Characterization of the bioactive conformation of the C-terminal tripeptide Gly-Leu-Met-NH2 of substance P using [3-prolinoleucine10]SP analogues.

Residue Leu10 of substance P (SP) is critical for NK-1 receptor recognition and agonist activity. In order to probe the bioactive conformation of this residue, cis- and trans-3-substituted prolinoleucines were introduced in position 10 of SP. The substituted SP analogues were tested for their affinity to human NK-1 receptor specific binding sites (NK-1M and NK-1m) and their potency to stimulate adenylate cyclase and phospholipase C in CHO cells transfected with the human NK-1 receptor. [trans-3-prolinoleucine10]SP retained affinity and potency similar to SP whereas [cis-3-prolinoleucine10]SP shows dramatic loss of affinity and potency. To analyze the structural implications of these biological results, the conformational preferences of the SP analogues were analyzed by NMR spectroscopy and minimum-energy conformers of Ac-cis-3-prolinoleucine-NHMe, Ac-trans-3-prolinoleucine-NHMe and model dipeptides were generated by molecular mechanics calculations. From NMR and modeling studies it can be proposed that residue Leu10 of SP adopts a gauche(+) conformation around the chi1 angle and a trans conformation around the chi2 angle in the bioactive conformation. Together with previously published results, our data indicate that the C-terminal SP tripeptide should preferentially adopt an extended conformation or a PPII helical structure when bound to the receptor.     

Incorporation of vinylogous scaffolds in the C-terminal tripeptide of substance P.

Glycine-9 and leucine-10 of substance P (SP) are critical for (NK)-1 receptor recognition and agonist activity. Propsi(Z)-CH=CH(CH3)-CONH)Leu (or Met) and Propsi((E)-CH=CH(CH3)-CONH)Leu (or Met) have been introduced in the sequence of SP, in order to restrict the conformational flexibility of the C-terminal tripeptide, Gly-Leu-Met-NH2, of SP. Propsi((Z)-CH=C(CH2CH(CH3)2)-CONH)Met-NH2, with an isobutyl substituent to mimic the Leu side-chain, was also incorporated in place of the C-terminal tripeptide. The substituted-SP analogs were tested for their affinity to human NK-1 receptor specific binding sites (NK-1M and NK-1m) and their potency to stimulate adenylate cyclase and phospholipase C in Chinese Hamster ovary (CHO) cells transfected with the human NK-1 receptor. The most potent SP analogs [Pro9psi((Z)CH=C(CH3)CONH)Leu10]SP and [Pro9psi ((E)CH=C(CH3)CONH)Leu10]SP, are about 100-fold less potent than SP on both binding sites and second messenger pathways. These vinylogous (Z)- or (E)-CH=C(CH3)- or (Z)-CH=C(CH2CH(CH3)2) moieties hamper the correct positioning of the C-terminal tripeptide of SP within both the NK-1M- and NK-1m-specific binding sites. The origin of these lower potencies is related either to an incorrect peptidic backbone conformation and/or an unfavorable receptor interaction of the methyl or isobutyl group.     

A novel bioassay for the NK-2 neurokinin receptor: the guinea pig gallbladder

Although three neurokinin receptors (NK-1, NK-2, NK-3) have been identified by radioligand binding assays, only the NK-1 and NK-3 types have been found in smooth muscle bioassays. In this study, evidence is presented demonstrating functional NK-2 type receptors in the guinea pig gallbladder (GPGB). The potencies of the following neurokinins were determined in the GPGB and the guinea pig ileum (GPI): substance P (SP), physalaemin (PH), eledoisin (EL), substance K (SK) and kassinin (KA). ED50 values were determined by linear regression analysis of the dose-related increases in the force generated by each peptide. In the GPI, the rank order of potency was SP = PH = EL greater than SK = KA, indicating NK-1 selectivity. In the GPGB, the relative potencies were SK greater than KA greater than EL much greater than PH greater than SP, which is similar to that reported for the NK-2 receptor in radioligand binding assays. These findings demonstrate the NK-2 receptor tissue selectivity of the GPGB.     

