Conformational features responsible for binding of cyclic analogues of enkephalin to opioid receptors. I. Low-energy peptide backbone conformers of analogues containing Phe4

Low-energy peptide backbone conformers were found by means of energy calculation for several cyclic analogues of enkephalin in an attempt to assess models for receptor-bound conformations for opioid receptors of the mu- and delta-types. They included [D-Cys2, L-Cys5]- and [D-Cys2, D-Cys5]-enkephalinamides showing moderate preference for mu-receptors, the delta-selective compounds [D-Pen2, L-Pen5] and [D-Pen2, D-Pen5]-enkephalins and Tyr-D-Lys-Gly-Phe- analogue possessing very high affinity to receptors of the mu-type. The low-energy conformers obtained for these analogues were in good agreement with the results of calculations by other authors and with experimental evidence. All of the analogues contain a Phe residue in position 4 of the peptide chain which facilitates the eventual search for geometrical similarity between the low-energy backbone conformers of different analogues in question.     

Conformational analysis of cyclic angiotensin II analogues

Summary Conformationally restricted cyclic analogues of angiotensin II (ANG II), Asp¹-Arg²-Val³-Tyr⁴-Val⁵-His⁶-Pro⁷-Phe⁸, with a link between positions 3 and 5 have considerable biological activity. It is proposed that the spatial arrangement of the pharmacophore groups of Tyr⁴, His⁶ and Phe⁸ side chains and the C-terminal carboxyl group in ANG II and active analogues is similar. Conformational analysis of ANG II and two cyclic analogues c[Sar¹, Lys³,Glu⁵]ANG II and c[Sar¹,Hcy³,Mpt⁵]ANG II was performed, and a geometrical comparison of the low-energy conformations of these compounds allowed one to propose a model of receptor-bound conformation in terms of the spatial arrangement of the pharmacophore groups. This model is characterised by the close spatial location of the His⁶-Phe⁸ side chains and the Tyr⁴ C-terminal carboxyl group and is stabilised by the electrostatic interaction of Arg² and the C-terminal carboxyl group.Abbreviations ANG II angiotensin II - Hcy homocysteine - Mpt trans-4-mercaptoproline     

The TIPP opioid peptide family: development of delta antagonists, delta agonists, and mixed mu agonist/delta antagonists

The discovery of the prototype delta opioid antagonists TIPP (H-Tyr-Tic-Phe-Phe-OH) and TIP (H-Tyr-Tic-Phe-OH) in 1992 was followed by extensive structure-activity relationship studies, leading to the development of analogues that are of interest as pharmacological tools or as potential therapeutic agents. Stable TIPP-derived delta opioid antagonists with subnanomolar delta receptor binding affinity and extraordinary delta receptor selectivity include TIPP[Psi] (H-Tyr-TicPsi[CH(2)NH]Phe-Phe-OH] and TICP[Psi] (H-Tyr-TicPsi[CH(2)NH]Cha-Phe-OH); Cha: cyclohexylalanine), which are widely used in opioid research. Theoretical conformational analyses in conjunction with the pharmacological characterization of conformationally constrained TIPP analogues led to a definitive model of the receptor-bound conformation of H-Tyr-Tic-(Phe-Phe)-OH-related delta opioid antagonists, which is characterized by all-trans peptide bonds. Further structure-activity studies revealed that the delta antagonist vs delta agonist behavior of TIP(P)-derived compounds depended on very subtle structural differences in diverse locations of the molecule and suggested a delta receptor model involving a number of different inactive receptor conformations. A further outcome of these studies was the identification of a new class of potent and very selective dipeptide delta agonists of the general formula H-Tyr-Tic-NH-X (X = arylalkyl), which are of interest for drug development because of their low molecular weight and lipophilic character. Most interestingly, TIPP analogues containing a C-terminal carboxamide group displayed a mixed mu agonist/delta antagonist profile, and thus were expected to be analgesics with a low propensity to produce tolerance and physical dependence. This turned out to be the case with the TIPP-derived mu agonist/delta antagonist DIPP-NH(2)[Psi] (H-Dmt-TicPsi[CH(2)NH]Phe-Phe-NH(2)); Dmt: 2',6'- dimethyltyrosine).     

