Conformational studies of parathyroid hormone (PTH)/PTH-related protein (PTHrP) point-mutated hybrids

Peggion, Evaristo; Mammi, Stefano; Schievano, Elisabetta; Behar, Vered; Rosenblatt, Michael; Chorev, Michael

doi:10.1002/(sici)1097-0282(19991015)50:5<525::aid-bip6>3.0.co;2-8

Cited by 12 publications

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References 22 publications

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“…26 Conformational analysis carried out by combined CD, nmr, and computer simulations in the DPC micellar system clearly showed that the highly potent (Val 21 )PTHrP(1-34)NH 2 analogue exhibits three-helical segments connected by two flexible points in the region 12-13 and 18 -19. In the weakly active hybrid (Glu 19 )PTHrP(1-34)-NH 2 , the second flexible region is shifted to the segment 20 -25, close to the principal receptor binding domain.…”

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confidence: 99%

Conformational studies of parathyroid hormone (PTH)/PTH-related protein (PTHrp) chimeric peptides

et al. 2000

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The N‐terminal 1–34 segments of both parathyroid hormone (PTH) and parathyroid hormone‐related protein (PTHrP) bind and activate the same membrane‐embedded G protein‐coupled receptor (PTH1 Rc) present on the surface of cells in target tissues such as bone and kidney. This binding occurs in spite of major differences between the two hormones in their amino acid sequence. Recently, it was shown that in (1–34) PTH/PTHrP hybrid peptides, the N‐terminal 1–14 segment of PTHrP is incompatible with the C‐terminal 15–34 region of PTH in terms of bioactivity. The sites of incompatibility were identified at positions 5 in PTHrP and 19 in PTH. In the present paper we describe the synthesis, biological evaluation, and conformational characterization of two segmental hybrids: PTHrP(1–27)‐[Tyr34]bPTH(28–34)‐NH2 (hybrid I) and PTHrP(1–18)‐[Nal23, Tyr34]bPTH(19–34)‐NH2 (hybrid II). Hybrid I is as active as PTH(1–34)NH2 and more than two orders of magnitude more active than hybrid II. The conformational properties of the hybrids were studied in water/trifluoroethanol (TFE) mixtures and in aqueous solutions containing dodecylphosphocholine (DPC) micelles by CD, two‐dimensional nmr and computer simulations. Upon addition of TFE to the aqueous solution, both hybrids undergo a coil–helix transition. The helix content in 1:1 water/TFE obtained by CD data is about 75% for both hybrids. In the presence of DPC, helix formation is observed at detergent concentrations above critical micellar concentration and the maximum helix content is of ∼35 and ∼30% for hybrid I and II, respectively. Combined nmr analysis, distance geometry, and molecular dynamics calculations suggest that, in both solvent systems, the biologically active hybrid I exhibits two flexible sites, centered at residues 12 and 19, connecting helical segments. The flexibility point at position 19 is not present in the poorly active hybrid II. Our findings support the hypothesis, proposed in our previous work, that in bioactive PTH analogues the presence and location of flexibility points between helical segments are essential for enabling them to fold into the bioactive conformation upon interaction with the PTH1 receptor. © 2000 John Wiley & Sons, Inc. Biopoly 54: 429–447, 2000

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Section: Discussionmentioning

confidence: 99%

Conformational studies of parathyroid hormone (PTH)/PTH-related protein (PTHrp) chimeric peptides

et al. 2000

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Structure–function relationship studies of bovine parathyroid hormone [bPTH(1–34)] analogues containing α‐amino‐iso‐butyric acid (Aib) residues

