Crystal structure of lactadherin C2 domain at 1.7 Å resolution with mutational and computational analyses of its membrane-binding motif
Crystal structure of lactadherin C2 domain at 1.7 Å resolution with mutational and computational analyses of its membrane-binding motif
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DOI：
10.1074/jbc.m705195200
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发表时间：
2008-03-14
影响因子：
4.8
通讯作者：
Gilbert, Gary E.
中科院分区：
文献类型：
--
作者：
Shao, Chenghua;Novakovic, Valerie A.;Gilbert, Gary E.
Lactadherin是一种磷脂酰-L-丝氨酸（Ptd-L-Ser）结合蛋白，其装饰乳脂球的膜。主要的Ptd-L- Ser结合功能的lactadherin已被定位到其C2结构域，这股同源性与凝血因子VIII和因子V的C2结构域。与这种同源性，纯化的lactadherin有效地竞争与凝血因子VIII和V的Ptd-L-Ser结合位点，作为一种有效的抗凝剂。我们已经确定了1.7埃分辨率的乳凝集素C2结构域（Lact-C2）的晶体结构。牛Lact-C2结构具有与因子VIII C2（fVIII-C2）和因子V C2（fV-C2）结构域同源的β-桶核心。在β-桶末端的两个环，指定为尖峰1和3，显示四个暴露于水的疏水氨基酸，使人想起fVIII-C2和fV-C2的膜相互作用残基。与fVIII-C2和fV-C2中的相应环相反，Lact-C2的尖峰1采用发夹转角，其中7-残基环通过内部氢键稳定。此外，中心甘氨酸残基在两个膜相互作用的环可以提高顺应性Lact-C2膜结合位点。致突变研究证实了膜相互作用的疏水性和/或甘氨酸残基的穗1和穗3。将fVIII-C2的尖峰1置换成Lact-C2也降低了结合。计算配体对接研究确定了两个前瞻性的Ptd-L-Ser相互作用位点。这些结果确定了Lact-C2的两个膜相互作用环，并为乳凝集素与因子VIII和因子V相比更有效的磷脂结合提供了结构基础。
Lactadherin is a phosphatidyl-L-serine ( Ptd-L-Ser)-binding protein that decorates membranes of milk fat globules. The major Ptd-L- Ser binding function of lactadherin has been localized to its C2 domain, which shares homology with the C2 domains of blood coagulation factor VIII and factor V. Correlating with this homology, purified lactadherin competes efficiently with factors VIII and V for Ptd-L-Ser binding sites, functioning as a potent anticoagulant. We have determined the crystal structure of the lactadherin C2 domain ( Lact-C2) at 1.7 angstrom resolution. The bovine Lact-C2 structure has a beta-barrel core that is homologous with the factor VIII C2 ( fVIII-C2) and factor V C2 ( fV-C2) domains. Two loops at the end of the beta-barrel, designated spikes 1 and 3, display four water-exposed hydrophobic amino acids, reminiscent of the membrane-interactive residues of fVIII-C2 and fV-C2. In contrast to the corresponding loops in fVIII-C2 and fV-C2, spike 1 of Lact-C2 adopts a hairpin turn in which the 7-residue loop is stabilized by internal hydrogen bonds. Further, central glycine residues in two membrane-interactive loops may enhance conformability of Lact-C2 to membrane binding sites. Mutagenesis studies confirmed a membrane-interactive role for the hydrophobic and/ or Gly residues of both spike 1 and spike 3. Substitution of spike 1 of fVIII-C2 into Lact-C2 also diminished binding. Computational ligand docking studies identified two prospective Ptd-L-Ser interaction sites. These results identify two membrane-interactive loops of Lact-C2 and provide a structural basis for the more efficient phospholipid binding of lactadherin as compared with factor VIII and factor V.