Search for alpha-helical propensity in the receptor-bound conformation of glucagon-like peptide-1

To elucidate the receptor-bound conformation of glucagon-like peptide-1 (GLP-1), a series of conformationally constrained GLP-1 analogues were synthesized by introducing lactam bridges between Lys(i) and Glu(i)(+4) to form alpha-helices at various positions. The activity and affinity of these analogues to GLP-1 receptors suggested that the receptor-bound conformation comprises two alpha-helical segments between residues 11-21 and 23-34. It is notable that the N-terminal alpha-helix is extended to Thr(11), and that Gly(22) plays a pivotal role in arranging the two alpha-helices. Based on these findings, a highly potent bicyclic GLP-1 analogue was synthesized which is the most conformationally constrained GLP-1 analogue reported to date.     

Towards a KCC2 blocker pharmacophore model

A multi-disciplinary approach was used to identify the first pharmacophore model for KCC2 blockers: several physico-chemical studies such as XRD and NMR were combined to molecular modelling techniques, SAR analysis and synthesis of constrained analogues in order to determine a minimal conformational space regrouping few potential bioactive conformations. These conformations were further compared to the conformational space of a different series of KCC2 blockers in order to identify the common pharmacophoric features. The synthesis of more potent analogues in this second series confirmed the usefulness of this KCC2 blocker pharmacophore model.     

Unsaturated Analogues of the Neurotransmitter GABA: <Emphasis Type="Italic">trans</Emphasis>-4-Aminocrotonic, <Emphasis Type="Italic">cis</Emphasis>-4-Aminocrotonic and 4-Aminotetrolic Acids

Analogues of the neurotransmitter GABA containing unsaturated bonds are restricted in the conformations they can attain. This review traces three such analogues from their synthesis to their use as neurochemicals. trans-4-Aminocrotonic acid was the first conformationally restricted analogue to be extensively studied. It acts like GABA across a range of macromolecules from receptors to transporters. It acts similarly to GABA on ionotropic receptors. cis-4-Aminocrotonic acid selectively activates bicuculline-insensitive GABA_C receptors. 4-Aminotetrolic acid, containing a triple bond, activates bicuculline-sensitive GABA_A receptors. These findings indicate that GABA activates GABA_A receptors in extended conformations and GABA_C receptors in folded conformations. These and related analogues are important for the molecular modelling of ionotropic GABA receptors and to the development of new agents acting selectively on these receptors.     

Conformational analysis of cysteine-containing peptides and prospects of their application to 213Bi chelating in antitumor therapy

The method of conformational analysis was applied to the spatial structures of peptide analogues of phytochelatins and some fragments of metallothioneins: (Cys-Gly)₃, (Cys-Gly)₃-Asp, (Cys-Gly)₃-Glu, (Cys-βAla)₃, (Cys-γGlu)₃, and (Cys-Gly-Gly)₃. All the possible low-energy conformations of the molecules were revealed and the role of intra-and interresidual interactions in the formation of their spatial structures was determined. A different tendency of the molecules under study for acceptance of conformations favorable for binding bismuth ions was shown. Low-energy structures providing an optimum binding of bismuth ion were shown to be most frequent for (Cys-βAla)₃ peptide. Among the analogues of peptide fragments of the metallothioneins, lacking in natural peptides, low-energy pentapeptide CCXXC fragments (where X = Gln, Asn, Phe, Tyr, or Gly) were revealed. In the α-helical conformations of these pentapeptides, the distance between the sulfur atoms corresponds to that in Bi₂S₃.     

Chemical conversion of aspartyl peptides to isoaspartyl peptides. A method for generating new methyl-accepting substrates for the erythrocyte D-aspartyl/L-isoaspartyl protein methyltransferase

Mammalian protein carboxyl methyltransferases have recently been proposed to recognize atypical configurations of aspartic acid and may possibly function in the metabolism of covalently altered cellular proteins. Consistent with this proposal, the tetrapeptide tetragastrin, containing a single "normal" L-aspartyl residue (L-Trp-L-Met-L-Asp-L-Phe-NH2) was found here not to be an in vitro substrate for erythrocyte carboxyl methyltransferase activity. However, chemical treatment of tetragastrin by methyl esterification and then de-esterification of the aspartic acid residue yielded a mixture of peptide products, the major one of which could now be enzymatically methylated. We show here that this new peptide species is the isomeric beta-aspartyl form of tetragastrin (L-iso-tetragastrin; L-Trp-L-Met-L-Asp-L-Phe-NH2), and it appears that isomerization proceeds via an intramolecular succinimide intermediate during the de-esterification procedure. L-iso-Tetragastrin is stoichiometrically methylated (up to 90% in these experiments) with a Km for the enzyme of 5.0 microM. Similar chemical treatment of several other L-aspartyl peptides also resulted in the formation of new methyltransferase substrates. This general method for converting normal aspartyl peptides to isoaspartyl peptides may have application in the reverse process as well.     

