Hoh François, Cerdan Rachel, Kaas Quentin, Nishi Yoshinori, Chiche Laurent, Kubo Shigeru, Chino Naoyoshi, Kobayashi Yuji, Dumas Christian, Aumelas André
Centre de Biochimie Structurale, UMR5048 CNRS-Université Montpellier I, UMR554 INSERM-Université Montpellier I, Faculté de Pharmacie, 15 avenue Charles Flahault, BP 14491, 34093 Montpellier-Cedex 5, France.
Biochemistry. 2004 Dec 7;43(48):15154-68. doi: 10.1021/bi049098a.
Previous structural studies on the [Lys((-2))-Arg((-1))]endothelin-1 peptide (KR-ET-1), 540-fold less potent than ET-1, strongly suggested the presence of an intramolecular Arg(-1)-Asp(8) (R(-1)-D(8)) salt bridge that was also observed in the shorter [Lys((-2))-Arg((-1))-des(17-21)]endothelin-1 derivative (KR-CSH-ET). In addition, for these two analogues, we have shown that the Lys-Arg dipeptide, which belongs to the prosequence, significantly improves the formation of the native disulfide bonds (>or=96% instead of approximately 70% for ET-1). In contrast to what was inferred from NMR data, molecular dynamics simulations suggested that such an intramolecular salt bridge would be unstable. The KR-CSH-ET peptide has now been crystallized at pH 5.0 and its high-resolution structure determined ab initio at 1.13 A using direct methods. Unexpectedly, KR-CSH-ET was shown to be a head-to-tail symmetric dimer, and the overall interface involves two intermolecular R(-1)-D(8) salt bridges, a two-stranded antiparallel beta-sheet, and hydrophobic contacts. Molecular dynamics simulations carried out on this dimer clearly showed that the two intermolecular salt bridges were in this case very stable. Sedimentation equilibrium experiments unambiguously confirmed that KR-ET-1 and KR-CSH-ET also exist as dimers in solution at pH 5.0. On the basis of the new dimeric structure, previous NMR data were reinterpreted. Structure calculations were performed using 484 intramolecular and 38 intermolecular NMR-derived constraints. The solution and the X-ray structures of the dimer are very similar (mean rmsd of 0.85 A). Since the KR dipeptide at the N-terminus of KR-CSH-ET is present in the prosequence, it can be hypothesized that similar intermolecular salt bridges could be involved in the in vivo formation of the native disulfide bonds of ET-1. Therefore, it appears to be likely that the prosequence does assist the ET-1 folding in a chaperone-like manner before successive cleavages that yield the bioactive ET-1 hormone.
先前对[赖氨酸（-2）-精氨酸（-1）]内皮素-1肽（KR-ET-1）的结构研究表明，其活性比ET-1低540倍，强烈提示存在分子内精氨酸（-1）-天冬氨酸（8）（R（-1）-D（8））盐桥，在较短的[赖氨酸（-2）-精氨酸（-1）-去（17-21）]内皮素-1衍生物（KR-CSH-ET）中也观察到该盐桥。此外，对于这两种类似物，我们已经表明，属于前序列的赖氨酸-精氨酸二肽显著改善了天然二硫键的形成（>或=96%，而ET-1约为70%）。与从核磁共振数据推断的结果相反，分子动力学模拟表明这种分子内盐桥不稳定。KR-CSH-ET肽现已在pH 5.0条件下结晶，并使用直接法从头确定了其1.13 Å的高分辨率结构。出乎意料的是，KR-CSH-ET被证明是一个头对尾对称二聚体，整体界面涉及两个分子间R（-1）-D（8）盐桥、一个双股反平行β-折叠片和疏水相互作用。对该二聚体进行的分子动力学模拟清楚地表明，在这种情况下，两个分子间盐桥非常稳定。沉降平衡实验明确证实，KR-ET-1和KR-CSH-ET在pH 5.0的溶液中也以二聚体形式存在。基于新的二聚体结构，对先前的核磁共振数据进行了重新解释。使用484个分子内和38个分子间核磁共振衍生约束进行了结构计算。二聚体的溶液结构和X射线结构非常相似（平均均方根偏差为0.85 Å）。由于KR-CSH-ET N端的KR二肽存在于前序列中，可以推测类似的分子间盐桥可能参与了ET-1天然二硫键的体内形成。因此，在前序列在连续切割产生生物活性ET-1激素之前，它似乎很可能以类似伴侣的方式协助ET-1折叠。
