complex lacking sHA3, indicating that the order of binding events is very important. Molecular modeling suggested a possible molecular mechanism in which sHA3 could potentially favor the association of TßR-I when added sequentially. For the first time bioactivity of TGF-ß1 in conjunction with sHA was investigated at the receptor level. TßR-I and, furthermore, Smad2 phosphorylation were decreased in the presence of sHA3 indicating the formation of an inactive signaling complex. The results contribute to an improved understanding of the interference of sHA3 with TGF-ß1:receptor complex formation and will help to further improve the design of functional biomaterials that interfere with TGF-ß1-driven skin fibrosis.
Assuntos
Adjuvantes Imunológicos/metabolismo , Ácido Hialurônico/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta1/metabolismo , Simulação de Dinâmica Molecular , Ligação Proteica , Ressonância de Plasmônio de SuperfícieRESUMO
Several pathologic conditions such as rheumatoid arthritis, ocular neovascularization, cancer, or atherosclerosis are often associated with abnormal angiogenesis, which requires innovative biomaterial-based treatment options to control the activity of angiogenic factors. Here, we studied how sulfated hyaluronan (sHA) and oversulfated chondroitin sulfate derivatives as potential components of functional biomaterials modulate vascular endothelial growth factor-A (VEGF-A) signaling and endothelial cell activity in vitro. Tissue inhibitor of metalloproteinase-3 (TIMP-3), an effective angiogenesis inhibitor, exerts its activity by competing with VEGF-A for binding to VEGF receptor-2 (VEGFR-2). However, even though TIMP-3 and VEGF-A are known to interact with glycosaminoglycans (GAGs), the potential role and mechanism by which GAGs alter the VEGF-A/TIMP-3 regulated VEGFR-2 signaling remains unclear. Combining surface plasmon resonance, immunobiochemical analysis, and molecular modeling, we demonstrate the simultaneous binding of VEGF-A and TIMP-3 to sHA-coated surfaces and identified a novel mechanism by which sulfated GAG derivatives control angiogenesis: GAG derivatives block the binding of VEGF-A and TIMP-3 to VEGFR-2 thereby reducing their biological activity in a defined, sulfation-dependent manner. This effect was stronger for sulfated GAG derivatives than for native GAGs. The simultaneous formation of TIMP-3/sHA complexes partially rescues the sHA inhibited VEGF-A/VEGFR-2 signaling and endothelial cell activation. These results provide novel insights into the regulation of angiogenic factors by GAG derivatives and highlight the potential of sHA derivatives for the treatment of diseases associated with increased VEGF-A and VEGFR-2 levels.
Assuntos
Ácido Hialurônico/química , Indutores da Angiogênese , Células Endoteliais , Neovascularização Patológica , Inibidor Tecidual de Metaloproteinase-3 , Fator A de Crescimento do Endotélio VascularRESUMO
Chemical synthesis of oligosaccharide conjugates is essential for studying the functional relevance of carbohydrates, and this task would be facilitated considerably if reliable methods for the anomeric ligation of unprotected sugars in water were available. Here, a method for the preparation of anomeric glycosyl thiols from complex unprotected mono-, di-, and oligosaccharides is presented. By exploiting the neighboring-group effect of the 2-acetamido-group, 1,2-oxazolines are generated and converted into 1-glycosyl thioesters through treatment with 1-thioacids. The unprotected anomeric glycosyl thiolates released inâ situ were conjugated to Michael acceptors, aliphatic halogenides, and aziridines to furnish versatile glycoconjugates. Conjugation of amino acids and proteins was accomplished using the thiol-ene reaction with terminal olefins. This method gives efficient access to anomeric glycosyl thiols and thiolates, which enables anomeric ligations of complex unprotected glycans in water.
