Towards a Structure for a TSG-6·Hyaluronan Complex by Modeling and NMR Spectroscopy

Abstract

The Link module from human TSG-6, a hyaladherin with roles in ovulation and inflammation, has a hyaluronan (HA)-binding groove containing two adjacent tyrosine residues that are likely to form CH-stacking interactions with sequential rings in the sugar. We have used this observation to construct a model of a protein⅐HA complex, which was then tested against existing experimental information and by acquisition of new NMR data sets of [ 13 C, 15 N]HA (8-mer) complexed with unlabeled protein. A major finding of this analysis was that acetamido side chains of two GlcNAc rings fit into hydrophobic pockets on either side of the adjacent tyrosines, providing a selectivity mechanism of HA over other polysaccharides. Furthermore, two basic residues have a separation that matches that of glucuronic acids in the sugar, consistent with the formation of salt bridges; NMR experiments at a range of pH values identified protein groups that titrate due to their proximity to a free carboxylate in HA. Sequence alignment and construction of homology models for all human Link modules in their HA-bound states revealed that many of these features are conserved across the superfamily, thus allowing the prediction of functionally important residues. In the case of cartilage link protein, its two Link modules were docked together (using bound HA as a guide), identifying hydrophobic residues likely to form an intra-Link module interface as well as amino acids that could be involved in supporting intermolecular interactions between link proteins and chondroitin sulfate proteoglycans. Here, we propose a mechanism for ternary complex formation that generates higher order helical structures, as may exist in cartilage aggregates.

Determining the Molecular Basis for the pH-dependent Interaction between the Link Module of Human TSG-6 and Hyaluronan

“…All NMR experiments were performed at 25°C on spectrometers at the Oxford Centre for Molecular Sciences (University of Oxford, UK) at a proton resonance frequency of 500 MHz (unless otherwise stated) following the methods detailed previously (26,28,29 (27) within the lowest energy HA 8 AN -bound Link_TSG6 structure (28). Aromatic (blue) and basic (red) residues are shown in their experimentally determined conformations (28), whereas the HA (gold) is derived from a model for the complex (29). The ␤1-␣1 and ␤4 -␤5 loops that rearrange upon HA binding are indicated.…”

“…The titration curves resulting from the pH-dependent changes in chemical shift were fitted by nonlinear least squares analysis to both one-and two-site models for the pK a based on the Henderson-Hasselbach equation (29). Two-site models for the data were only accepted where the 2 value for the fit was more than 10 times better than that for the one-site model.…”

“…Since Link_TSG6 showed no unfolding or gross perturbation to the structure between pH 6.0 and 7.5, it was concluded that a change in ionization state of one (or more) charged residues was likely to be responsible for the loss of activity above pH 6.0 (36). The four histidine residues (His 4 , His 29 , His 45 , His 96 ) within Link_TSG6 are reasonable candidates for this activity, as is an aspartate residue (Asp 89 ) buried within the protein (see Fig. 1C) (21,36,37).…”

“…1B) (29). 6 Therefore, it should now be possible to come to a molecular understanding of the pH dependence of HA binding.…”

TSG-6 Protein Is Crucial for the Development of Pulmonary Hyaluronan Deposition, Eosinophilia, and Airway Hyperresponsiveness in a Murine Model of Asthma

“…TSG-6 is a 35-kDa secreted protein that is involved in both physiological and pathophysiological tissue responses. In addition to HA (23)(24)(25)(26)(27)(28), TSG-6 has been shown to interact with a wide range of ECM components such as the GAGs (29), chondroitin 4-sulfate (25), and heparan sulfate and heparin (30). TSG-6 hyaladherin properties are endowed by virtue of the link module (24) and have been shown to be important for mediating HA interaction with CD44, which is also a link module-containing hyaladherin (29,31).…”