Conformational Analysis of the Tachykinins in Solution: Substance P and Physalaemin

Abstract

A determination of the solution conformational behavior of two tachykinins, substance P and physalaemin, is described. Two-dimensional homonuclear Hartmann-Hahn (HOHAHA) and rotating-frame cross relaxation spectroscopy (ROESY) are used to obtain complete proton resonance assignments. Interproton distance restraints obtained from ROESY spectroscopy are used to characterize the conformational behavior. These data show that in solution both substance P and physalaemin exist in a mixture of conformational states, rather than as a single three-dimensional structure. In water both peptides prefer to be in an extended chain structure. In methanol, their behavior is described as a mixture of β-turn conformations in dynamic equilibrium. Solvent titration data and chemical shift temperature coefficients complement the NMR estimate of interproton distances by locating hydrogen bonds and serving to identify predominant conformational states. The C-terminal tetrapeptide segment has the same conformational behavior for both substance P and physalaemin. In physalaemin, the midsegment of the peptide may also be constrained by formation of a salt bridge. The conformational behavior of substance P and physalaemin is discussed in relation to potency and receptor binding properties.     

Conformation of the tripeptide Cbz-Pro-Leu-Trp-OBzl(CF3)2 deduced from two-dimensional 1H-NMR and conformational energy calculations is related to its affinity for NK1-receptor.

Chemical modifications of dual NK1/NK2 ligand Cbz-Gly-Leu-Trp-OBzl(CF3)2 (1) enabled us to create a high NK1 selective ligand Cbz-Pro-Leu-Trp-OBzl(CF3)2 (2). A determination of the conformational behavior of tripeptide 2 in solution is described. The 1D and 2D 1H-NMR techniques (COSY and ROESY) were used to assign resonances. Observed interproton distance restraints were considered to characterize conformational behavior. Spectral data indicate that tripeptide 2 presents a rigidified structure in DMSO stabilized by H-bond in two gamma-turns. Agreement with experimental data was obtained by averaging the 1H-NMR parameters over several combinations of low-energy conformations.     

Synthesis, conformational studies, and molecular dynamics calculations of two cyclic tetrapeptides with 17- and 18-membered rings

Two cyclic tetrapeptides [Boc-cyclo(-Xxx-Pro-Asn-Lys-)OMe (Xxx = Asp or Glu)] were synthesized and investigated by NMR spectroscopy. They were designed in order to mimic the salt bridge found in physalaemin. Isomers of the urethane bond were observed in DMSO solution. The ROESY spectrum allowed the assignment of many signals of the minor isomer of both compounds. Conformational studies based on the temperature gradients of the NH chemical shifts, coupling constants, and ROEs revealed a similar conformation for the Asp analogue as proposed for physalaemin. A beta I turn with Pro and Asn in the corner positions was found for the major isomer. No hydrogen bonds were detected for the major isomer of the cyclic Glu analogue. Molecular dynamics calculations, using the NMR based initial structures, yielded sets of conformations in agreement with the experimental data. It is concluded that the salt bridge in physalaemin is best approximated by a lactam formed from the original amino acids.     

Conformational analogy between substance P and physalaemin

The three-dimensional structure of physalaemin, pGlu-Ala-Asp-Pro-Asn-Lys-Phe-Tyr-Gly-Leu-Met-NH2, has been studied by one- and two-dimensional 500 MHz NMR spectroscopies in two solvents: methanol and dimethyl sulfoxide. As previously observed for substance P in methanol, the core of physalaemin 4----8 is folded into an helical conformation. This structure is stabilized by the presence of a salt bridge between Asp-3 and Lys-6 in both solvents. The only differences observed reside in the N-terminal sequences; the N-terminal tripeptide of substance is flexible whereas that of physalaemin is in an extended conformation.     

