Determining the molecular basis for the pH-dependent interaction between the link module of human TSG-6 and hyaluronan

TSG-6 is an inflammation-associated hyaluronan (HA)-binding protein that has anti-inflammatory and protective functions in arthritis and asthma as well as a critical role in mammalian ovulation. The interaction between TSG-6 and HA is pH-dependent, with a marked reduction in affinity on increasing the pH from 6.0 to 8.0. Here we have investigated the mechanism underlying this pH dependence using a combined approach of site-directed mutagenesis, NMR, isothermal titration calorimetry and microtiter plate assays. Analysis of single-site mutants of the TSG-6 Link module indicated that the loss in affinity above pH 6.0 is mediated by the change in ionization state of a histidine residue (His4) that is not within the HA-binding site. To understand this in molecular terms, the pH-dependent folding profile and the pKa values of charged residues within the Link module were determined using NMR. These data indicated that His4 makes a salt bridge to one side-chain oxygen atom of a buried aspartate residue (Asp89), whereas the other oxygen is simultaneously hydrogen-bonded to a key HA-binding residue (Tyr 12). This molecular network transmits the change in ionization state of His4 to the HA-binding site, which explains the loss of affinity at high pH. In contrast, simulations of the pH affinity curves indicate that another histidine residue, His45, is largely responsible for the gain in affinity for HA between pH 3.5 and 6.0. The pH-dependent interaction of TSG-6 with HA (and other ligands) provides a means of differentially regulating the functional activity of this protein in different tissue microenvironments. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.