Aumelas A, Kubo S, Chino N, Chiche L, Forest E, Roumestand C, Kobayashi Y
Faculté de Pharmacie, Centre de Biochimie Structurale, CNRS UMR C9955, INSERM U414, 34060 Montpellier Cedex 2, France.
Biochemistry. 1998 Apr 14;37(15):5220-30. doi: 10.1021/bi9723764.
The [Lys-Arg]-endothelin-1 analogue (KR-ET-1) yields almost selectively the native disulfide pattern (96%), in contrast to endothelin-1 (ET-1) that gives at least 25% of the non-native disulfide pattern. We have previously shown that the carboxylate-state structure of KR-ET-1 is more constrained and stabilized by a salt bridge between Arg(-1) and the Asp8 or Glu10 side chain [Aumelas et al. (1995) Biochemistry 34, 4546-4561]. To identify this salt bridge and its potential involvement in the disulfide bond formation, [E10Q], [D18N], and [D8N] carboxamide analogues were studied, which led to the unambiguous identification of the Arg(-1)-Asp8 salt bridge. Furthermore, while [E10Q] and [D18N] analogues gave a high yield of the native isomer (>/=90%), the [D8N] analogue afforded a ratio of the two isomers close to that observed for ET-1 (68%) [Kubo et al. (1997) Lett. Pept. Sci. 4, 185-192]. Assuming that the formation of disulfide bonds occurs in a thermodynamically controlled step, we have hypothesized that the Arg(-1)-Asp8 salt bridge and concomitant interactions could be responsible for the increase in yield of the native isomer of KR-ET-1. In the present work, we describe the structural studies of the carboxamide analogues and of the minor non-native KR-ET-1 isomer. On the basis of 1H NMR and CD spectra as a function of pH, [E10Q] and [D18N] analogues display a conformational change similar to that of the parent peptide, whereas the structure of the [D8N] analogue is unchanged. For the non-native isomer, we measured a lower helical content than for the native isomer and observed a marked difference in the orientation of the KRCSC backbone. In addition, no salt bridge was experimentally observed. Altogether, these results allow us to hypothesize that the salt bridge between two highly conserved residues, one belonging to the prosequence [Arg(-1)] and the other to the mature sequence [Asp8], is involved in the formation of the native disulfide isomer of ET-1. The involvement of the prosequence in the formation of the native disulfide isomer strongly suggests that, in the maturation pathway of ET-1, cleavage of the Arg52-Cys53 amide bond occurs after native disulfide bond formation.
与内皮素 -1（ET-1）相比，[赖氨酸 - 精氨酸] - 内皮素 -1类似物（KR-ET-1）几乎选择性地产生天然二硫键模式（96%），而ET-1至少有25%的非天然二硫键模式。我们之前已经表明，KR-ET-1的羧酸盐态结构通过精氨酸（-1）与天冬氨酸8或谷氨酸10侧链之间的盐桥而更加受限和稳定[Aumelas等人（1995年）《生物化学》34卷，4546 - 4561页]。为了确定这个盐桥及其在二硫键形成中的潜在作用，对[E10Q]、[D18N]和[D8N]羧酰胺类似物进行了研究，从而明确鉴定出了精氨酸（-1）-天冬氨酸8盐桥。此外，虽然[E10Q]和[D18N]类似物产生高产量的天然异构体（≥90%），但[D8N]类似物得到的两种异构体的比例接近ET-1所观察到的比例（68%）[Kubo等人（1997年）《肽科学快报》4卷，185 - 192页]。假设二硫键的形成发生在热力学控制步骤中，我们推测精氨酸（-1）-天冬氨酸8盐桥及伴随的相互作用可能是KR-ET-1天然异构体产量增加的原因。在本工作中，我们描述了羧酰胺类似物和次要的非天然KR-ET-1异构体的结构研究。基于1H NMR和作为pH函数的CD光谱，[E10Q]和[D18N]类似物显示出与亲本肽类似的构象变化，而[D8N]类似物的结构未发生变化。对于非天然异构体，我们测得其螺旋含量低于天然异构体，并观察到KRCSC主链方向存在显著差异。此外，实验未观察到盐桥。总之，这些结果使我们推测，两个高度保守残基之间的盐桥，一个属于前序列[精氨酸（-1）]，另一个属于成熟序列[天冬氨酸8]，参与了ET-1天然二硫键异构体的形成。前序列参与天然二硫键异构体的形成强烈表明，在ET-1的成熟途径中，精氨酸52 - 半胱氨酸53酰胺键的切割发生在天然二硫键形成之后。
