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J. Biol. Chem., Vol. 282, Issue 17, 12976-12988, April 27, 2007

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

Charles D. Blundell¹², David J. Mahoney¹, Martin R. Cordell, Andrew Almond³, Jan D. Kahmann, András Perczel^¶, Jonathan D. Taylor, Iain D. Campbell, and Anthony J. Day^||4

From the Medical Research Council Immunochemistry Unit and the Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom, the ^¶Protein Modelling Group, Institute of Chemistry, Eötvös Loránd University, P.O.B. 32, H-1117 Budapest, Hungary, and the ^||Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, United Kingdom

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 pK_a 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 (Tyr¹²). 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.

Received for publication, December 21, 2006 , and in revised form, February 15, 2007.

The chemical shifts at each pH value have been deposited in the BioMagRes-Bank under accession codes 7221 and 7222.

^* This work was funded in part by Grants D0525, D0540, D0562, D0569, M0625, and 16539 from the Arthritis Research Campaign and Grants 052830/Z/97/ZMEP/RC/CRD and 063822/Z/01/Z from the Wellcome Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental information and Figs. S1-S3.

This article was selected as a Paper of the Week.

¹ These authors contributed equally to this work.

² Recipient of a Yamanouchi Research Institute scholarship.

³ Supported by a Wellcome Trust Prize Traveling Research Fellowship (Grant 058154).

⁴ To whom correspondence should be addressed: Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University of Manchester, Michael Smith Bldg., Oxford Rd., Manchester M13 9PT, United Kingdom. Tel.: 44-161-2751495; Fax: 44-161-2751505; E-mail: anthony.day{at}manchester.ac.uk.

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