Conformational analysis of cysteine-containing peptides and prospects of their application to 213Bi chelating in antitumor therapy

The method of conformational analysis was applied to the spatial structures of peptide analogues of phytochelatins and some fragments of metallothioneins: (Cys-Gly)3, (Cys-Gly)3Asp, (Cys-Gly)3Glu, (Cys-betaAla)3, (Cys-gammaGlu)3, and (Cys-Gly-Gly)3. All the possible low-energy conformations of the molecules were revealed and the role of intra- and inter-residual interactions in the formation of their spatial structures was determined. A different tendency of the molecules under study for acceptance of conformations favorable for binding bismuth ions was shown. Low-energy structures providing an optimum binding of bismuth ion were shown to be most frequent for (Cys-betaAla)3 peptide. Among the analogues of peptide fragments of the metallothioneins, lacking in natural peptides, low-energy pentapeptide CCXXC fragments (where X = Gln, Asn, Phe, Tyr, and Gly) were revealed. In the alpha-helical conformations of these pentapeptides, the distance between the sulfur atoms corresponds to that in Bi2S3. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2006, vol. 32, no. 3; see also http://www.maik.ru.     

Conformational Recognition of an Intrinsically Disordered Protein

There is a growing interest in understanding the properties of intrinsically disordered proteins (IDPs); however, the characterization of these states remains an open challenge. IDPs appear to have functional roles that diverge from those of folded proteins and revolve around their ability to act as hubs for protein-protein interactions. To gain a better understanding of the modes of binding of IDPs, we combined statistical mechanics, calorimetry, and NMR spectroscopy to investigate the recognition and binding of a fragment from the disordered protein Gab2 by the growth factor receptor-bound protein 2 (Grb2), a key interaction for normal cell signaling and cancer development. Structural ensemble refinement by NMR chemical shifts, thermodynamics measurements, and analysis of point mutations indicated that the population of preexisting bound conformations in the free-state ensemble of Gab2 is an essential determinant for recognition and binding by Grb2. A key role was found for transient polyproline II (PPII) structures and extended conformations. Our findings are likely to have very general implications for the biological behavior of IDPs in light of the evidence that a large fraction of these proteins possess a specific propensity to form PPII and to adopt conformations that are more extended than the typical random-coil states.     

Conformational Recognition of an Intrinsically Disordered Protein

There is a growing interest in understanding the properties of intrinsically disordered proteins (IDPs); however, the characterization of these states remains an open challenge. IDPs appear to have functional roles that diverge from those of folded proteins and revolve around their ability to act as hubs for protein-protein interactions. To gain a better understanding of the modes of binding of IDPs, we combined statistical mechanics, calorimetry, and NMR spectroscopy to investigate the recognition and binding of a fragment from the disordered protein Gab2 by the growth factor receptor-bound protein 2 (Grb2), a key interaction for normal cell signaling and cancer development. Structural ensemble refinement by NMR chemical shifts, thermodynamics measurements, and analysis of point mutations indicated that the population of preexisting bound conformations in the free-state ensemble of Gab2 is an essential determinant for recognition and binding by Grb2. A key role was found for transient polyproline II (PPII) structures and extended conformations. Our findings are likely to have very general implications for the biological behavior of IDPs in light of the evidence that a large fraction of these proteins possess a specific propensity to form PPII and to adopt conformations that are more extended than the typical random-coil states.     

