High-resolution X-ray structure of the unexpectedly stable dimer of the [Lys(-2)-Arg(-1)-des(17-21)]endothelin-1 peptide.
High-resolution X-ray structure of the unexpectedly stable dimer of the [Lys(-2)-Arg(-1)-des(17-21)]endothelin-1 peptide.
复制标题
[Lys(-2)-Arg(-1)-des(17-21)]endothelin-1 肽出乎意料稳定的二聚体的高分辨率 X 射线结构。
DOI：
10.1021/bi049098a
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发表时间：
2004
期刊：
影响因子：
2.9
通讯作者：
A. Aumelas
中科院分区：
文献类型：
--
作者：
F. Hoh;R. Cerdan;Q. Kaas;Y. Nishi;L. Chiche;S. Kubo;N. Chino;Yuji Kobayashi;C. Dumas;A. Aumelas
先前对[Lys（（-2））-Arg（（-1））]内皮素-1肽（KR-ET-1）的结构研究，其效力比ET-1低540倍，强烈提示分子内存在Arg（-1）-Asp（8）（R（-1）-D（8））盐桥，在较短的[Lys（（-2））-Arg（（-1））-des（17-21）]内皮素-1衍生物中也观察到这种盐桥（KR-CSH-ET）。此外，对于这两种类似物，我们已经表明，属于前序列的Lys-Arg二肽显著改善了天然二硫键的形成（>或=96%，而不是ET-1的约70%）。与从NMR数据推断的相反，分子动力学模拟表明，这种分子内盐桥是不稳定的。KR-CSH-ET肽现已在pH 5.0下结晶，并使用直接方法在1.13 A下从头开始测定其高分辨率结构。出乎意料的是，KR-CSH-ET显示为头-尾对称二聚体，并且整个界面包括两个分子间R（-1）-D（8）盐桥、双链反平行β折叠和疏水接触。对这种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。沉降平衡实验明确地证实，KR-ET-1和KR-CSH-ET也作为二聚体存在于pH 5.0的溶液中。在新的二聚体结构的基础上，重新解释了以前的NMR数据。使用484个分子内和38个分子间NMR衍生的约束进行结构计算。二聚体的溶液和X射线结构非常相似（平均rmsd为0.85 A）。由于KR二肽的N-末端的KR-CSH-ET是存在于prosequence，它可以假设，类似的分子间盐桥可能参与在体内形成的天然二硫键的ET-1。因此，在产生生物活性ET-1激素的连续裂解之前，前序列似乎确实以伴侣样方式帮助ET-1折叠。
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.