Jalbert L R, Chan J C, Christiansen W T, Castellino F J
Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556, USA.
Biochemistry. 1996 Jun 4;35(22):7093-9. doi: 10.1021/bi960290p.
We have previously proposed that a cluster of surface-exposed hydrophobic amino acids, viz., F4, L5, and L8, present at the amino-terminus of the Ca(2+)-bound form of gamma-carboxyglutamic acid domain (GD) of human protein C (PC), contributes a substantial portion of the total functional binding energy of PC and its activated form, APC, to acidic phospholipid (PL) vesicles. A deeper understanding of the importance of the hydrophobic nature of sequence position 5, and the particular relevance of leucine at that location, was sought by examination of the properties of a series of mutant proteins containing A5, V5, I5, and W5 as replacements for L5 in recombinant (r)-PC and APC. The Ca(2+)- and PL-dependent plasma-based anticoagulant activities of [L5A]r-APC, [L5V]r-APC, [L5I]r-APC, and [L5W]r-APC were determined to be approximately 28%, 51%, 98%, and 105%, respectively, of that of wild-type r-APC. A similar trend in activities of the mutant enzymes was observed in in vitro factor V/Va and factor VIII/VIIIa inactivation assays. Apparently normal Ca(2+)-dependent conformations were adopted by each of the mutant proteins, but the Ca(2+)-bound form of [L5A]r-PC was relatively the most defective of the mutants in its binding to PL. These results confirm the importance of the hydrophobic character at sequence position 5 as critical to the functional binding of PC to PL.
我们之前提出，人蛋白C（PC）的γ-羧基谷氨酸结构域（GD）的Ca(2+)结合形式的氨基末端存在一组表面暴露的疏水氨基酸，即F4、L5和L8，它们对PC及其活化形式活化蛋白C（APC）与酸性磷脂（PL）囊泡的总功能结合能贡献了很大一部分。通过检查一系列突变蛋白的特性，我们试图更深入地了解序列位置5的疏水性质的重要性，以及该位置亮氨酸的特殊相关性，这些突变蛋白在重组（r）-PC和APC中用A5、V5、I5和W5取代L5。[L5A]r-APC、[L5V]r-APC、[L5I]r-APC和[L5W]r-APC的基于血浆的Ca(2+)和PL依赖性抗凝活性分别测定为野生型r-APC的约28%、51%、98%和105%。在体外因子V/Va和因子VIII/VIIIa失活试验中观察到突变酶活性有类似趋势。每个突变蛋白显然都采用了正常的Ca(2+)依赖性构象，但[L5A]r-PC的Ca(2+)结合形式在与PL结合方面相对是突变体中缺陷最大的。这些结果证实了序列位置5的疏水特性对PC与PL功能结合的关键重要性。