RESUMO
Sulfated glycosaminoglycans (sGAGs) modulate cellular processes via their interaction with extracellular matrix (ECM) proteins. We revealed a direct binding of tissue inhibitor of metalloproteinase-3 (TIMP-3) to the endocytic receptor low-density lipoprotein receptor-related protein (LRP-1) clusters II and IV using surface plasmon resonance. Sulfated hyaluronan (sHA) and chondroitin sulfate (sCS) derivatives interfered with TIMP-3/LRP-1 complex formation in a sulfation-dependent manner stronger than heparin. Electrostatic potential calculations suggested a competition between negatively charged GAGs and highly negatively charged complement-like domains of LRP-1 for the binding to a positively charged area of TIMP-3 as an underlying mechanism. In vitro studies revealed increased amounts of pericellular TIMP-3 in the presence of sHA as a consequence of the blocked protein uptake. GAG derivatives as part of biomaterials might post-translationally modulate TIMP-3 levels stronger than native GAGs, thus exhibiting catabolic effects on the ECM, which could prevent extensive pathological matrix degradation and promote wound healing.
Assuntos
Glicosaminoglicanos/administração & dosagem , Ácido Hialurônico/administração & dosagem , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/biossíntese , Inibidor Tecidual de Metaloproteinase-3/biossíntese , Sulfatos de Condroitina/administração & dosagem , Sulfatos de Condroitina/química , Endocitose/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Glicosaminoglicanos/química , Humanos , Ácido Hialurônico/química , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/química , Células-Tronco Mesenquimais/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Ressonância de Plasmônio de Superfície , Inibidor Tecidual de Metaloproteinase-3/química , Cicatrização/efeitos dos fármacosRESUMO
An imbalance between tissue-degrading matrix metalloproteinases (MMPs) and their counterparts' tissue inhibitors of metalloproteinases (TIMPs) causes pathologic extracellular matrix (ECM) degradation in chronic wounds and requires new adaptive biomaterials that interact with these regulators to re-establish their balance. Sulfated glycosaminoglycans (GAGs) and TIMP-3 are key modulators of tissue formation and remodeling. However, little is known about their molecular interplay. GAG/TIMP-3 interactions were characterized combining surface plasmon resonance, ELISA, molecular modeling and hydrogen/deuterium exchange mass spectrometry. We demonstrate the potential of solute and surface-bound sulfated hyaluronan (sHA) and chondroitin sulfate (sCS) derivatives to manipulate GAG/TIMP-3 interactions by varying GAG concentration, sulfation degree and chain length. Three GAG binding sites in the N- and C-terminal domains of TIMP-3 were identified. We reveal no overlap with the matrix metalloproteinases (MMP)-binding site, elucidating why GAGs did not change MMP-1/-2 inhibition by TIMP-3 in enzyme kinetics. Since we prove that GAGs alone have a low impact on MMP activity, sHA and sCS offer a promising strategy to possibly control ECM remodeling via stabilizing and accumulating TIMP-3 by maintaining its MMP inhibitory activity under GAG-bound conditions. Whether GAG-based functional biomaterials can be applied to foster chronic wound healing by shifting the MMP/TIMP balance to a healing promoting state needs to be evaluated in vivo. STATEMENT OF SIGNIFICANCE: Increased levels of tissue-degrading matrix metalloproteinases (MMPs) lead to pathologic matrix degradation in chronic wounds. Therefor functional biomaterials that restore the balance between MMPs and tissue inhibitors of metalloproteinases (TIMPs) are required to promote wound healing. Since sulfated glycosaminoglycan (GAG) derivatives demonstrated already to be e.g. anti-inflammatory and immunomodulatory, and native GAGs interact with TIMP-3 the former are promising candidates for functionalizing biomaterials. We identified the GAG binding sites of TIMP-3 by combining experimental and molecular modeling approaches and revealed that GAG derivatives have a higher capacity to sequester TIMP-3 than native GAGs without altering its inhibitory potential towards MMPs. Thus GAG derivative-containing biomaterials could protect tissue from excessive proteolytic degradation e.g. in chronic wounds by re-establishing the MMP/TIMP balance.