et al. 2003

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The N-terminal 1-34 fragments of the parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) elicit the full spectrum of bone-related biological activities of the intact native sequences. It has been suggested that the structural elements essential for bioactivity are two helical segments located at the N-terminal and C-terminal sequences, connected by hinges or flexible points around positions 12 and 19. In order to assess the relevance of the local conformation around Gly(12) upon biological function, we synthesized and characterized the following PTH(1-34) analogues containing Aib residues: (I) A-V-S-E-I-Q-F-nL-H-N-Aib-G-K-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-Y-NH(2) ([Nle(8,18), Aib(11), Nal(23),Tyr(34)]bPTH(1-34)-NH(2)); (II) A-V-S-E-I-Q-F-nL-H-N-L-Aib-K-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-Y-NH(2) ([Nle(8,18), Aib(12),Nal(23),Tyr(34)]bPTH(1-34)-NH(2)); (III) A-V-S-E-I-Q-F-nL-H-N-L-G-Aib-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-Y-NH(2) ([Nle(8,18), Aib(13), Nal(23),Tyr(34)]bPTH(1-34)-NH(2)); (IV) A-V-S-E-I-Q-F-nL-H-N-Aib-Aib-K-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-YNH(2) ([Nle(8,18), Aib(11,12), Nal(23),Tyr(34)]bPTH(1-34)-NH(2)); (V) A-V-S-E-I-Q-F-nL-H-N-L-Aib-Aib-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-Y-NH(2) ([Nle(8,18), Aib(12,13),Nal(23),Tyr(34)]bPTH(1-34)-NH(2)). (nL= Nle; Nal= L-(2-naphthyl)-alanine; Aib= alpha-amino-isobutyric acid.) The introduction of Aib residues at position 11 in analogue I or at positions 11 and 12 in analogue IV resulted in a 5-20-fold lower efficacy and a substantial loss of binding affinity compared to the parent compound [Nle(8,18), Nal(23),Tyr(34)]bPTH(1-34)-NH(2). Both binding affinity and adenylyl cyclase stimulation activity are largely restored when the Aib residues are introduced at position 12 in analogue II, 13 in analogue III, and 12-13 in analogue V. The conformational properties of the analogues in aqueous solution containing dodecylphosphocholine micelles were studied by CD, two-dimensional (2D) NMR and computer simulations. The results indicated the presence of two helical segments in all analogues, located at the N-terminal and C-terminal sequences. Insertion of Aib residues at positions 12 and 13, or of Aib dyads at positions 11-12 and 12-13, enhances the stability of the N-terminal helix of all analogues. In all analogues the Aib residues are included in the helical segments. These results confirmed the importance of the helical structure in the N-terminal activation domain, as well as of the presence of the Leu(11) hydrophobic side chain in the native sequence, for PTH-like bioactivity.

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Conformational and biological characterization of human parathyroid hormone hPTH(1–34) analogues containing β‐amino acid residues in positions 17–19

et al. 2003

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The N-terminal 1-34 fragment of parathyroid hormone (PTH) elicits the full spectrum of bone-related biological activities of the intact native sequences. It has been suggested that the structural elements essential for bioactivity are two helical segments located at the N-terminal and C-terminal sequences, connected by hinges or flexible points around positions 12 and 19. In order to assess the relevance of the local conformation around Gly(18) upon biological function, we synthesized and characterized the following human (h) PTH(1-34) analogues containing beta-amino acid residues: [analogues: see text]. Biological activity and binding affinity of analogue I are one order of magnitude lower than those of the parent compound. In analogue II, both binding affinity and biological activity are partially recovered. Analogues III and V have no binding affinity and very low biological activity. Both bioactivity and binding affinity are partially recovered in analogue IV. The conformational properties of the analogues in aqueous solution containing dodecylphosphocholine micelles were studied by CD, 2D-nuclear magnetic resonance and molecular dynamics calculations. The results confirmed the presence in all analogues of two helical segments located at the N-terminal and C-terminal sequences. The insertion of beta-amino acid residues around position 18 does not cause appreciable conformational differences in the five analogues. The differences in biological activity and binding affinity among the five analogues cannot be related to structural differences in the membrane mimetic environment reported in this study. Our results stress the importance of the side-chain functionalities in the sequence 17-19 for biological function.

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Conformational studies of parathyroid hormone (PTH)/PTH-related protein (PTHrP) point-mutated hybrids

Cited by 12 publications

References 22 publications

Conformational studies of parathyroid hormone (PTH)/PTH-related protein (PTHrp) chimeric peptides

Conformational studies of parathyroid hormone (PTH)/PTH-related protein (PTHrp) chimeric peptides

Structure–function relationship studies of bovine parathyroid hormone [bPTH(1–34)] analogues containing α‐amino‐iso‐butyric acid (Aib) residues

Conformational and biological characterization of human parathyroid hormone hPTH(1–34) analogues containing β‐amino acid residues in positions 17–19

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