Membrane-Induced Secondary Structures of Neuropeptides:  A Comparison of the Solution Conformations Adopted by Agonists and Antagonists of the Mammalian Tachykinin NK₁ Receptor

Abstract

We present what we believe to be the first documented example of an inducement of distinctly different secondary structure types onto agonists and antagonists selective for the same G-coupled protein receptor using the same membrane-model matrix wherein the induced structures are consistent with those suggested to be biologically active by extensive analogue studies and conventional binding assays. 1H NMR chemical shift assignments for the mammalian NK1 receptor-selective agonists alpha-neurokinin (NKA) and beta-neurokinin (NKB) as well as the mammalian NK1 receptor-selective antagonists [d-Pro2,d-Phe7,d-Trp9]SP and [d-Arg1, d-Pro2,d-Phe7,d-His9]SP have been determined at 600 MHz in sodium dodecyl sulfate (SDS) micelles. The SDS micelle system simulates the membrane-interface environment the peptide experiences when in the proximity of the membrane-embedded receptor, allowing for conformational studies that are a rough approximation of in vivo conditions. Two-dimensional NMR techniques were used to assign proton resonances, and interproton distances were estimated from the observed nuclear Overhauser effects (NOEs). The experimental distances were used as constraints in a molecular dynamics and simulated annealing protocol using the modeling package DISCOVER to generate three-dimensional structures of the two agonists and two antagonists when present in a membrane-model environment to determine possible prebinding ligand conformations. It was determined that (1) NKA is helical from residues 6 to 9, with an extended N-terminus; (2) NKB is helical from residues 4 to 10, with an extended N-terminus; (3) [d-Pro2,d-Phe7,d-Trp9]SP has poorly defined helical properties in the midregion and a beta-turn structure in the C-terminus (residues 6-9); and (4) [d-Arg1,d-Pro2, d-Phe7,d-His9]SP has a helical structure in the midregion (residues 4-6) and a well-defined beta-turn structure in the C-terminus (residues 6-10). Attempts have been made to correlate the observed conformational differences between the agonists and antagonists to their binding potencies and biological activity.

The Tachykinin Peptide Neurokinin B Binds Copper Forming an Unusual [CuII(NKB)2] Complex and Inhibits Copper Uptake into 1321N1 Astrocytoma Cells

“…These peaks are characteristic of an α-helical structure, which is consistent with previous structural studies and is thought to be a physiologically relevant state. 4,37,38 The addition of 0.5, 1.0, and 1.5 equiv of copper results in a loss of intensity of all peaks, and a plot of the absolute intensity of the negative peaks as a function of the copper equivalents added shows that copper addition beyond 0.5 equiv has little effect on the intensity ( Figure 6B). In accord with the visible CD and electronic absorption spectroscopy, this confirms a stoichiometry of 1:2 Cu II /NKB.…”

Molecular Characterization of the Substance P·Neurokinin-1 Receptor Complex

“…The ligand⅐receptor complexes that best fulfilled the experimental restraints were utilized as starting structures for the following simulations. Based on numerous NMR studies of SP, an ␣-helix in the center of the sequence, from Gln 4 to Phe 8 , was assumed (25)(26)(27)(28)(29) …”

Pharmacophore Pattern Identification of Tachykinin Receptor Selective Peptide Agonists: Implications in Receptor Selectivity

“…Bioactive conformation of tachykinin neuropeptides has been extensively investigated using high-resolution nuclear magnetic resonance (NMR), circular dichroism (CD) and Infrared (IR) spectroscopy. Solution structure for SP, NKA, NKB, Physalaemin, Eledoisin and various naturally derived or synthetic analogues has been reported in various membrane mimetic solvents [12][13][14][15][16][17][18][19][20][21][22][23][24][25] . We present here a brief summary of threedimensional structure of tachykinin neuropeptides (NK1, NK2 and NK3 receptor agonists) bound to dodecylphosphocholine (DPC) micelles, one of the well-characterized model membrane systems, using two-dimensional NMR spectroscopy.…”