Assuntos
Matriz Extracelular/metabolismo , Glicosaminoglicanos/metabolismo , Homeostase , Sulfatos/metabolismo , Inibidor Tecidual de Metaloproteinase-3/metabolismo , Sítios de Ligação , Glicosaminoglicanos/química , Humanos , Modelos Moleculares , Sulfatos/químicaRESUMO
The stromal cell-derived factor 1α (CXCL12) belongs to the CXC chemokine family and plays an important role in tissue regeneration and the recruitment of stem cells. Here, a stable chemotactic gradient is essential that is formed by the interaction of CXCL12 with the extracellular matrix. Binding properties of CXCL12 to naturally occurring glycosaminoglycans (GAGs) as well as to the artificial highly sulfated hyaluronic acid (HA) are investigated by using a combination of NMR spectroscopy, molecular modeling and molecular dynamics simulations. Our results demonstrate a preferred protein binding for the sulfated GAGs heparin (HE) and highly sulfated HA. Furthermore, we could demonstrate that the orientation of the sulfate is crucial for binding. All sulfated GAGs interact with the CXCL12 GAG-binding motif (K24-H25-L26-K27-R41-K43-R47), where K27 and R41 represent the anchor points. Furthermore, differences could be observed in the second interaction interface of CXCL12: both HE and highly sulfated HA interfere with the receptor-binding motif, while chondroitin sulfate binds different amino acids in close proximity to this motif. CXCL12 does not interact with HA, which was directly demonstrated by NMR spectroscopy and molecular modeling and explained by the lack of sulfate groups of the HA molecule.
Assuntos
Quimiocina CXCL12/química , Glicosaminoglicanos/química , Sítios de Ligação , Configuração de Carboidratos , Humanos , Modelos MolecularesRESUMO
The increasing need for site-specific protein decorations that mimic natural posttranslational modifications requires access to a variety of noncanonical amino acids with moieties enabling bioorthogonal conjugation chemistry. Here we present the incorporation of long-chain olefinic amino acids into model proteins with rational variants of pyrrolysyl-tRNA synthetase (PylRS). Nε-heptenoyl lysine was incorporated for the first time using the known promiscuous variant PylRS(Y306A/Y384F), and Nε-pentenoyl lysine was incorporated in significant yields with the novel variant PylRS(C348A/Y384F). This is the only example of rational modification at position C348 to enlarge the enzyme's binding pocket. Furthermore, we demonstrate the feasibility of our chosen amino acids in the thiol-ene conjugation reaction with a thiolated polysaccharide.
Assuntos
Alcenos/química , Aminoácidos/química , Proteínas/química , Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/metabolismo , Sítios de Ligação , Modelos Moleculares , Processamento de Proteína Pós-Traducional , Especificidade por SubstratoRESUMO
Implants and artificial biomaterials containing sulfated hyaluronans have been shown to improve the healing of injured skin and bones. It is hypothesized that these effects are mediated by the binding of sulfated glycosaminoglycans (GAGs) to growth factors and cytokines, resulting in the sequestering of proteins to the wound healing site and in modulated protein activity. Given that no direct synthetic access to sulfated oligohyaluronans has been available, little is known about their protein binding and the structure of the resulting protein complexes. Here, the chemoenzymatic preparation of oligohyaluronans on the gram scale is described. Oligohyaluronans are converted into anomeric azides at the reducing end, enabling the attachment of analytical labels through an anomeric ligation reaction. A nonasulfated tetrahyaluronan-ethylenediaminetetraacetic acid derivative has been produced and used as a paramagnetic tag for the elucidation of the complex of this ligand with interleukin-10 using paramagnetic relaxation enhancement NMR analysis. The metal ion position is resolved with 1.0 Å, enabling a refined structural model of the complex.
Assuntos
Materiais Biocompatíveis/química , Glicosaminoglicanos/química , Ácido Hialurônico/química , Ácido Hialurônico/síntese química , Interleucina-10/química , Glicosaminoglicanos/metabolismo , Ligantes , Ressonância Magnética Nuclear Biomolecular/métodos , Ligação ProteicaRESUMO
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.