Studies of conformational isomerism in α-melanocyte stimulating hormone by design of cyclic analogues

Results of energy calculations for α-MSH (α-melanocyte stimulating hormone, Ac-Ser¹-Tyr²-Ser³-Met⁴-Glu⁵-His⁶-Phe⁷-Arg⁸-Trp⁹-Gly¹⁰-Lys¹¹-Pro¹²-Val¹³-NH₂) and [D -Phe⁷]α-MSH were used for design of cyclic peptides with the general aim to stabilize different conformational isomers of the parent compound. The minimal structural modifications of the conformationally flexible Gly¹⁰ residue, as substitutions for L -Ala, D -Ala, or Aib (replacing of hydrogen atoms by methyl groups), were applied to obtain octa- and heptapeptide analogues of α-MSH(4–11) and α-MSH(5–11), which were cyclized by lactam bridges between the side chains in positions 5 and 11. Some of these analogues, namely those with substitutions of the Gly¹⁰ residue with L -Ala or Aib, showed biological activity potencies on frog skin comparable to the potency of the parent tridecapeptide hormone. Additional energy calculations for designed cyclic analogues were used for further refinement of the model for the biologically active conformations of the His-Phe-Arg-Trp “message” sequence within the sequences of α-MSH and [D -Phe⁷]α-MSH. In such conformations the aromatic moieties of the side chains of the His⁶, L/D -Phe⁷, and Trp⁹ residues form a continuous hydrophobic “surface,” presumably interacting with a complementary receptor site. This feature is characteristic for low-energy conformers of active cyclic analogues, but it is absent in the case of inactive analogues. This particular spatial arrangement of functional groups involved in the message sequence is very close for α-MSH and [D -Phe⁷]α-MSH, as well as for biologically active cyclic analogues despite differences of dihedral angle values for corresponding low-energy conformations. © 1998 John Wiley & Sons, Inc. Biopoly 46: 155–167, 1998     

Spatial structure of BAM-12P dodecapeptide and its analogues

Theoretical conformational analysis was used to study the spatial structure and conformational properties of the bovine adrenal medulla dodecapeptide BAM-12P (Tyr1-Gly2-Gly3-Phe4-Met5-Arg6-Arg7-Val8-Gly9-Arg10-Pro11-Glu12). Twenty-three low-energy conformations of the BAM-12P backbone were shown to represent the spatial structure of the peptide. The inverse structural problem was solved, and synthetic analogues of BAM-12P were proposed, the spatial structures of which correspond to a set of low-energy potentially physiologically active conformations of the natural dodecapeptide. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 3; see also http://www.maik.ru.     

Studies of the receptor-bound conformation of alphaIIbbeta3 antagonists by 15N-edited NMR spectroscopy

We report the results of NMR studies and computer simulations of potent antagonists reflective of the alpha(IIb)beta(3) receptor-bound conformations. The peptides c[Mpa-(15)N-Arg(1)-(15)N-Gly(2)-(15)N-Asp(3)-(15)N-Phe(4)-(15)N-Arg(5)-Cys]-NH(2) (Phe-Arg analog) (Mpa: 3-mercaptopropionic acid) and c[Mpa-(15)N-Arg(1)-(15)N-Gly(2)-(15)N-Asp(3)-(15)N-Asp(4)-(15)N-Val(5)-Cys]-NH(2) (Asp-Val analog) were subjected to (15)N-edited NMR experiments to study the conformations of these peptides in the absence and in the presence of alpha(IIb)beta(3) receptor. The NMR studies of the Phe-Arg analog, a selective alpha(IIb)beta(3) antagonist, resulted in distinctly different experimental data in the presence and absence of the receptor. The computer simulations for this peptide resulted in one large family of structures consistent with the experimental data. This conformation suggests a type I beta-turn spanning residues Arg(1) and Gly(2) when bound to the receptor and we were able to establish a model for the three dimensional arrangement of the pharmacophores. The studies on the Asp-Val analog, an alpha(v)beta(3) antagonist that binds to the alpha(IIb)beta(3) with moderate affinity, resulted in conformations that are not as well defined as those for the Phe-Arg analog but are consistent with the model established for this analog. These results are important for the design of novel alpha(IIb)beta(3) antagonists.     