The two NK-1 binding sites are distinguished by one radiolabelled substance P analogue

Two non-stoichiometric binding sites had previously been characterized for the NK-1 receptor using two different types of radiolabelled analogues of substance P. However, the question remained on their eventual conformational interconversion induced or not by the ligand. In this study, kinetic, saturation, and competition studies using [3H]propionyl[Pro(9)]SP demonstrate the existence of two independent binding components in CHO cells transfected with the human NK-1 receptor, with K(d) values of 0.040 nM ( approximately 20% of total sites) and 5.9 nM ( approximately 80% of total sites) that correspond to those of the two previously described binding sites. These two binding sites do not seem to interconvert since the minor one can be selectively extinguished in saturation studies in the presence of a SP analogue specific of this binding site.     

The two NK-1 binding sites correspond to distinct, independent, and non-interconvertible receptor conformational states as confirmed by plasmon-waveguide resonance spectroscopy

Two nonstoichiometric ligand binding sites have been previously reported for the NK-1 receptor, with the use of classical methods (radioligand binding and second messenger assays). The most populated (major, NK-1M) binding site binds substance P (SP) and is related to the adenylyl cyclase pathway. The less populated (minor, NK-1m) binding site binds substance P, C-terminal hexa- and heptapeptide analogues of SP, and the NK-2 endogenous ligand, neurokinin A, and is coupled to the phospholipase C pathway. Here, we have examined these two binding sites with plasmon-waveguide resonance (PWR) spectroscopy that allows the thermodynamics and kinetics of ligand-receptor binding processes and the accompanying structural changes of the receptor to be monitored, through measurements of the anisotropic optical properties of lipid bilayers into which the receptor is incorporated. The binding of the three peptides, substance P, neurokinin A, and propionyl[Met(O(2))(11)]SP(7-11), to the partially purified NK-1 receptor has been analyzed by this method. Substance P and neurokinin A bind to the reconstituted receptor in a biphasic manner with two affinities (K(d1) = 0.14 +/- 0.02 nM and K(d2) = 1.4 +/- 0.18 nM, and K(d1) = 5.5 +/- 0.7 nM and K(d2) = 620 +/- 117 nM, respectively), whereas only one binding affinity (K(d) = 5.5 +/- 0.4 nM) could be observed for propionyl[Met(O(2))(11)]SP(7-11). Moreover, binding experiments in which one ligand was added after another one has been bound to the receptor have shown that the binding of these ligands to each binding site was unaffected by the fact that the other site was already occupied. These data strongly suggest that these two binding sites are independent and non-interconvertible on the time scale of these experiments (1-2 h).     

Further Demonstration that [Pro9]-Substance P Is a Potent and Selective Ligand of NK-1 Tachykinin Receptors

Previous studies have indicated that [Pro9]-substance P ([Pro9]-SP) possesses very good affinity for NK-1 binding sites and that, in contrast to substance P, it interacts selectively with these sites. Therefore, [3H][Pro9]-SP (75 Ci/mmol) was synthesized in order to study its binding to membranes of the rat brain. Specific binding of [3H][Pro9]-SP (75% of total binding) was temperature-dependent, saturable, and reversible. Scatchard analysis and Hill plots revealed the existence of a single population of noninteracting binding sites (KD and Bmax values: 1.48 nM and 29.7 fmol/mg of protein, respectively). Competition studies with several tachykinins and analogues indicated that the pharmacological profile of [3H][Pro9]-SP binding sites is identical to that of NK-1 binding sites. Rat brain sections labeled with either [3H][Pro9]-SP or [3H]SP, revealed a close similarity in the topographical distribution of [3H][Pro9]-SP and [3H]SP binding sites. Biochemical, pharmacological, and autoradiographic data obtained with [3H][Pro9]-SP did not provide any evidence for the existence of subtypes of NK-1 binding sites. [Pro9]-SP had neither agonist nor antagonist properties on NK-2 and NK-3 receptors. Indeed, it did not stimulate phosphoinositide turnover on the hamster urinary bladder (NK-2 assay) and was devoid of activity on the contraction of the rabbit pulmonary artery (NK-2 assay) and of the rat portal vein (NK-3 assay). As a result of its high selectivity, [Pro9]-SP thus appears an excellent tool for investigating the functional properties of NK-1 receptors.     

Photoaffinity labeling the substance P receptor using a derivative of substance P containing p-benzoylphenylalanine.

