Solution structure by 2D proton NMR of a chimeric peptide recognized by galanin and neuropeptide Y receptors

Abstract

The 25 amino acid residue chimeric peptide M32, galanin(1-13)-neuropeptide Y(25-36)-amide, was synthesized. The peptide was found to be recognized by both galanin and NPY receptors. The solution structure in 30% (v/v) 1,1,1,3,3,3-hexafluoro-2-propanol was examined by 2-D 1H-NMR and by CD. Proton resonance assignments were made, and structures were calculated using DIANA and refined by restrained energy minimization and molecular dynamics. The obtained structures contain an alpha-helical part in the NPY portion of the peptide including residues 13-20, and in some structures it continues to the C-terminal Tyr25. The more flexible N-terminal portion of the peptide has the freedom to approach the C-terminal alpha-helix, via a reverse turn or a nascent alpha-helix, which permits the N-terminus with Trp2 to come into close contact with the C-terminus with Tyr25. Among the ten NMR structures with lowest energy, there are structures reminiscent of the horseshoe shape of aPP, a close relative of NPY with known crystal structure. It appears that the strong alpha-helical character of the NPY (25-36) amide fragment of M32 helps to stabilize structural features in the galanin-derived part of the peptide. It is noteworthy that this rigid NPY portion of M32 does not prevent the recognition of the peptide by galanin receptors; rather, the peptide has unusually high affinity: IC50 = 0.1 nM at galanin receptors. The chimeric peptide M32 is also recognized by NPY receptors with submicromolar affinity (IC50 = 0.25 microM). The availability of a solution structure for peptide M32, which is recognized by two peptide receptors that are both members of the family of G-protein-coupled receptors, may be useful in understanding peptide receptor-ligand interactions and in designing new galanin and NPY receptor ligands.

A Determination of the Solution Conformation of Secretoneurin, a Neuropeptide Originating from the Processing of Secretogranin II, by 1H‐NMR and Restrained Molecular Dynamics

“…7. For most of the slowly exchanging amide protons the hydrogen bonds fulfill the criteria described earlier by Arvidsson et al [49]. The large number of (+4) hydrogen bonds and the slowly exchanging amide protons of residues Gln3-Gln8 and Pro1 1 -Gly25 indicates the existence of stable a-helices in these two regions.…”

Stimulation of Porcine Pituitary Luteinizing Hormone Release by Galanin: Putative Auto/Paracrine Regulation

“…However, because of the NPY component 25–36 in the M32 molecule and because we know that NPY inhibits LH secretion in the pig [this paper; Elsaesser et al, in preparation], we suspected that the attenuation of GnRH-induced LH release by this compound might be due as well to some inherent NPY activity. In fact, significant effects of M32 on GnRH-induced LH release were only observed when M32 was used in micromole per liter concentrations, concentrations of M32 which are also recognized by NPY receptors in rat cerebral cortex membranes (IC 50 = 0.25 µ M ) [42]. Furthermore the inhibition of GnRH-induced LH release by M32 could partially be compensated by the NPY antagonist BIBP, again questioning the specificity of the galanin antagonist M32 at the pituitary level.…”

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“…The secondary and tertiary structures of NPY have been investigated using circular dichroism and NMR, and a 3D model has been proposed from these studies (11,12,13). The x-ray structure of the homologous peptide APP, has been reported (14); however, to date neither the parent peptide nor any analogs have been crystallized.…”