[LYS(-2)-ARG(-1)]ENDOTHELIN-1 SOLUTION STRUCTURE BY 2-DIMENSIONAL H-1-NMR - POSSIBLE INVOLVEMENT OF ELECTROSTATIC INTERACTIONS IN NATIVE DISULFIDE BRIDGE FORMATION AND IN BIOLOGICAL-ACTIVITY DECREASE

Addition of the Lys(-2)-Arg(-1) dipeptide, present in the precursor pr otein, to the N-terminus of endothelin-1 (ET-1), to form a 23-residue peptide (KR-ET-1) has been shown to greatly improve formation of nativ e disulfide bridges and to dramatically decrease biological activity. Conformational analysis was carried out on this peptide. During proton ation of the carboxyl groups, CD spectra showed a decrease in the heli cal contribution, and NMR spectra displayed strong chemical shift modi fications, suggesting the importance of electrostatic interactions in the KR-ET-1 conformation. CD spectra and two-dimensional NMR experimen ts were performed to investigate the KR-ET-1 three-dimensional structu re in water in the carboxylic acid and carboxylate states. Distance an d angle constraints were used as input for distance geometry calculati ons. The KR-ET-1 carboxylic acid conformation was found to be very sim ilar to ET-I, with a helix spanning residues 9-15 and an unconstrained C-terminal part. In contrast, in the carboxylate state, large changes in Arg(-1) and Phe14 chemical shifts and long-range NOEs were consist ent with a conformation characterized by a helix extension to Leu17 an d a stabilized C-terminal section folded back toward the N-terminus. I n addition, thanks to NOEs with Cys11 and Phe14, the Arg(-1) side chai n appeared well-defined. Simulated annealing and molecular dynamics ca lculations, supported an Arg(-1)-Glu10 salt bridge and an electrostati c network involving the charged groups of Trp21, Asp18, and Lys(-2). M oreover, stabilization of the KR-ET-1 C-terminal part is probably rein forced by hydrophobic interactions involving the Val12, Tyr13, Phe14, Leu17, Ile19, Ile20, and Trp21 side chains. In vitro, native disulfide bond formation improvement observed for KR-ET-1 could be ascribed to electrostatic interactions and more specifically to the Arg(-1)-Glu10 salt bridge. In vivo, similar interactions could play an important rol e in the native folding of the ET-1 precursor protein. On the other ha nd, modification in the environment and a reduced mobility of the KR-E T-1 Trp21 key residue, when compared to ET-1, could explain, at least in part, the strong decrease in biological activity.