Analysis of tachykinin-binding site interactions using NMR and energy calculation data of potent cyclic analogues of substance P

The three-dimensional structures of [Cys3,6,Tyr8]-, [Gly2,Cys3,6,Tyr8]- and [DCys3,Cys6]substance P, designed as conformational analogues of substance P, have been studied by 1H-NMR (500 MHz) in different solvents and by energy calculations. As previously observed for substance P and physalaemin, two tachykinins acting via the NK-1 receptor, [Cys3,6,Tyr8]substance P presents an alpha-helical structure of the 4----8 sequence in methanol. This structure is stabilized by a beta-turn III via the formation of three hydrogen bonds involving the Cys-6, Phe-7 and Tyr-8 NH groups. In contrast to substance P, two of these hydrogen bonds are still present in dimethyl sulfoxide and in water the Cys-6 NH hydrogen bond is the only one remaining, such that a beta-turn structure inside the ring can be envisaged. In close agreement with the NMR data, the energy calculations lead to three types of folding for the core of [Cys3,6,Tyr8]substance P: a beta-turn III, a less stable beta-turn I (delta E = 3 kcal), and a beta-turn II (delta E = 4.6 kcal). The structure of Gly-Leu-Met-NH2 is strongly affected by changing the hydrophobicity of the medium. The most stable calculated conformation is the helix; however, numerous unrelated structures are destabilized by about 2-3 kcal/mol. These data are analyzed and discussed in connection with the high potency of [Cys3,6,Tyr8]substance P for both the NK-1 and NK-3 binding sites; that is the internal region of tachykinins (non-homologous amino acids) might present a similar three-dimensional structure when bound to the receptors (which may be at the origin of some lack of selectivity), whereas paradoxically the selectivity may be due to the common C-terminal sequence.     

NMR studies of peptide T, an inhibitor of HIV infectivity, in an aqueous environment.

The synthetic octapeptide peptide T (ASTTTNYT) has been shown to interfere with binding of the HIV-1 envelope glycoprotein gp120 to the chemokine receptor R5, thus preventing viral infection. This study investigated the degree of conformational order of two analogs of peptide T, one biologically active (D-Ala peptide T amide) and one inactive (D-Ala, D-Tyr peptide T amide) using nuclear magnetic resonance (NMR) spectroscopy in an aqueous environment, both in solution and in the frozen solid state. Standard solution NMR techniques such as DQFCOSY, HMQC, ROESY and inversion recovery measurements have been utilized to characterize these peptides. Solid state NMR experiments were likewise employed to study the peptides in a frozen glycerol:water mixture. The NMR results indicate that the monomeric form of both peptide T analogs have considerable conformational heterogeneity. Solid state NMR studies indicate aggregation of D-Ala peptide T, possibly into a beta-sheet structure, at concentrations higher than 10 mM.     

Substance K: a novel mammalian tachykinin that differs from substance P in its pharmacological profile

The primary structure of one of the bovine brain substance P precursors has shown the existence of a second mammalian tachykinin sequence, named substance K, that is remarkably homologous to that of the amphibian peptide kassinin. In this study, three substance K sequences were chemically synthesized and were submitted to parallel bioassays with kassinin, substance P and physalaemin. The results show that the three substance K peptides all possess biological activities characteristic of the tachykinin family and that their biological activities more closely resemble those of kassinin than those of substance P or physalaemin. This suggests that substance K may have a physiological role which is related to but different from that of substance P in mammalian organisms.     

Preferential conformation of substance P in solution

The three-dimensional structure of substance P has been studied by 1H-NMR, (500 MHz), and by circular dichroism (CD) in different solvents. The analysis of the different NMR parameters suggest that substance P adopts a rather extended structure in dimethylsulfoxide and pyridine. In water, besides the aggregation phenomenon, the monomeric substance P presents a complex conformational equilibrium. The addition of sodium dodecylsulfate to the aqueous solution induces, as shown by CD spectroscopy, a preferential alpha-helical conformation. And in methanol three structural conclusions may be drawn: the flexibility of the N-terminal Arg-Pro-Lys, the alpha-helical structure of Pro4-Gln5-Gln6-Phe7-Phe8 and the interaction of the C-terminal carboxamide with the primary amides from both glutamines.     

Membrane-assisted molecular mechanism of neurokinin receptor subtype selection.

