Determining the molecular basis for the pH-dependent interaction between the link module of human TSG-6 and hyaluronan
Determining the molecular basis for the pH-dependent interaction between the link module of human TSG-6 and hyaluronan
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DOI：
10.1074/jbc.m611713200
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发表时间：
2007-04-27
影响因子：
4.8
通讯作者：
Day, Anthony J.
中科院分区：
文献类型：
--
作者：
Blundell, Charles D.;Mahoney, David J.;Day, Anthony J.
TSG-6是一种炎症相关的透明质酸（HA）结合蛋白，在关节炎和哮喘中具有抗炎和保护功能，在哺乳动物排卵中也起关键作用。TSG-6和HA之间的相互作用是pH依赖性的，当pH从6.0增加到8.0时，亲和力显著降低。在这里，我们已经调查了潜在的机制，这种pH值依赖性使用的组合方法的定点诱变，NMR，等温滴定量热法和微量滴定板测定。对TSG-6连接模块的单位点突变体的分析表明，pH 6.0以上亲和力的丧失是由不在HA结合位点内的组氨酸残基（His（4））的电离状态变化介导的。为了从分子角度理解这一点，使用NMR测定了Link模块内带电残基的pH依赖性折叠谱和pK（a）值。这些数据表明，His（4）与隐藏的天冬氨酸残基（Asp（89））的一个侧链氧原子形成盐桥，而另一个氧原子同时与关键的HA结合残基（Tyr（12））氢键结合。该分子网络将His（4）的电离状态的变化传递到HA结合位点，这解释了在高pH下亲和力的损失。相反，pH亲和力曲线的模拟表明，另一个组氨酸残基His（45）在pH 3.5和6.0之间对HA的亲和力的增加主要是由于。TSG-6与HA（和其他配体）的pH依赖性相互作用提供了在不同组织微环境中差异调节该蛋白质的功能活性的手段。
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 (His(4)) 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 His(4) makes a salt bridge to one side-chain oxygen atom of a buried aspartate residue (Asp(89)), 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 His(4) 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, His(45), 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.