Tertiary structure of endothelin-1 in water by 1H NMR and molecular dynamics studies

Ragg, Enzio; Mondelli, R.; Penco, Sergio; Bolis, Giorgio; Baumer, Luca; Guaragna, A.

doi:10.1039/p29940001317

Cited by 10 publications

(6 citation statements)

References 21 publications

(8 reference statements)

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“…The helical character was further enhanced by addition of SDS as membrane mimetic. This is in agreement with previous results on ET‐1 folding, which report on the induction of secondary structures by cell membranes 27 . Similar effects were demonstrated for other peptides hormones like the neuropeptide Y, which displays different folding patterns depending on the absence or presence of hydrophobic environments 28 .…”

Section: Discussionsupporting

confidence: 92%

“…This is in agreement with previous results on ET-1 folding, which report on the induction of secondary structures by cell membranes. 27 Similar effects were demonstrated for other peptides hormones like the neuropeptide Y, which displays different folding patterns depending on the absence or presence of hydrophobic environments. 28 Contrary to the wt peptide, the linearized ET-1 (peptide 1) showed only random coil structures in aqueous solution, although addition of the membrane mimetic induced an ET-1-like helical fold.…”

Section: Discussionsupporting

confidence: 55%

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The ring size of monocyclic ET‐1 controls selectivity and signaling efficiency at both endothelin receptor subtypes

Wolf

Beck‐Sickinger

2021

Journal of Peptide Science

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Cardiovascular diseases (CVDs) like hypertension are a major cause for death worldwide. In the cardiovascular tissue, the endothelin system—consisting of the receptor subtypes A (ETAR) and B (ETBR) and the mixed agonist endothelin 1 (ET‐1)—is a major key player in the regulation of vascular tone and blood pressure. Tight control of this system is required to maintain homeostasis; otherwise, the endothelin system can cause severe CVDs like pulmonary artery hypertension. The high sequence homology between both receptor subtypes limits the development of novel and selective ligands. Identification of small differences in receptor–ligand interactions and determination of selectivity constraints are crucial to fine‐tune ligand properties and subsequent signaling events. Here, we report on novel ET‐1 analogs and their detailed pharmacological characterization. We generated simplified ET‐1‐derived monocyclic peptides to provide an accessible synthesis route. By detailed in vitro characterization, we demonstrated that both G protein signaling and the subsequent arrestin recruitment of activated ETBR remain intact, whereas activation of the ETAR depends on the intramolecular ring size. Increasing of the intramolecular ring structure reduces activity at the ETAR and shifts the peptide toward ETBR selectivity. All ET‐1 analogs displayed efficient ETBR‐mediated signaling by G protein activation and arrestin 3 recruitment. Our study provides in‐depth characterization of the ET‐1/ETAR and ET‐1/ETBR interactions, which has the potential for future development of endothelin‐based drugs for CVD treatment. By identification of Lys9 for selective labeling, novel analogs for peptide‐mediated shuttling by ET‐1 are proposed.

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Section: Discussionsupporting

confidence: 92%

Section: Discussionsupporting

confidence: 55%

The ring size of monocyclic ET‐1 controls selectivity and signaling efficiency at both endothelin receptor subtypes

Wolf

Beck‐Sickinger

2021

Journal of Peptide Science

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“…We have now completed NMR structure elucidations for several additional endothelin analogues, , including ones in which each of the disulfides were absent (C → A) or altered (C → Pen) and in which an endothelin-like sequence was present but the disulfide linkages were those of apamin . Other groups have reported both NH exchange data and temperature gradients for endothelin mutants (including other monocyclic species) and for monocyclic analogues of apamin. With both sequences and linkage pattern included, this is a diverse collection of peptides.…”