Based on the observed membrane structures of substance P, physalaemin, and eledoisin, preferred conformations, orientations and accumulations of 13 mammalian neurokinins and non-mammalian tachykinins were estimated and compared with pharmacologic and selective binding data taken from the literature. Principal site affinities and relative affinities supported the view that neurokinins bind to three principal mammalian sites: the NK-1 (preferring substance P), the NK-2 (preferring neurokinin A), and the NK-3 site (preferring neurokinin B). Strong hydrophobic membrane interaction of the C-terminal message segment as a perpendicularly oriented alpha-helical domain correlated with NK-1 selection. Electrostatic accumulation of the peptide at the anionic fixed charge layer of the membrane without hydrophobic interaction through a helix correlated with NK-2 preference. Electrostatic repulsion by the anionic fixed charge layer correlated with NK-3 selection. Thus, neurokinin receptor selection is guided by the same principles as opioid receptor selection. Membrane catalysis of specific agonist--receptor interactions may prove to be a quite general phenomenon, and the membrane structure of a peptide more important for its structure--activity relationship than its crystal structure or its mixture of conformers in solution or in vacuo.     

Conformational behaviour of some tachykinin C-terminal heptapeptides

The conformational behaviour of substance P, physalaemin and eledoisin C-terminal heptapeptides was investigated using empirical energy calculations. Although the conformational distributions of the three heptapeptides are somewhat different, they have a few common low energy conformations. Some of them, which satisfy the structure-activity relationships, may fit the well known SP-P receptors.     

Assignment of the 1H-NMR resonances of the four rotamers of beta-casomorphin-5 in DMSO.

We report the complete assignment of the 1H-nmr spectrum of beta-casomorphin-5 in DMSO-d6 solution. With a combination of one-dimensional, double quantum filtered correlated spectroscopy, homonuclear Hartmann-Hahn, and rotating frame nuclear Overhauser enhancement spectroscopy (ROESY) spectra, we were able to differentiate the four conformers originating from two Xxx-Pro bonds present in the sequence. Exchange peaks in the ROESY spectra confirmed the presence of four interchanging conformational isomers. Based on integrations, the relative populations of the four species were estimated, while characteristic sequential nuclear Overhauser enhancements (NOEs) were used to determine the orientation of the Xxx-Pro bonds. This orientation was also shown to correlate with the chemical shift changes for the alpha protons of both the Xxx and Pro residues. Finally, interresidue NOEs indicate conformational preferences for the aromatic side chains, especially in the all-trans conformer.     

Characterization of a conformationally sensitive TOAC spin-labeled substance P

To probe the binding of a peptide agonist to a G-protein coupled receptor in native membranes, the spin-labeled amino acid analogue 4-amino-4-carboxy-2,2,6,6-tetramethylpiperidino-1-oxyl (TOAC) was substituted at either position 4 or 9 within the substance P peptide (RPKPQQFFGLM-NH2), a potent agonist of the neurokinin-1 receptor. The affinity of the 4-TOAC analog is comparable to the native peptide while the affinity of the 9-TOAC derivative is approximately 250-fold lower. Both peptides activate receptor signaling, though the potency of the 9-TOAC peptide is substantially lower. The utility of these modified ligands for reporting conformational dynamics during the neurokinin-1 receptor activation was explored using EPR spectroscopy, which can determine the real-time dynamics of the TOAC nitroxides in solution. While the binding of both the 4-TOAC substance P and 9-TOAC substance P peptides to isolated cell membranes containing the neurokinin-1 receptor is detected, a bound signal for the 9-TOAC peptide is only obtained under conditions that maintain the receptor in its high-affinity binding state. In contrast, 4-TOAC substance P binding is observed by solution EPR under both low- and high-affinity receptor states, with evidence of a more strongly immobilized peptide in the presence of GDP. In addition, to better understand the conformational consequences of TOAC substitution into substance P as it relates to receptor binding and activation, atomistic models for both the 4- and 9-TOAC versions of the peptide were constructed, and the molecular dynamics calculated via simulated annealing to explore the influence of the TOAC substitutions on backbone structure.     

Conformations of neurotensin in solution and in membrane environments studied by 2-D NMR spectroscopy

Two-dimensional HOHAHA and ROESY nuclear magnetic resonance techniques are used to obtain complete proton resonance assignments and to perform a conformational investigation of the neuropeptide neurotensin (pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu) in aqueous solution, methanol, and membrane-mimetic [deuterated sodium dodecylsulfate (SDS)] environments. Results suggest the absence of discernible elements of secondary structure in water and methanol. ROESY spectra confirm that Lys-Pro and Arg-Pro peptide bonds are all-trans, but that a significant population of cis Arg-Pro bonds arises in aqueous solution, which increases in the environment of SDS micelles. The conformational ensemble of the peptide is observed to narrow as it becomes bound through its cationic mid-region to SDS micelles, with the accompanying advent of local extended structure. The overall results indicate the inherent conformational flexibility of neurotensin, and emphasize the environmental dependence of conformation in peptides of medium length.     

