Plasticity of the TSG-6 HA-binding Loop and Mobility in the TSG-6-HA Complex Revealed by NMR and X-ray Crystallography

Tumour necrosis factor-stimulated gene-6 (TSG-6) is a glycosaminoglycan-binding protein expressed during inflammatory and inflammation-like processes. Previously NMR structures were calculated for the Link module of TSG-6 (Link_TSG6) in its free state and when bound to an octasaccharide of hyaluronan (HA8). Heparin was found to compete for HA binding even though it interacts at a site that is distinct from the HA-binding surface. Here we present crystallography data on the free protein, and 15N NMR relaxation data for the uncomplexed and HA8-bound forms of Link_TSG6. Although the Link module is comparatively rigid overall, the free protein shows a high degree of mobility in the β4/β5 loop and at the Cys47-Cys68 disulfide bond, both of which are regions involved in HA binding. When bound to HA8, this dynamic behaviour is dampened, but not eliminated, suggesting a degree of dynamic matching between the protein and sugar that may decrease the entropic penalty of complex formation. A further highly dynamic residue is Lys54, which is distant from the HA-binding site, but was previously shown to be involved in heparin binding. When HA is bound, Lys54 becomes less mobile, providing evidence for an allosteric effect linking the HA and heparin-binding sites. A mechanism is suggested involving the β2-strand and α2-helix. The crystal structure of free Link_TSG6 contains five molecules in the asymmetric unit that are highly similar to the NMR structure and support the dynamic behaviour seen near the HA-binding site: they show little or no electron density for the β4/β5 loop and display multiple conformations for the Cys47-Cys68 disulfide bond. The crystal structures were used in docking calculations with heparin. An extended interface between a Link_TSG6 dimer and heparin 11-mer was identified that is in excellent agreement with previous mutagenesis and calorimetric data, providing the basis for further investigation of this interaction. © 2007 Elsevier Ltd. All rights reserved.