Knott V, Downing A K, Cardy C M, Handford P
Sir William Dunn School of Pathology, Oxford, UK.
J Mol Biol. 1996 Jan 12;255(1):22-7. doi: 10.1006/jmbi.1996.0003.
Ca2+ binding epidermal growth factor-like (EGF-like) domains are found in a large number of extracellular proteins with diverse functions, including those involved in blood coagulation, determination of cell fate, cell adhesion and connective tissue architecture. Their importance is emphasised by the identification of mutations in these domains in patients with haemophilia B (defective in coagulation factor IX) and the Marfan syndrome (defective in the connective tissue protein fibrillin-1). The X-ray crystal structure of a single Ca2+ binding EGF-like domain from human coagulation factor IX has recently been solved. It shows that the Ca2+ ligands form a pentagonal bipyramid, where one ligand is provided by an adjacent (N-terminal) EGF-like domain in the crystal. The N and C termini of the neighbouring domains are only approximately 4 angstrum apart, hence the crystal packing has been proposed as a model for the association of contiguous EGF-like domains in proteins. Since the adjacent EGF-like domain in the crystal, although close, is not covalently linked to its neighbour, this model requires verification. In this study we have expressed and purified a Ca2+ binding EGF-like domain pair from human fibrillin-1 and used an in vitro refolding system to obtain protein with the correct EGF fold. The Ca2+ binding properties of the protein have been investigated by two-dimensional NMR. The affinity of the C-terminal domain for Ca2+ is approximately 25-fold higher than that of the N-terminal domain, consistent with the two Ca2+ binding sites having different local environments. In addition, these data provide the first direct experimental evidence that Ca2+ plays a major role in defining the interdomain linkage in multiple repeats of Ca2+ binding EGF-like domains.
钙离子结合表皮生长因子样（EGF样）结构域存在于大量具有多种功能的细胞外蛋白质中，这些功能包括参与血液凝固、细胞命运决定、细胞黏附以及结缔组织结构形成。血友病B（凝血因子IX缺陷）和马凡综合征（结缔组织蛋白原纤维蛋白-1缺陷）患者中这些结构域的突变被发现，这凸显了它们的重要性。最近已解析出人类凝血因子IX单个钙离子结合EGF样结构域的X射线晶体结构。结果表明，钙离子配体形成一个五角双锥，其中一个配体由晶体中相邻的（N端）EGF样结构域提供。相邻结构域的N端和C端仅相距约4埃，因此晶体堆积被认为是蛋白质中连续EGF样结构域缔合的模型。由于晶体中相邻的EGF样结构域尽管距离很近，但并未与其相邻结构域共价连接，所以该模型需要验证。在本研究中，我们表达并纯化了来自人类原纤维蛋白-1的一对钙离子结合EGF样结构域，并使用体外重折叠系统获得具有正确EGF折叠的蛋白质。通过二维核磁共振研究了该蛋白质的钙离子结合特性。C端结构域对钙离子的亲和力比N端结构域高约25倍，这与两个钙离子结合位点具有不同的局部环境一致。此外，这些数据首次提供了直接的实验证据，表明钙离子在确定钙离子结合EGF样结构域多个重复序列中的结构域间连接方面起主要作用。
