The structural investigation of glycosaminoglycan binding to CXCL12 displays distinct interaction sites.

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.

NMR characterization of the binding properties and conformation of glycosaminoglycans interacting with interleukin-10.

The cytokine interleukin-10 (IL-10) is an important regulator of the host immune system with both pro- and anti-inflammatory functions. Glycosaminoglycans (GAGs) play a decisive role in the biology of many growth factors, e.g., for receptor binding or protection from proteolytic degradation. GAGs of the extracellular matrix inhibit IL-10 signaling, however, the molecular mechanism is so far unknown. Here, we studied the interaction between GAGs and IL-10 using a combination of nuclear magnetic resonance (NMR) spectroscopy and computer simulations. The binding region of a set of heparin and chondroitin sulfate GAG disaccharides with varying sulfation pattern were determined by saturation transfer difference (STD) NMR spectroscopy. From the initial growth rate of the STD amplification factor binding affinities were determined and KD values in the low millimolar to micromolar range were obtained. We observed the highest binding affinity to IL-10 with fully sulfated heparin; however, a hyaluronan hexasaccharide did not exhibit binding, which suggests that GAG sulfation is necessary for interaction with IL-10. For octasaccharides or longer GAGs, a cooperative binding behavior was observed, which could indicate simultaneous interaction with both dimer subunits of IL-10. Finally, structural information about the bound GAG was exemplarily obtained for a heparin tetrasaccharide fragment (ΔUA,2S-GlcNS,6S-IdoA,2S-GlcNS,6S) using transferred NOESY experiments, proton-proton scalar couplings and molecular dynamics simulations. The overall backbone conformation is only slightly changed in the presence of IL-10 and the conformational equilibrium between (1)C4 chair and (2)So skew-boat structure of the internal iduronic acid residue is preserved.

Flexibility and explicit solvent in molecular-dynamics-based docking of protein-glycosaminoglycan systems.

We present Dynamic Molecular Docking (DMD), a novel targeted molecular dynamics-based protocol developed to address ligand and receptor flexibility as well as the inclusion of explicit solvent in local molecular docking. A class of ligands for which docking performance especially benefits from overcoming these challenges is the glycosaminoglycans (GAGs). GAGs are periodic, highly flexible, and negatively charged polysaccharides playing an important role in the extracellular matrix via interaction with proteins such as growth factors and chemokines. The goal of our work has been to develop a proof of concept for an MD-based docking approach and to analyze its applicability for protein-GAG systems. DMD exploits the electrostatics-driven attraction of a ligand to its receptor, treats both as entirely flexible, and considers solvent explicitly. We show that DMD has high predictive significance for systems dominated by electrostatic attraction and demonstrate its capability to reliably identify the receptor residues contributing most to binding.

Binding of chondroitin 4-sulfate to cathepsin S regulates its enzymatic activity.

Human cysteine cathepsin S (catS) participates in distinct physiological and pathophysiological cellular processes and is considered as a valuable therapeutic target in autoimmune diseases, cancer, atherosclerosis, and asthma. We evaluated the capacity of negatively charged glycosaminoglycans (heparin, heparan sulfate, chondroitin 4/6-sulfates, dermatan sulfate, and hyaluronic acid) to modulate the activity of catS. Chondroitin 4-sulfate (C4-S) impaired the collagenolytic activity (type IV collagen) and inhibited the peptidase activity (Z-Phe-Arg-AMC) of catS at pH 5.5, obeying a mixed-type mechanism (estimated Ki = 16.5 ± 6 µM). Addition of NaCl restored catS activity, supporting the idea that electrostatic interactions are primarly involved. Furthermore, C4-S delayed in a dose-dependent manner the maturation of procatS at pH 4.0 by interfering with the intermolecular processing pathway. Binding of C4-S to catS was demonstrated by gel-filtration chromatography, and its affinity was measured by surface plasmon resonance (equilibrium dissociation constant Kd = 210 ± 40 nM). Moreover, C4-S induced subtle conformational changes in mature catS as observed by intrinsic fluorescence spectroscopy analysis. Molecular docking predicted three specific binding sites on catS for C4-S that are different from those found in the crystal structure of the cathepsin K-C4-S complex. Overall, these results describe a novel glycosaminoglycan-mediated mechanism of catS inhibition and suggest that C4-S may modulate the collagenase activity of catS in vivo.

Identification and characterization of a glycosaminoglycan binding site on interleukin-10 via molecular simulation methods.

The biological function of the pleiotropic cytokine interleukin-10 (IL-10), which has an essential role in inflammatory processes, is known to be affected by glycosaminoglycans (GAGs). GAGs are essential constituents of the extracellular matrix with an important role in modulating the biological function of many proteins. The molecular mechanisms governing the IL-10-GAG interaction, though, are unclear so far. In particular, detailed knowledge about GAG binding sites and recognition mode on IL-10 is lacking, despite of its imminent importance for understanding the functional consequences of IL-10-GAG interaction. In the present work, we report a GAG binding site on IL-10 identified by applying computational methods based on Coulomb potential calculations and specialized molecular dynamics simulations. The identified GAG binding site is constituted of several positively charged residues, which are conserved among species. Exhaustive conformational space sampling of a series of GAG ligands binding to IL-10 led to the observation of two GAG binding modes in the predicted binding site, and to the identification of IL-10 residues R104, R106, R107, and K119 as being most important for molecular GAG recognition. In silico mutation as well as single-residue energy decomposition and detailed analysis of hydrogen-bonding behavior led to the conclusion that R107 is most essential and assumes a unique role in IL-10-GAG interaction. This structural and dynamic characterization of GAG-binding to IL-10 represents an important step for further understanding the modulation of the biological function of IL-10.

Structural and functional insights into sclerostin-glycosaminoglycan interactions in bone.

In order to improve bone defect regeneration, the development of new adaptive biomaterials and their functional and biological validation is warranted. Glycosaminoglycans (GAGs) are important extracellular matrix (ECM) components in bone and may display osteogenic properties that are potentially useful for biomaterial coatings. Using hyaluronan (HA), chondroitin sulfate (CS) and chemically modified highly sulfated HA and CS derivatives (sHA3 and sCS3; degree of sulfation ∼3), we evaluated how GAG sulfation modulates Wnt signaling, a major regulator of osteoblast, osteoclast and osteocyte biology. GAGs were tested for their capability to bind to sclerostin, an inhibitor of Wnt signaling, using surface plasmon resonance and molecular modeling to characterize their interactions. GAGs bound sclerostin in a concentration- and sulfate-dependent manner at a common binding region. These findings were confirmed in an LRP5/sclerostin interaction study and an in vitro model of Wnt activation. Here, pre-incubation of sclerostin with different GAGs led to a sulfate- and dose-dependent loss of its bioactivity. Using GAG-biotin derivatives in a competitive ELISA approach sclerostin was shown to be the preferred binding partner over Wnt3a. In conclusion, highly sulfated GAGs might control bone homeostasis via interference with sclerostin/LRP5/6 complex formation. Whether these properties can be utilized to improve bone regeneration needs to be validated in vivo.