A novel photoreactive substance P (SP) analogue has been synthesized by solid-phase peptide synthesis methodology to incorporate the amino acid p-benzoyl-L-phenylalanine [L-Phe(pBz)] in place of the Phe8 residue of SP. [Phe8(pBz)]SP was equipotent with SP in competing for SP binding sites on rat submaxillary gland membranes and had potent sialagogic activity in vivo. In the absence of light, the 125I-labeled Bolton-Hunter conjugate of [Phe8(pBz)]SP bound in a saturable and reversible manner to an apparently homogeneous class of binding sites (Bmax = 0.2 pmol/mg of membrane protein) with an affinity KD = 0.4 nM. The binding of 125I-[Phe8(pBz)]SP was inhibited competitively by various tachykinin peptides and analogues with the appropriate specificity for SP/NK-1 receptors. Upon photolysis, up to 70% of the specifically bound 125I-[Phe8(pBz)]SP underwent covalent linkage to two polypeptides of Mr = 53,000 and 46,000, identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Quantitative analysis of the inhibitory effects of SP and related peptides on 125I-[Phe8(pBz)]SP photoincorporation indicated that the binding sites of the two photolabeled polypeptides have the same peptide specificity, namely, that typical of NK-1-type SP receptors. In addition, the labeling of the two polypeptides was equally sensitive to inhibition by guanyl-5'-yl imidodiphosphate, a nonhydrolyzable analogue of GTP. Further information on the relationship between the two labeled SP binding sites was provided by enzymatic digestion studies: the Mr = 46,000 polypeptide contains N-linked carbohydrates and is derived most likely from the higher molecular weight species by proteolytic nicking.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fetal exposure to cocaine causes programming of Prkce gene repression in the left ventricle of adult rat offspring

Previous studies demonstrated that maternal cocaine administration caused a significant decrease in protein kinase C epsilon (PRKCE) abundance in the left ventricle and an increase in susceptibility of the heart to ischemic injury in adult male offspring. The present study tested the hypothesis that epigenetic modification has a key role in cocaine-mediated programming of cardiac Prkce gene repression. Pregnant Sprague-Dawley rats were administered saline or cocaine (30 mg/kg/day i.p.) from Days 15 to 21 of gestational age, and hearts of 3-mo-old adult offspring were studied. Cocaine exposure significantly decreased Prkce mRNA levels in the left ventricle of male but not female offspring. CpG dinucleotides identified in Bhlhb2, Pparg, E2f, and Egr1 binding sites at the Prkce gene promoter were densely methylated in males and females and were unaffected by cocaine exposure. In contrast, methylation of CpGs in the two Sp1 binding sites (-346 and -268) was low and was significantly increased by cocaine exposure in male offspring. In females, methylation of the Sp1 binding site at -268 but not -346 was increased. Reporter gene assays showed that both Sp1 binding sites had a strong stimulatory role in Prkce gene activity. Methylation of the Sp1 binding sites significantly decreased SP1 binding to the Prkce promoter. Cocaine exposure did not affect nuclear SP1 protein levels but decreased the SP1 binding affinity to its binding site at -268. The results demonstrate an epigenetic mechanism of DNA methylation in programming of cardiac Prkce gene repression, linking fetal cocaine exposure and pathophysiological consequences in the heart of adult male offspring in a gender-dependent manner.     

Possible existence of a new tachykinin receptor subtype in the guinea pig ileum.

The guinea pig ileum possesses NK-1 and NK-3 tachykinin receptors. As expected, [Pro9]SP and senktide, which are selective agonists of NK-1 and NK-3 receptors, respectively, were found to be highly potent in contracting the guinea pig ileum. Surprisingly, similar observations were made with septide, SP-O-CH3, [Apa9-10]SP, or [Pro9,10]SP although, in contrast to [Pro9]SP, these four peptides showed a low affinity for 3H-[Pro9]SP-specific NK-1 binding sites on membranes from the guinea pig ileum. They were also devoid of affinity for NK-2 and NK-3 binding sites. GR 71251, a compound which has been described as a NK-1 antagonist, was more potent in inhibiting the septide- than the [Pro9]SP-evoked contracting response. Altogether, these results suggest that septide, [Apa9-10]SP, and [Pro9,10]SP exert their high contracting activity in the guinea pig ileum by acting on a new subtype of tachykinin receptors.     