Spatial Structure of BAM-12P Dodecapeptide and Its Analogues

Theoretical conformational analysis was used to study the spatial structure and conformational properties of the bovine adrenal medulla dodecapeptide BAM-12P (Tyr1-Gly2-Gly3-Phe4-Met5-Arg6-Arg7-Val8-Gly9-Arg10-Pro11-Glu12). Twenty-three low-energy conformations of the BAM-12P backbone were shown to represent the spatial structure of the peptide. The inverse structural problem was solved, and synthetic analogues of BAM-12P were proposed, the spatial structures of which correspond to a set of low-energy potentially physiologically active conformations of the natural dodecapeptide.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 3, 2005, pp. 245–250.Original Russian Text Copyright © 2005 by Akhmedov, Tagiev, Hasanov, Makhmudova.     

Structure-activity relationships of simplified resiniferatoxin analogues with potent VR1 agonism elucidates an active conformation of RTX for VR1 binding

We previously described a series of N-(3-acyloxy-2-benzylpropyl) homovanillate and N'-(4-hydroxy-3-methoxybenzyl) thiourea derivatives that were potent VR1 agonists with high-affinities and excellent analgesic profiles. The design of these simplified RTX analogues was based on our RTX-derived pharmacophore model which incorporates the 4-hydroxy-3-methoxyphenyl (A-region), C(20)-ester (B-region), orthophenyl (C1-region) and C(3)-keto (C2-region) groups of RTX. For the purpose of optimizing the spatial arrangement of the four principal pharmacophores on the lead agonists (1-4), we have modified the distances in the parent C-region, 3-acyloxy-2-benzylpropyl groups, by lengthening or shortening one carbon to vary the distances between the pharmacophores. We find that two of the amides, 4 and 19, possess EC(50) values <1 nM for induction of calcium influx in the VR1-CHO cells. As observed previously, the structure-activity relations for inhibition of RTX binding to VR1 and for induction of calcium uptake were distinct, presumably reflecting both intrinsic and methodological factors. In order to find the active conformation of VR1 ligands, the energy-minimized conformations of seven selected agonists were determined and the positions of their four pharmacophores were matched with those of five low energy RTX conformations. The rms values for the overlaps in the pharmacophores were calculated and correlated with the measured binding affinities (K(i)) and calcium influx (EC(50)) values. The binding affinities of the agonists correlated best with the RMS values derived from RTX conformation E (r(2)=0.92), predicting a model of the active conformation of RTX and related vanilloids for binding to VR1. Poorer correlation was obtained between any of the conformations and the EC(50) values for calcium influx.     

Structural modelling and mutation analysis of a nociceptin receptor and its ligand complexes

We recently modelled and proposed four ligand-bound conformations for a G-protein-coupled receptor, namely, forms I, II, III and IV, based on the 3D structure and functional evidences for rhodopsin. In this study, the same strategy was applied to a human nociceptin receptor (NR), in order to predict ligand-bound receptor structures. Additionally, site-directed mutagenesis studies were carried out to evaluate these structures. A Thr138Ala mutant demonstrated the same affinity for [Phe(1)Psi(CH(2)-NH)Gly(2)]nociceptin(1-13)NH(2) as the wild-type receptor; however, the affinity of this mutant for nociceptin was 20-fold lower than that of the wild type. A Ser223Ala mutation showed the same characteristics as those of the wild type. On the other hand, a Gln280Ala mutation reduced the affinity to nociceptin by more than 60-folds. These results suggested that a change in the conformation of NR following agonist binding did not accompany the rigid-body rotation of the sixth transmembrane segment that was reported for an adrenergic receptor and a kappa-opioid receptor. NR is potently activated not only by nociceptin but also a synthetic peptide, i.e. Ac-RYYRIK-NH(2), although the amino acid sequences of both these ligands are completely different. The model explains why both the ligands activate NR and shows that their receptor-bound conformations have similar 3D structures.     

Molecular docking and 3-D-QSAR studies on the possible antimalarial mechanism of artemisinin analogues

Artemisinin (Qinghaosu) is a natural constituent found in Artemisia annua L, which is an effective drug against chloroquine-resistant Plasmodium falciparum strains and cerebral malaria. The antimalarial activities of artemisinin and its analogues appear to be mediated by the interactions of the drugs with hemin. In order to understand the antimalarial mechanism and the relationship between the physicochemical properties and the antimalarial activities of artemisinin analogues, we performed molecular docking simulations to probe the interactions of these analogues with hemin, and then performed three-dimensional quantitative structure-activity relationship (3-D-QSAR) studies on the basis of the docking models employing comparative molecular force fields analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA). Molecular docking simulations generated probable 'bioactive' conformations of artemisinin analogues and provided a new insight into the antimalarial mechanism. The subsequent partial least squares (PLS) analysis indicates that the calculate binding energies correlate well with the experimental activity values. The CoMFA and CoMSIA models based on the bioactive conformations proved to have good predictive ability and in turn match well with the docking result, which further testified the reliability of the docking model. Combining these results, that is molecular docking and 3-D-QSAR, together, the binding model and activity of new synthesized artemisinin derivatives were well explained.     

