Plasticity of the TSG-6 HA-binding loop and mobility in the TSG-6-HA complex revealed by NMR and X-ray crystallography
Plasticity of the TSG-6 HA-binding loop and mobility in the TSG-6-HA complex revealed by NMR and X-ray crystallography
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DOI：
10.1016/j.jmb.2007.05.073
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发表时间：
2007-08-17
影响因子：
5.6
通讯作者：
Day, Anthony J.
中科院分区：
文献类型：
--
作者：
Higman, Victoria A.;Blundell, Charles D.;Day, Anthony J.
肿瘤坏死因子刺激基因-6（TSG-6）是一种糖胺聚糖结合蛋白，在炎症和炎症样过程中表达。先前计算了TSG-6的连接模块（Link_TSG 6）在其游离状态下以及当与透明质酸的八糖（HA（8））结合时的NMR结构。发现肝素竞争HA结合，即使它在不同于HA结合表面的位点相互作用。在此，我们提供了游离蛋白的晶体学数据，以及Link_TSG6的未复合和HA（8）结合形式的N-15 NMR弛豫数据。尽管连接模块总体上相对刚性，但游离蛋白在β 4/β 5环和Cys 47-Cys 68二硫键处显示出高度的移动性，这两个区域都参与HA结合。当与HA（8）结合时，这种动态行为被抑制，但没有消除，表明蛋白质和糖之间的动态匹配程度可能降低复合物形成的熵罚。另一个高度动态的残基是Lys 54，其远离HA结合位点，但先前显示参与肝素结合。当HA被结合时，Lys 54变得不那么移动的，提供了连接HA和肝素结合位点的变构效应的证据。提出了一种涉及β 2-链和α 2-螺旋的机制。游离Link-TSG 6的晶体结构在不对称单元中含有5个分子，这些分子与NMR结构高度相似，并支持在HA结合位点附近观察到的动态行为：它们显示β 4/β 5环的电子密度很小或没有电子密度，并显示Cys 47-Cys 68二硫键的多种构象。晶体结构用于与肝素的对接计算。Link_TSG6二聚体和肝素11聚体之间的扩展界面被确定为与先前的诱变和量热数据非常一致，为进一步研究这种相互作用提供了基础。(c)2007爱思唯尔有限公司保留所有权利。
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 (HA(8)). Heparin was found to compete for HA binding even though it interacts at a site that is distinct from the HA-binding sur Here we present crystallography data on the free protein, and N-15 NMR relaxation data for the uncomplexed and HA(8)-bound forms of Link_TSG6. Although the Link module is comparatively rigid overall, the free protein shows a high degree of mobility in the beta 4/beta 5 loop and at the Cys47-Cys68 disulfide bond, both of which are regions involved in HA binding. When bound to HA(8), 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 beta 2-strand and alpha 2-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 beta 4/beta 5 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. (c) 2007 Elsevier Ltd. All rights reserved.