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

The [Lys-Arg]-endothelin-1 analogue (KR-ET-1) yields almost selectivel y the native disulfide pattern (96%), in contrast to endothelin-1 (ET- 1) that gives at least 25% of the nan-native disulfide pattern. We hav e previously shown that the carboxylate-state structure of KR-ET-1 is more constrained and stabilized by a salt bridge between Arg(-1) and t he Asp8 or Glu10 side chain [Aumelas et al. (1995) Biochemistry 34, 45 46-4561], To identify this salt bridge and its potential involvement i n the disulfide bond formation, [E1OQ], [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] an alogues gave a high yield of the native isomer (>90%), the [D8N] analo gue 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 tha t the formation of disulfide bonds occurs in a thermodynamically contr olled step, we have hypothesized that the Arg(-1)-Asp8 salt bridge and concomitant interactions could be responsible for the increase in yie ld of the native isomer of KR-ET-1, In the present work, we describe t he structural studies of the carboxamide analogues and of the minor no n-native KR-ET-1 isomer. On the basis of H-1 NMR and CD spectra as a f unction of pH, [E10Q] and [D18N] analogues display a conformational ch ange similar to that of the parent peptide, whereas the structure of t he [D8N] analogue is unchanged. For the non-native isomer, we measured a lower helical content than for the native isomer and observed a mar ked 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 conser ved residues, one belonging to the prosequence [Arg(-1)] and the other to the mature sequence [Asp8], is involved in the formation of the na tive disulfide isomer of ET-1. The involvement of the prosequence in t he formation of the native disulfide isomer strongly suggests that, in the maturation pathway of ET-1, cleavage of the Arg52-Cys53 amide bon d occurs after native disulfide bond formation.