Tachykinin NK-1 receptor probed with constrained analogues of substance P

The action of rotameric probes introduced either in position 7 or 8 in the sequence of substance P (SP) was investigated, i.e. -tetrahydroisoquinoleic acid (Tic), -fluorenylglycine (Flg), -diphenylalanine (Dip), the diastereoisomers of -1-indanylglycine (Ing) and -benz[ƒ]indanylglycine (Bfi), the Z- and E-isomers of dehydrophenylalanine and dehydronaphthylalanine (Δ^ZPhe, Δ^EPhe, Δ^ZNal, Δ^ENal) and (Dmp). The aim of this study was the topographical characterization of the binding subsites of human NK-1 receptor expressed in CHO cells, especially the S₇ and S₈ subsites, corresponding to residues Phe⁷ and Phe⁸ of substance P. According to the binding potencies of these substituted-SP analogues, the S₇ binding subsite is smaller than the S₈ subsite: the S₇ subsite accepts only one aromatic nucleus, while the S₈ can accommodate three coplanar nuclei altogether. These findings are compatible with the idea that the S₈ binding subsite may reside in the extracellular loops of the hNK-1 receptor. NK-1 agonists bind to human NK-1 receptor and activate the production of both inositol phosphates and cyclic AMP. As already quoted for septide, [pGlu⁶, Pro⁹]SP(6–11), discrepancies are observed between affinity (K_i) and activity (EC₅₀) values for IPs production. While a weak correlation between K_i and EC₅₀ values for IPs production could be found (r = 0.70), an excellent correlation could be demonstrated between their affinities (K_i) and their potencies (EC₅₀) for cAMP production (r = 0.97). The high potency (EC₅₀) observed for ‘septide-like’ molecules on PI hydrolysis, compared to their affinity is not an artefact related to the high level of NK-1 receptors expressed on CHO cells since a good correlation was found between EC₅₀ values obtained for PI hydrolysis and those measured for spasmogenic activity in guinea pig ileum bioassay (r = 0.94).

According to the binding potencies of constrained analogues of phenylalanine, the S₇ binding subsite of human NK-1 receptor is small, whereas the S₈, which can accommodate three coplanar nuclei, might probably reside in the extracellular loop. The discrepancies observed between affinity (K_i) and activity (EC₅₀) values for IPs production are not an artefact of CHO cells since a good correlation was found between EC₅₀ for PI hydrolysis and those measured in guinea pig ileum bioassay.     

Evidence for spatial proximity of two distinct receptor regions in the substance P (SP)*neurokinin-1 receptor (NK-1R) complex obtained by photolabeling the NK-1R with p-benzoylphenylalanine3-SP

Substance P (SP) belongs to the tachykinin family of bioactive peptides and exerts its many biological effects through functional interaction with its cell-surface, G protein-coupled neurokinin-1 receptor (NK-1R). Previous studies from our laboratory have shown that (125)I-Bolton-Hunter reagent-labeled p-benzoylphenylalanine(8)-SP (Bpa(8)SP) covalently attaches to Met(181), whereas (125)I-Bolton-Hunter reagent-labeled Bpa(4)SP covalently attaches to Met(174), both of which are located on the second extracellular loop (EC2) of the NK-1R. In this study, evidence has been obtained that at equilibrium, the photoreactive SP analogue (125)I-[D-Tyr(0)]Bpa(3)SP covalently labels residues in two distinct extracellular regions of the NK-1R. One site of (125)I-[D-Tyr(0)]Bpa(3)SP photoinsertion is located on EC2 within a segment of the receptor extending from residues 173 to 177; a second site of (125)I-[D-Tyr(0)]Bpa(3)SP photoinsertion is located on the extracellular N terminus within a segment of the receptor extending from residues 11 to 21, a sequence that contains both potential sites for N-linked glycosylation. Since competition binding data presented in this study do not suggest the existence of multiple peptide.NK-1R complexes, it is reasonable to assume that the receptor sequences within EC2 and N terminus identified by peptide mapping are in close proximity in the equilibrium complex.     

A new tachykinin receptor revealed by substance P analogues in the guinea pig ileum]

[Pro9]SP and septide have been described as selective agonists for the SP receptor (NK-1 type). These two peptides contract with a great efficacy the guinea-pig ileum, but unexpectedly septide was practically devoid of affinity for the NK-1 site labelled by 3H-[Pro9]SP. Like septide, SP analogues like SP-O-CH3, [Apa9-10]SP and [Pro9,10]SP share the same peculiar properties. In addition, the contracting activity of these peptides is not explained by an interaction with NK-2 or NK-3 sites. GR 71,251, a compound which has been described as NK-1 antagonist, was more potent in inhibiting the septide- and the [Apa9-10]SP- than the [Pro9]SP-evoked contracting responses. Altogether, these results suggest that septide, SP-O-CH3, [Apa9-10]SP and [Pro9,10]SP exert their high contracting activity in the guinea-pig ileum by acting on a new type of tachykinin receptor.     

