Conformational isomerism of endothelin in acidic aqueous media: a quantitative NOESY analysis

Abstract

The conformational features of endothelin-1 (ET-1) in mixed water/ethylene glycol media have been studied by two-dimensional 1H NMR experiments throughout the pH range 3.2-7.2. At pH less than 5 all backbone NH signals can be observed, and NOESY experiments provided a large set of dipolar cross-peaks. Cross-peak intensities from each experiment (different mixing times and H2O versus D2O) were converted to distance constraints using a novel algorithm (program DISCON) for removing spin diffusion effects and thus obtain cross-rates rather than cross-peak intensities. A set of 168 nonstereospecific distance bounds (average experimental precision, +/- 0.38 A) was used in dynamics simulated annealing refinements. Two consensus structural features were found--a reverse turn at Ser5----Asp8 and an alpha-helical stretch from Lys9 to Cys15; however, after constraint-free minimization, structures generated using XPLOR-1.5, CONGEN, and DISCOVER all violated at least 32% of the bounds by more than 0.2 A, which we ascribe to conformational isomerism. When the constraints were modified to reflect subsequent experimental data and to eliminate constraints that could not be obeyed by any single conformer structure, the relaxed structures still violated at least 15% of this more limited and looser set of constraints. Therefore, a modified procedure for constrained dynamics refinement (using XPLOR-2.1), which allows for conformational isomerism outside of the central helical core region, was developed. This "conformer search procedure" produced structures which fell into five tightly defined conformational clusters. The two most populated clusters correspond to a rotation of the 8,9-amide unit. The conformer which we propose as the major contributor at pH 3.2-5.8 was defined to a backbone rmsd of 0.51 A over residues 1----15. An alternative description of the motional averaging in segments of the endothelin structure as extensive randomization rather than rapid interconversion between a small number of discreet conformers was ruled out by an analysis of NH shift-temperature gradients and exchange rates. This analysis suggests that small delta delta/delta T values need not correlate with H-bonding for conformational mixtures. In ET-1 the greatest motional averaging occurs from Ser2 through Ser5 (not in the C-terminus) and may be so extensive as to approximate a flexible random coil population as high as 30%. The C-terminus shows less rapid and less extensive conformational averaging, but no definitive structures for individual conformers could be derived in the absence of stereospecific constraints. The pharmacological implications of the consensus structural features are discussed.

A virtual-system coupled multicanonical molecular dynamics simulation: Principles and applications to free-energy landscape of protein–protein interaction with an all-atom model in explicit solvent

“…[32][33][34][35][36] Because of its two intra-peptide disulfide bonds, ET1 has a compact tertiary structure in solution. In both the NMR [37][38][39] and X-ray 40 structures, the N-terminal region of ET1 adopts a somewhat distorted strand and the middle region forms a helix. This strand and helix are tightly linked by the disulfide bonds, constituting a cystine-stabilized helical (CSH) motif.…”

Development of agonists of endothelin‐1 exhibiting selectivity towards ETA receptors

“…A direct function at the receptor level, involving a disulfide exchange mechanism, has even been proposed for the exterior bridge (Spinella et al, 1991). Conformational studies agree that the central core of ET and possibly the segment 9 -21 adopts a helical organization (Endo et al, 1989;Saudek et al, 1989;Perkins et al, 1990;Aumelas et al, 1991;Andersen et al, 1992;Wallace & Janes, 1995;Orry & Wallace, 2000). This secondary structure would be stabilized by the cystine residues (Heitz et al, 1999) and would be, for instance, characterized by the alignment of the Tyr 13 and His 16 side chains (Orry & Wallace, 2000).…”

“…Thus, structural analyses using circular dichroism or nuclear magnetic resonance (NMR) spectroscopies, as well as X-ray crystallography, have described, for instance, an a-helical arrangement located between residues 9 and 15 (or 16) (Endo et al, 1989;Saudek et al, 1989;Perkins et al, 1990;Aumelas et al, 1991;Andersen et al, 1992;Wallace & Janes, 1995;Katahira et al, 1998;Van Der Walle & Barlow, 1998;Boulanger et al, 1999;Hewage et al, 1999;Orry & Wallace, 2000;Hewage et al, 2002). Furthermore, some of these studies have reported that the helical backbone would be stabilized by hydrogen bonds involving more particularly the amino-acid pairs Lys 9 -Tyr 13 and Glu 10 -Phe…”

“…The conformational investigations also indicated that the N-terminal segment prevails in an extended geometry followed by type I b-turn between residues 5 and 8, while the C-terminal tail would be rather elongated and mobile. Nevertheless, no unambiguous conclusions are available about the C-terminal stretch, as Janes et al (1994), (by means of X-ray diffraction) depicted for ET-1 an irregular a-helix extended up to the C-terminal residue, whereas other authors (using NMR spectroscopy) rather observed a random coil arrangement able to bend back and lie in close proximity with the core residues (Saudek et al, 1989;Andersen et al, 1992). Worthy of note is the revealing of defined secondary structures at the C-terminus of ET analogues, such as a g-turn in IRL-1620 (Katahira et al, 1998) and a b-bend in biological active truncated ET derivatives .…”

Molecular modelling of the structures of endothelin antagonists Identification of a possible structural determinant for ET‐A receptor binding

“…In the following study, the 3D structure of these two antagonists are predicted, and the resulting models are then compared against the various solution structures for ET-1 that have been determined by means of 2D NMR and distance geometry calculations [17][18][19]. On the basis of these analyses a possible structural determinant of ET-A receptor binding is identified, and novel peptide designs are proposed for consideration as synthetic ET-A receptor antagonists.…”