Conformational analysis of β-methyl-para-nitrophenylalanine stereoisomers of cyclo[D-Pen2, D-Pen5]enkephalin by NMR spectroscopy and conformational energy calculations

Solution conformations of β-methyl-para-nitrophenylalanine⁴ analogues of the potent δ-opioid peptide cyclo[D-Pen², D-Pen⁵]enkephalin (DPDPE) were studied by combined use of nmr and conformational energy calculations. Nuclear Overhauser effect connectivities and ³J_HNC^α_H coupling constants measured for the (2S, 3S)-, (2S, 3R)-, and (2R, 3R)-stereoisomers of[β-Me-p-NO₂Phe⁴]DPDPE in DMSO were compared with low energy conformers obtained by energy minimization in the Empirical Conformational Energy Program for Peptides #2 force field. The conformers that satisfied all available nmr data were selected as probable solution conformations of these peptides. Side-chain rotamer populations, established using homonuclear (³J_H^α_H^β) and heteronuclear (³J_H^αC^γ) coupling constants and ¹³C chemical shifts, show that the β-methyl substituent eliminates one of the three staggered rotamers of the torsion angle x¹ for each stereoisomer of the β-Me-p-NO₂Phe⁴. Similar solution conformations were suggested for the L-Phe⁴-containing (2S, 3S)- and (2S, 3R)-stereoisomers. Despite some local differences, solution conformations of L- and D-Phe⁴-containing analogues have a common shape of the peptide backbone and allow similar orientations of the main δ-opioid pharmacophores. This type of structure differs from several models of the solution conformations of DPDPE, and from the model of biologically active conformations of DPDPE suggested earlier. The latter model is allowed for the potent (2S, 3S)- and (2S, 3R)-stereoisomers of [β-Me-p-NO₂Phe⁴] DPDPE, but it is forbidden for the less active (2R, 3R)- and (2R, 3S)-stereoisomers. It was concluded that the biologically active stereoisomers of [β-Me-p-No₂Phe⁴] DPDPE in the δ-receptor-bound state may assume a conformation different from their favorable conformations in DMSO. © 1996 John Wiley & Sons, Inc.     

The mid-region of parathyroid hormone (1-34) serves as a functional docking domain in receptor activation

Elucidating the bimolecular interface between parathyroid hormone (PTH) and its cognate G protein-coupled receptor (PTHR1) should yield insights into the basis of molecular recognition and the mechanism of ligand-mediated intracellular signaling for a system that is critically important in regulating calcium levels in blood. We used photoaffinity scanning (PAS) to identify key ligand-receptor interactions for residues from the unstructured mid-region domain of PTH-(1-34). Four PTH analogues, containing a single photoreactive p-benzoylphenylalanine (Bpa) residue in position 11, 15, 18, or 21, were found to photo-cross-link within receptor regions [165-176], [183-189], [190-298], and [165-176], respectively. Addition of these mid-region contacts as constraints to our previously proposed model of the PTH-PTHR1 complex and extensive molecular simulation experiments enables substantial refinement of the model. Specifically, (1) the overall receptor-bound conformation of the hormone is not extended, but bent; (2) helix [169-176] of the N-terminal extracellular domain (N-ECD) of the receptor is redirected toward the heptahelical bundle; and (3) the hormone traverses between the top of transmembrane (TM) helices 1 and 2, rather than between TM-7 and TM-1. This significantly alters the model of both the receptor-bound tertiary structure of the hormone and the topological orientation of the C-terminus of the N-ECD in the hormone-receptor bimolecular complex. We propose that the mid-region of PTH-(1-34) has a role in fixing, by extensive contacts with the receptor, the entry of the N-terminal helix of the hormone into the heptahelical bundle between TM-1 and TM-2. This anchorage would orient the amino terminus into position to activate the receptor.