CRYSTALLOGRAPHIC STRUCTURES OF THROMBIN COMPLEXED WITH THROMBIN RECEPTOR PEPTIDES - EXISTENCE OF EXPECTED AND NOVEL BINDING MODES
CRYSTALLOGRAPHIC STRUCTURES OF THROMBIN COMPLEXED WITH THROMBIN RECEPTOR PEPTIDES - EXISTENCE OF EXPECTED AND NOVEL BINDING MODES
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DOI：
10.1021/bi00177a018
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发表时间：
1994-03-22
期刊：
影响因子：
2.9
通讯作者：
FENTON, JW
中科院分区：
文献类型：
--
作者：
MATHEWS, II;PADMANABHAN, KP;FENTON, JW
凝血酶对血小板和其他细胞的许多重要作用似乎是由最近克隆的七跨膜结构域凝血酶受体介导的。凝血酶通过一种新的蛋白水解机制激活该受体。该受体的氨基末端胞外结构域含有序列LDPRSFLLRNPNDKYEPF。突变受体和受体肽的结构-活性研究表明，该序列在两个位点与凝血酶结合：LDPR与凝血酶的活性中心和KYEPF与凝血酶的纤维蛋白原识别外位点。然后凝血酶切割Arg 41-Ser 42键以暴露新的氨基末端，其作为束缚的肽配体结合到受体体内尚未确定的位点以实现受体活化。我们已经确定了八个晶体结构的凝血酶与受体为基础的肽。双齿对接模型的两个组件中的每一个都被捕获在单独的共晶中。在一种晶体类型中，LDPR序列以类似于d-PheProArg氯甲基酮的方式停靠在凝血酶的活性中心。在其他晶体中，KYEPF序列以类似于水蛭素羧基末端肽的DFEEI序列的方式结合在凝血酶的纤维蛋白原阴离子结合外位点中。然而，引人注目的是，没有实现包括预期的双齿结合模式的两种组分的单晶的产生，显然是因为肽具有在活性中心不以生产性方式结合的主导溶液S样构象。这种肽结构显然有利于受体肽-凝血酶相互作用的一种新的替代模式，其中受体肽在相邻的凝血酶分子之间形成分子间桥，导致晶体中的无限肽凝血酶链。在这种结构中，KYEPF序列以预期的方式停靠在一个凝血酶分子的外部位点，但LDPR序列以不寻常的非生产性模式与相邻分子的活性中心停靠。去除S样受体肽结构的重要决定因素的突变并没有显著改变凝血酶信号传导。此外，受体密度与细胞反应性的比较不支持受体寡聚化在信号传导中的作用。这种意想不到的分子间结合模式的生理作用（如果有的话）仍有待确定。受体肽的KYEPF序列的对接与凝血酶的活性中心和自溶环中的构象变化相关;前者可解释据报道由外位点结合诱导的底物特异性的变化，并且可能对受体活化具有潜在的重要性。
Many of the vital actions of thrombin on platelets and other cells appear to be mediated by the recently cloned seven-transmembrane-domain thrombin receptor. Thrombin activates this receptor by a novel proteolytic mechanism. The amino-terminal exodomain of the receptor contains the sequence LDPRSFLLRNPNDKYEPF. Structure-activity studies with mutant receptors and receptor peptides suggest that this sequence binds to thrombin at two sites: LDPR with the active center of thrombin and KYEPF with the fibrinogen recognition exosite of thrombin. Thrombin then cleaves the Arg41-Ser42 bond to unmask a new amino terminus, which functions as a tethered peptide ligand binding to as yet undefined sites within the body of the receptor to effect receptor activation. We have determined eight crystal structures of thrombin complexed with receptor-based peptides. Each of the two components of the bidentate docking model was captured in individual cocrystals. In one crystal type, the LDPR sequence docked in the active center of thrombin in a manner analogous to d-PheProArg chloromethyl ketone. In other crystals, the KYEPF sequence bound in the fibrinogen anion binding exosite of thrombin in a manner analogous to the DFEEI sequence of the carboxylate-terminal peptide of hirudin. Strikingly, however, generation of a single crystal that includes both components of the anticipated bidentate binding mode was not achieved, apparently because the peptides have a dominant solution S-like conformation that does not bind in a productive way at the active center. This peptide structure apparently favored a novel alternative mode of receptor peptide-thrombin interaction in which the receptor peptides formed an intermolecular bridge between neighboring thrombin molecules, resulting in an infinite peptide thrombin chain in crystals. In this structure, the KYEPF sequence docked in the expected manner at the exosite of one thrombin molecule, but the LDPR sequence docked in an unusual nonproductive mode with the active center of a neighboring molecule. Mutations that removed important determinants of the S-like receptor peptide structure underlying the bridging mode in the receptor itself did not significantly alter thrombin signaling. Additionally, a comparison of receptor density to the responsiveness of a cell did not support a role for receptor oligomerization in signaling. The physiological role for this unexpected intermolecular binding mode, if any, remains to be identified. Docking of the KYEPF sequence of receptor peptides was associated with conformational changes in the active center and the autolysis loop of thrombin; the former may account for changes in substrate specificity reported to be induced by exosite binding and may be potentially important for receptor activation.