The rotating frame nuclear Overhauser effect spectroscopy experiment as an aid to determining solution structures of DNA oligomers

Important intrinsic characteristics of the rotating frame nuclear Overhauser effect spectroscopy (ROESY) experiment were found to be advantageous in DNA solution structure determination. In a ROESY experiment, the different mechanisms of relaxation result in different signs of cross peaks, enabling a clear distinction between H2' resonances and H2" resonances of the DNA sugar backbone. This method is of particular importance in crowded spectra, for purine resonances whose H2', H2" protons typically resonate closely, as well as in conditions where line broadening makes coupling constants in a correlated spectroscopy experiment impossible to determine. By observing the signs of cross peaks in the base proton to H2', H2" sugar proton region, the ROESY spectrum can be used to distinguish A-form, B-form, and Z-form DNA.     

Possible influence of tachykinins on body fluid homeostasis in the rat

The effect on water intake, urine flow and vasopressin release of intracranial injections of substance P, physalaemin and eledoisin was studied in Wistar and Brattleboro, homozygous and heterozygous, rats. The tachykinins strongly inhibited water intake both in Wistar and in Brattleboro, homozygous and heterozygous, rats. Physalaemin and eledoisin reduced urine flow in Wistar and heterozygous, but not in homozygous, Brattleboro rats. Substance P never affected urine elimination. Physalaemin and eledoisin produced a dose-dependent, long lasting release of vasopressin in Wistar rats. Substance P did not affect the release of vasopressin. The results suggest that both substance P and physalaemin could influence brain mechanisms which control water intake, acting as thirst inhibitors, and that physalaemin could also participate in body fluid control by conserving water through vasopressin release.     

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 biologically relevant conformation of substance P is likely to be dictated by the lipid milieu wherein the hormone would interact with its receptor. Assuming that specific constraints to the hormone structure may be imparted by its interaction with Ca2+ ions in the low dielectric lipid medium, the interaction of substance P and its inactive analog, Ala7-substance P, has been characterized in a lipid-mimetic solvent. Circular dichroism (CD) and NMR spectral methods were employed to study the conformation of the free and Ca2+-bound forms of the peptides and the conformational changes that occur on Ca2+ binding. The results show that both peptides assume a helical structure in the non-polar solvent used, a mixture of acetonitrile and trifluoroethanol. The N-terminal region is, however, less ordered in the analog peptide compared with the native hormone. Ca2+ addition causes significant conformational changes in both the peptides. However, while substance P binds two Ca2+ ions in a cooperative manner, Ala7-substance P binds only one Ca2+ ion with a relatively weaker affinity. Computations of the minimum-energy conformations of the free and Ca2+-bound peptides were performed using interproton distances derived from nuclear Overhauser enhancement spectra of the two peptides, as well as the information provided by changes in proton chemical shifts caused by Ca2+ addition. Taken together, the results of this study suggest that differences in the interaction of substance P and Ala7-substance P with Ca2+ in the non-polar milieu, which in turn leads to differences in their Ca2+-bound conformations, may be the basis for the differences in their biological potencies.     

Chemical and immunochemical characterisation of substance P-like immunoreactivity and physalaemin-like immunoreactivity in a carcinoid tumour

Extracts of a carcinoid tumour, resected from the mid-portion of the ileum of a patient with no symptoms of endocrine disorder, were associated with a high concentration of substance P-like immunoreactivity. Using reverse-phase high performance liquid chromatography and antisera specifically directed against the C-terminal and N-terminal regions of substance P and against the N-terminal region of physalaemin, the following components were isolated and identified: substance P and its oxidised form, [pGlu⁵]substance P-(5–11) peptide and its oxidised form, and the oxidised form of physalaemin. The identity of tumour substance P with the undecapeptide was confirmed by amino acid analysis and high performance ion-exchange chromatography. In vitro incubation of tumour tissue from a lymph node metastasis from the same patient with [³H]leucine resulted in incorporation of radioactivity into newly synthesised substance P.     