Section: Resultsmentioning

confidence: 99%

Extracting Information from the Temperature Gradients of Polypeptide NH Chemical Shifts. 1. The Importance of Conformational Averaging

et al. 1997

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A detailed analysis of backbone amide NH chemical shift temperature gradients (∆δ/∆T values) for proteins and highly cross-linked peptides reveals that hydrogen-bonded exchange-protected NHs are characterized by ∆δ/∆T values of -2.0 ( 1.4 ppb/°C while exposed NHs typically display gradients of -6.0 f -8.5 ppb/°C; however, numerous exceptions to these generalizations occur. For partially folded peptides (rather than proteins), exceptions are more common than concordance with this rule; ∆δ/∆T values ranging from -28 to +12 ppb/°C have been observed. In the case of the peptide systems for which exchange protection data is available, the common practice of assuming that a ∆δ/∆T value less negative than -4 ppb/°C indicates that the NH is sequestered from solvent is shown to have zero predictive validity. The analysis of the data for partially folded peptides, protein fragments, and other peptides which are expected to display minimal structuring reveals a significant correlation between ∆δ/∆T and the deviation of δ NH from the random coil reference shift. The analysis was facilitated by plotting NH chemical shift deviations (NH-CSD) Versus the ∆δ/∆T values. Using such plots, slow-exchanging hydrogen-bonded sites in proteins can be determined with much higher confidence than using the value of the gradient alone. For peptides, the occurrence of large shift deviations and abnormal gradients are diagnostic for partial structuring at lower temperatures which becomes increasingly randomized on warming. A good correlation coefficient (R g 0.75) for NH-CSD and ∆δ/∆T values indicates that essentially all of the NH shift deviation from reference values is due to the concerted formation of a single structured state on cooling. Correlation coefficients greater than 0.95 were observed for both helix and -hairpin forming peptides. The slope of the correlation plot (parts per thousand/°C) is a measure of the decrease in the population of the structured state upon warming. A detailed model which rationalizes the effects of conformational equilibria upon NH shifts is presented. A positive ∆Cp for unfolding is required to rationalize the linearity of δ NH with temperature that is routinely observed for partially structured peptides. This analysis suggests that ordered states of short peptides achieve significant populations in water only when the hydrophobic effect favors the structured state. This conclusion is pertinent to the current questions concerning the temporal sequence of secondary Versus tertiary structure formation during protein folding. Further, it is suggested that the use of NMR parameters (scalar and dipolar couplings) to derive the structural preferences of protein fragments which might serve a "seeding" role in the folding pathway is justified only when the CSD/gradient plot displays both a correlation coefficient greater than 0.70 and significant NH-CSD values (|CSD| > 0.3).With the development of 2D NMR methods, peptide/protein structure elucidation has been dominated by methods based on NOE-derived distance const...

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“…In addition, lower RMSD values for KR-ET-1 in the carboxylate state are observed all along the sequence (Figure 10A). Such a pH-dependent C-terminal folding has not been reported as yet for ET-1, although a recent study of ET-1 in water at neutral pH showed a C-terminus folded toward the helical moiety with Ilel9 and Trp21 side chains lying close to Tyrl3 (Ragg et al, 1994). This folding was ascribed to hydrophobic interactions.…”

Section: Chemical Shift (Ppm)mentioning

confidence: 88%

[Lys(-2)-Arg(-1)]Endothelin-1 Solution Structure by Two-Dimensional 1H-NMR: Possible Involvement of Electrostatic Interactions in Native Disulfide Bridge Formation and in Biological Activity Decrease

Aumelas¹,

Chiche²,

Kubo³

et al. 1995

Biochemistry

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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.

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Tertiary structure of endothelin-1 in water by 1H NMR and molecular dynamics studies

Abstract: Leu6 and Cys' NH signals are very close and often overlap. t The stereospecific assignment of Ser5 P-protons, although nonequivalent, is uncertain.

Cited by 10 publications

References 21 publications

The ring size of monocyclic ET‐1 controls selectivity and signaling efficiency at both endothelin receptor subtypes

The ring size of monocyclic ET‐1 controls selectivity and signaling efficiency at both endothelin receptor subtypes

Extracting Information from the Temperature Gradients of Polypeptide NH Chemical Shifts. 1. The Importance of Conformational Averaging

[Lys(-2)-Arg(-1)]Endothelin-1 Solution Structure by Two-Dimensional 1H-NMR: Possible Involvement of Electrostatic Interactions in Native Disulfide Bridge Formation and in Biological Activity Decrease

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