Secondary structure of substance P bound to liposomes in organic solvents and in solution from Raman and CD spectroscopy.

The membrane lipid phase may be an important mediator of the peptide-receptor interaction. In order to understand the mechanism of this interaction, it is important to know the peptide structure, not only in the hydrophobic lipid bilayer environment, but also at the bilayer surface and in solution. To investigate this problem we have measured the secondary structure of the 11-residue neuropeptide substance P (SP) and its fragments in aqueous solutions, in membrane mimetic solvents, and associated with lipid bilayers using Raman and CD spectroscopy. Raman and CD spectra of SP bound to liposomes indicate a less than 20% helix content. We interpret these results to indicate that SP contains virtually no helix when bound to negatively charged liposomes. These spectra are similar to spectra of peptides in type I and III beta-turns. SP forms between 10 and 30% (1-3 residues) helical structure in sodium dodecyl sulfate micelles and less than 10% helix in methanol and trifluoroethanol. The binding of SP to negatively charged liposomes significantly changes the structure of the lipid acyl chains, decreasing order in some cases and increasing it in others. Raman spectra of SP in water indicates that SP near 30 mM forms an ensemble of structures in water that is distinct from completely unfolded peptide and from the aggregated beta-sheet form observed in saline solutions. We conclude from our CD results that methods used to quantitate secondary structure from CD spectra of short peptides cannot be used to distinguish between very short helical segments and beta-turns.     

NK-1 receptors are the only class of tachykinin receptors found on mouse cortical astrocytes.

Extending our previous studies, our results indicate that cultured cortical astrocytes from the mouse possess only NK-1 receptors coupled to phospholipase C. An excellent correlation was found in the potency of tachykinins and selective analogs at inhibiting 125I-BHSP binding and at stimulating phospholipase C activity, their rank order being that of NK-1 receptors. No binding sites could be found with ligands of NK-2 or NK-3 receptors. No additive effect could be shown with NK-2 or NK-3 agonists when phospholipase C activity was estimated with high concentrations of NK-1 agonists. C- or N-terminal SP fragments did not modify SP- or [Pro9]SP-evoked responses.     

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.     

Analogs of Substance P modified at the C-terminus which are both agonist and antagonist of the NK-1 receptor depending on the second messenger pathway.

The initial goal of this study was to analyze, using photolabeling, the interactions between Substance P and its tachykinin NK-1 receptor. Therefore, the photoreactive amino acid para-benzoyl-phenylalanine (pBzl)Phe was incorporated into the Substance P sequence from position 4 to 11 leading to Bapa0[(pBzl)Phex]SP analogs. Biotinyl sulfone-5-aminopentanoic acid (Bapa) was introduced in order to purify the covalent complex. These photoreactive SP analogs were first assayed for their affinity for the two binding sites associated with the NK-1 receptor, as well as for their potency in activating the phospholipase C and adenylate cyclase pathways. All analogs photoreactive from position 4 to 11 have moderate to high affinity for the two NK-1 receptor-binding sites, except for the analog modified at position 7. This affinity could be correlated to their potency to activate the phospholipase C and adenylate cyclase pathways, except for the analog photoreactive at position 11. Bapa0[(pBzl)Phe11]SP was found to be an agonist in the phospholipase C pathway and an antagonist in the adenylate cyclase pathway, other analogs modified at position 11 were therefore analyzed. Among these, Bapa0[Pro9, (pBzl)Hcy(O2)11]SP is a partial agonist, whereas Bapa0[Hcy(ethylaminodansyl)11]SP is a full agonist in the phospholipase C pathway, the two analogs being antagonist in the adenylate cyclase pathway. These results show that analogs of SP can be simultaneously agonist at one binding site and antagonist at the other binding site associated with the NK-1 receptor.     