Action of the newly discovered mammalian tachykinins, substance K and neuromedin K, on gastroduodenal motility of anesthetized dogs

The present experiments examined the local effects of two new mammalian tachykinins isolated from porcine spinal cord, substance K and neuromedin K, on gastroduodenal motility of anesthetized dogs. Tachykinins were injected through the gastroepiploic and cranial pancreaticoduodenal arteries at concentrations ranging from 1 to 100 ng/ml. Substance K, neuromedin K and substance P increased gastroduodenal smooth muscle contractions in a dose-dependent manner. The contractile response of the gastric antrum to newly discovered tachykinins was not as long-lasting as that to substance P. The potencies of various tachykinins on contractile responses showed the following rank order of potencies: physalaemin = eledoisin = substance P greater than substance K = neuromedin K in gastric smooth muscle; physalaemin = substance P = eledoisin greater than substance K = neuromedin K in the duodenal smooth muscle. Administration of atropine (100-200 micrograms/kg) inhibited the effect of tachykinins both in the gastric antrum and in the proximal duodenum. These results indicate that substance K and neuromedin K could act as transmitters or as modulators of neuronal activity influencing gastroduodenal motility.     

Structural Characterization of Neurokinin-3 Receptor Selective Peptide Agonist Scyliorhinin II Bound to DPC Micelles

Abstract

Scyliorhinin II, a cyclic Tachykinin peptide, is a potent NK3 receptor agonist. The pharmacology of NK3 receptor is least characterized out of the three tachykinin receptor subtypes cloned and characterized for Tachykinins. To understand the structural basis of peptide-receptor interaction, the three-dimensional structure of the Scyliorhinin II in aqueous and micellar environments has been studied by two-dimensional proton nuclear magnetic resonance (2D ¹H-NMR spectroscopy) and distance geometry calculations. Proton NMR assignments have been carried out with the aid of correlation spectroscopy (gradient-COSY and TOCSY) and nuclear Overhauser effect spectroscopy (NOESY and ROESY) experiments. The inter proton distances and dihedral angle constraints obtained from the NMR data have been used in torsion angle dynamics algorithm for NMR applications (DYANA) to generate a family of structures, which have been refined using restrained energy minimization and dynamics. The results show that in an aqueous environment, Scyliorhinin II lacks a definite secondary structure. The structure is well-defined in presence of dodecyl phosphocholine micelles. The global fold of Scyliorhinin II bound to DPC micelles consists of a well-defined helix in the C-terminal region from residue 12–18 and a series of turns towards N-terminus. The structure is further stabilized by disulfide bond between Cys7 and Cys13. The conformational range of the peptide revealed by NMR and CD studies has been analyzed in terms of characteristic secondary features. Observed conformational features have been compared with those of Substance P, Neurokinin A and Neurokinin B, potent NK1, NK2, and NK3 agonists, respectively.     

Structural characterization of neurokinin-3 receptor selective peptide agonist scyliorhinin II bound to DPC micelles

Scyliorhinin II, a cyclic Tachykinin peptide, is a potent NK3 receptor agonist. The pharmacology of NK3 receptor is least characterized out of the three tachykinin receptor subtypes cloned and characterized for Tachykinins. To understand the structural basis of peptide-receptor interaction, the three-dimensional structure of the Scyliorhinin II in aqueous and micellar environments has been studied by two-dimensional proton nuclear magnetic resonance (2D 1H-NMR spectroscopy) and distance geometry calculations. Proton NMR assignments have been carried out with the aid of correlation spectroscopy (gradient-COSY and TOCSY) and nuclear Overhauser effect spectroscopy (NOESY and ROESY) experiments. The inter proton distances and dihedral angle constraints obtained from the NMR data have been used in torsion angle dynamics algorithm for NMR applications (DYANA) to generate a family of structures, which have been refined using restrained energy minimization and dynamics. The results show that in an aqueous environment, Scyliorhinin II lacks a definite secondary structure. The structure is well-defined in presence of dodecyl phosphocholine micelles. The global fold of Scyliorhinin II bound to DPC micelles consists of a well-defined helix in the C-terminal region from residue 12-18 and a series of turns towards N-terminus. The structure is further stabilized by disulfide bond between Cys7 and Cys13. The conformational range of the peptide revealed by NMR and CD studies has been analyzed in terms of characteristic secondary features. Observed conformational features have been compared with those of Substance P, Neurokinin A and Neurokinin B, potent NK1, NK2, and NK3 agonists, respectively.