Formation of Native Disulfide Bonds in Endothelin-1. Structural Evidence for the Involvement of a Highly Specific Salt Bridge between the Prosequence and the Endothelin-1 Sequence
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- 作者：André ; Aumelas ; Shigeru Kubo ; Naoyoshi Chino ; Laurent Chiche ; Eric Forest ; Christian Roumestand ; and Yuji Kobayashi
- 刊名：Biochemistry
- 出版年：1998
- 出版时间：April 14, 1998
- 年：1998
- 卷：37
- 期：15
- 页码：5220 - 5230
- 全文大小：233K
- 年卷期：v.37,no.15(April 14, 1998)
- ISSN：1520-4995
文摘
The [Lys-Arg]-endothelin-1 analogue (KR-ET-1) yields almostselectively the native disulfidepattern (96%), in contrast to endothelin-1 (ET-1) that gives at least25% of the non-native disulfide pattern.We have previously shown that the carboxylate-state structure ofKR-ET-1 is more constrained andstabilized by a salt bridge between Arg(-1) and the Asp8 orGlu10 side chain [Aumelas et al. (1995)Biochemistry 34, 4546-4561]. To identify this saltbridge and its potential involvement in the disulfidebond formation, [E10Q], [D18N], and [D8N] carboxamide analogueswere studied, which led to theunambiguous 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 twoisomers close to that observed for ET-1 (68%) [Kubo et al. (1997)Lett. Pept. Sci. 4, 185-192]. Assumingthat the formation of disulfide bonds occurs in a thermodynamicallycontrolled step, we have hypothesizedthat the Arg(-1)-Asp8 salt bridge and concomitant interactionscould be responsible for the increase inyield of the native isomer of KR-ET-1. In the present work, wedescribe the structural studies of thecarboxamide analogues and of the minor non-native KR-ET-1 isomer.On the basis of 1H NMR and CDspectra as a function of pH, [E10Q] and [D18N] analogues display aconformational change similar tothat of the parent peptide, whereas the structure of the [D8N]analogue is unchanged. For the non-nativeisomer, we measured a lower helical content than for the native isomerand observed a marked differencein the orientation of the KRCSC backbone. In addition, no saltbridge was experimentally observed.Altogether, these results allow us to hypothesize that the saltbridge between two highly conserved residues,one belonging to the prosequence [Arg(-1)] and the other tothe mature sequence [Asp8], is involved inthe formation of the native disulfide isomer of ET-1. Theinvolvement of the prosequence in the formationof the native disulfide isomer strongly suggests that, in thematuration pathway of ET-1, cleavage of theArg52-Cys53 amide bond occurs after native disulfide bondformation.
90%), the [D8N]analogue afforded a ratio of the twoisomers close to that observed for ET-1 (68%) [Kubo et al. (1997)Lett. Pept. Sci. 4, 185-192]. Assumingthat the formation of disulfide bonds occurs in a thermodynamicallycontrolled step, we have hypothesizedthat the Arg(-1)-Asp8 salt bridge and concomitant interactionscould be responsible for the increase inyield of the native isomer of KR-ET-1. In the present work, wedescribe the structural studies of thecarboxamide analogues and of the minor non-native KR-ET-1 isomer.On the basis of 1H NMR and CDspectra as a function of pH, [E10Q] and [D18N] analogues display aconformational change similar tothat of the parent peptide, whereas the structure of the [D8N]analogue is unchanged. For the non-nativeisomer, we measured a lower helical content than for the native isomerand observed a marked differencein the orientation of the KRCSC backbone. In addition, no saltbridge was experimentally observed.Altogether, these results allow us to hypothesize that the saltbridge between two highly conserved residues,one belonging to the prosequence [Arg(-1)] and the other tothe mature sequence [Asp8], is involved inthe formation of the native disulfide isomer of ET-1. Theinvolvement of the prosequence in the formationof the native disulfide isomer strongly suggests that, in thematuration pathway of ET-1, cleavage of theArg52-Cys53 amide bond occurs after native disulfide bondformation.