Enzymatic methylations: III. Cadaverine-induced conformational changes of E.coli tRNAfMet as evidenced by the availability of a specific adenosine and a specific cytidine residue for methylation+

A partially purified tRNA methylase fraction from rat liver, containing m(2)G- m(1)A- and m(5)C-methylase, was used to study the influence of Mg(++) and of the biogenic polyamine cadaverine on the enzymatic methylation of E.coli tRNA(fMet)in vitro. In presence of 1 or 10 mM Mg(++), guanosine no. 27 was methylated to m(2)G. In 1 mM Mg(++) plus 30 mM cadaverine, guanosine in position 27 and adenosine in position 59 were methylated. In presence of 30 mM cadaverine alone tRNA(fMet) accepted three methyl groups: in addition to guanosine no. 27 and adenosine no. 59 cytidine no. 49 was methylated. In order to correlate tRNA(fMet) tertiary structure changes with the methylation patterns, differentiated melting curves of tRNA(fMet) were measured under the methylation conditions. It was shown that the thermodynamic stability of tRNA(fMet) tertiary structure is different in presence of Mg(++), or Mg(++) plus cadaverine, or cadaverine alone. From the differentiated melting curves and from the methylation experiments one can conclude that at 37 degrees in the presence of Mg(++) tRNA(fMet) has a compact structure with the extra loop and the TpsiC-loop protected by tertiary structure interactions. In Mg(++) plus cadaverine, the TpsiC-loop is available, while the extra loop is yet engaged in teritary structure (G-15: C-49) interactions. In cadaverine alone, the TpsiC-loop and the extra loop are free; hence under these conditions the open tRNA(fMet) clover leaf may be the substrate for methylation. In general, cadaverine destabilizes tRNA tertiary structure in the presence of Mg(++), and stabilizes tRNA(fMet) tertiary structure in the absence of Mg(++). This may be explained by a competition of cadaverine with Mg(++) for specific binding sites on the tRNA. On the basis of these experiments a possible role of biogenic polyamines in vivo may be discussed: as essential components of procaryotic and eucaryotic ribosomes they may together with ribosomal factors facilitate tRNA-ribosome binding during protein biosynthesis by opening the tRNA tertiary structure, thus making the tRNA's TpsiC-loop available for interaction with the complementary sequence of the ribosomal 5S RNA.     

Molecular characterization of the substance P*neurokinin-1 receptor complex: development of an experimentally based model

Molecular models for the interaction of substance P (SP) with its G protein-coupled receptor, the neurokinin-1 receptor (NK-1R), have been developed. The ligand.receptor complex is based on experimental data from a series of photoaffinity labeling experiments and spectroscopic structural studies of extracellular domains of the NK-1R. Using the ligand/receptor contact points derived from incorporation of photolabile probes (p-benzoylphenylalanine (Bpa)) into SP at positions 3, 4, and 8 and molecular dynamics simulations, the topological arrangement of SP within the NK-1R is explored. The model incorporates the structural features, determined by high resolution NMR studies, of the second extracellular loop (EC2), containing contact points Met(174) and Met(181), providing important experimentally based conformational preferences for the simulations. Extensive molecular dynamics simulations were carried out to probe the nature of the two contact points identified for the Bpa(3)SP analogue (Bremer, A. A., Leeman, S. E., and Boyd, N. D. (2001) J. Biol. Chem. 276, 22857-22861), examining modes of ligand binding in which the contact points are fulfilled sequentially or simultaneously. The resulting ligand.receptor complex has the N terminus of SP projecting toward transmembrane helix (TM) 1 and TM2, exposed to the solvent. The C terminus of SP is located in proximity to TM5 and TM6, deeper into the central core of the receptor. The central portion of the ligand, adopting a helical loop conformation, is found to align with the helices of the central regions EC2 and EC3, forming important interactions with both of these extracellular domains. The model developed here allows for atomic insight into the biochemical data currently available and guides targeting of future experiments to probe specific ligand/receptor interactions and thereby furthers our understanding of the functioning of this important neuropeptide system.     

Structural basis of the methylation specificity of R.DpnI

R.DpnI consists of N-terminal catalytic and C-terminal winged helix domains that are separately specific for the Gm6ATC sequences in Dam-methylated DNA. Here we present a crystal structure of R.DpnI with oligoduplexes bound to the catalytic and winged helix domains and identify the catalytic domain residues that are involved in interactions with the substrate methyl groups. We show that these methyl groups in the Gm6ATC target sequence are positioned very close to each other. We further show that the presence of the two methyl groups requires a deviation from B-DNA conformation to avoid steric conflict. The methylation compatible DNA conformation is complementary with binding sites of both R.DpnI domains. This indirect readout of methylation adds to the specificity mediated by direct favorable interactions with the methyl groups and solvation/desolvation effects. We also present hydrogen/deuterium exchange data that support ‘crosstalk’ between the two domains in the identification of methylated DNA, which should further enhance R.DpnI methylation specificity.