Interaction of substance P and its N‐ and C‐terminal fragments with Ca²⁺: Implications for hormone action

Abstract

In an attempt to understand the role of Ca2+ on the bioactive conformation of peptide hormones, we have examined the interaction between Ca2+ and the neuropeptide substance P. Using CD spectroscopy to monitor conformational changes caused by Ca2+ binding, we found no significant binding of the cation by substance P in water. However, a substantial conformational change occurred in the hormone on Ca2+ addition in trifluoroethanol or an 80:20 (v/v) mixture of acetonitrile and trifluoroethanol. A biphasic binding of Ca2+ was observed in these solvents with saturation at 2 cations per hormone molecule. Mg2+ caused a relatively smaller conformational change in the hormone. A peptide corresponding to residues 1-7 at the N-terminal fragment of substance P showed a weak nonsaturating binding of Ca2+ in the nonpolar solvents whereas the 7-11 C-terminal fragment peptide displayed a binding indicative of an 1:1 Ca2+/peptide complex. Ca2+ binding by the hormone and the 7-11 fragment was also monitored by changes in fluorescence of the phenylalanyl residues. The results support the conclusion drawn from the CD data about a distinct Ca2+ binding site in the C-terminal part of substance P. The Kd values obtained from fluorescence data were 160 microM for Ca2+ and 1 mM for Mg2+ binding by substance P. The hormone and the two peptide fragments were also tested for their effect on the stability of dimyristoyl lecithin vesicles. Substance P and the N-terminal fragment caused no significant leakage of either fluorescent dyes or K+ trapped in the vesicles. Nor did they cause membrane fusion as monitored by the fluorescence quenching method.(ABSTRACT TRUNCATED AT 250 WORDS)

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.…”

CD,1H NMR and molecular modeling studies of the interaction of Ca2+ with substance P and Ala7-substance P in a non-polar solvent

“…The far-uv CD spectra of SP in ACN-TFE in the native and Ca 2 + -bound forms have been reported earlier [17]. They were repeated in this study with a view to comparing the structures and Ca 2 + interactions of SP and Ala 7 -SP under identical conditions.…”

“…One possible reason for this may be that the Ca 2 + -bound conformation of SP in the lipid environment may be required for receptor activation. Based on this rationale, we have, in an earlier study [17], examined the interaction of SP and its N-and C-terminal peptide fragments with Ca 2 + in a lipid-mimetic solvent (a mixture of 80:20 (v:v) acetonitrile (ACN) and TFE; we will refer to this mixture from now on as ACN-TFE). Circular dichroism (CD) and fluorescence spectral data showed stoichiometric interaction of SP and its C-terminal peptide (residues 7 -11) with Ca 2 + .…”

“…It has been suggested that the lipid milieu in which most peptide hormones would interact with membrane-bound receptors may impose specific constraints on hormone structure [3,14,15], so that the structure determined in lipid bilayers or micelles is generally regarded as a closer approximation to the bioactive conformation of SP than the structure prevailing in water. With this in mind, several researchers have determined the structure of SP in solvents that are regarded as lipid-mimetics [16], such as methanol [8][9][10][11][12] and trifluoroethanol (TFE) [8,11,17], as well as in lipid micelles and bilayers [8,11,[18][19][20][21][22][23][24][25]. While the results of these studies differ in specific details, they show that, in general, SP is capable of adopting a helical structure involving, in particularly, residues 7-11 in the C-terminal.…”

A Surface Plasmon Resonance Sensor for Substance P Using Gold-Modified Calmodulin and Melittin

“…38 The effect of the Ca 2+ concentration on the background SPR signals was examined by running a buffer solution containing Ca 2+ in the concentration range from 0 to 10 mM. The background SPR response was not affected by Ca 2+ if its concentration was below 1 mM, but at Ca 2+ concentration above 10 mM, sensorgrams became unstable and no steady SPR values were attained, indicating an interaction of Ca 2+ with the immobilized melittin on the sensor surface.…”