Addition of the Lys(-2)-Arg(-1) dipeptide, present in the precursor protein, 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 native disulfide bridges and to dramatically
decrease biological activity. Conformational analysis was carried out on this peptide.
During protonation of the carboxyl groups, CD spectra showed a decrease in the helical
contribution, and NMR spectra displayed strong chemical shift modifications, suggesting
the importance of electrostatic interactions in the KR-ET-1 conformation. CD spectra
and two-dimensional NMR experiments were performed to investigate the KR-ET-1 three-dimensional
structure in water in the carboxylic acid and carboxylate states. Distance and angle
constraints were used as input for distance geometry calculations. The KR-ET-1 carboxylic
acid conformation was found to be very similar to ET-1, 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 consistent
with a conformation characterized by a helix extension to Leu17 and a stabilized C-terminal
section folded back toward the N-terminus. In addition, thanks to NOEs with Cys11
and Phe14, the Arg(-1) side chain appeared well-defined. Simulated annealing and molecular
dynamics calculations, supported an Arg(-1)-Glu10 salt bridge and an electrostatic
network involving the charged groups of Trp21, Asp18, and Lys(-2). Moreover, stabilization
of the KR-ET-1 C-terminal part is probably reinforced 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 role in the native folding of
the ET-1 precursor protein. On the other hand, modification in the environment and
a reduced mobility of the KR-ET-1 Trp21 key residue, when compared to ET-1, could
explain, at least in part, the strong decrease in biological activity.