Nordling K, Björk I
Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, Uppsala Biomedical Center, Sweden.
Biochemistry. 1996 Aug 13;35(32):10436-40. doi: 10.1021/bi9603579.
Previous work has shown that insertion of the reactive-bond loop of antithrombin into the main beta-sheet of the inhibitor, the A sheet, leads to exposure of epitopes that are not present in intact antithrombin. Identical epitopes are exposed in antithrombin-proteinase complexes, inferring that the reactive-bond loop is inserted into the A beta-sheet also in these complexes. Loop insertion thus presumably is involved in the mechanism of inhibition of target proteinases. In this work, we have identified a linear epitope in bovine antithrombin that reacts with antibodies specific for loop-inserted forms of the inhibitor. This epitope is a hexapeptide sequence comprising residues 342-347, Glu-Asp-Leu-Phe-Ser-Pro, and is located on the surface of the protein just carboxy-terminal of helix I. The Phe residue of this epitope is highly conserved in members of the serpin superfamily and appears to stabilize the region of the epitope in antithrombin and other serpins by interacting with the protein core. The conformational change involving expansion of the A beta-sheet following insertion of the reactive-bond loop is presumably transmitted through this Phe residue to the epitope region, thereby rendering this region accessible to antibodies.
先前的研究表明，将抗凝血酶的反应性键环插入抑制剂的主要β折叠（即A折叠）中，会导致完整抗凝血酶中不存在的表位暴露。在抗凝血酶-蛋白酶复合物中也会暴露相同的表位，这表明反应性键环在这些复合物中也插入到了Aβ折叠中。因此，环插入可能参与了对靶蛋白酶的抑制机制。在这项研究中，我们在牛抗凝血酶中鉴定出了一个线性表位，它能与针对抑制剂环插入形式的特异性抗体发生反应。这个表位是一个六肽序列，由342 - 347位残基组成，即Glu-Asp-Leu-Phe-Ser-Pro，位于蛋白质表面螺旋I的羧基末端。该表位的苯丙氨酸残基在丝氨酸蛋白酶抑制剂超家族成员中高度保守，并且似乎通过与蛋白质核心相互作用来稳定抗凝血酶和其他丝氨酸蛋白酶抑制剂中表位的区域。反应性键环插入后涉及Aβ折叠扩展的构象变化可能通过这个苯丙氨酸残基传递到表位区域，从而使该区域能够被抗体识别。
