Cooperative interaction of arginine-19 and the N-terminal signaling domain in the affinity and potency of parathyroid hormone |
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Authors: | Tsomaia Natia Shimizu Masaru Shimizu Naoto Gardella Thomas J Mierke Dale F |
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Institution: | Department of Molecular Pharmacology, Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912, USA. |
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Abstract: | Residue 19 of parathyroid hormone (PTH) plays a unique role in the interaction process with the PTH1 receptor. A Glu(19) --> Arg(19) substitution, based on the Arg(19) of the PTH-related protein (PTHrP), increases the binding affinity when incorporated into the N-terminus of PTH i.e., PTH(1-20)] and has no effect when introduced into the C-terminus of PTH i.e., PTH(15-31)]. To explore Arg(19) and the midregion (residues 10-15), we designed the novel PTH scaffold peptide, PG5, which has the PTH(1-9) domain linked to the PTH(15-31) segment via a pentaglycine spacer. Substitution of Glu(19) with Arg(19) in PG5 resulted in a 9-fold increase in binding affinity. Additionally, the substitution enhanced stimulated cAMP formation in cells expressing PTH1-delNt, a PTH1 receptor construct lacking most of the N-terminus, confirming that residue 19 is interacting with the juxtamembrane portion of PTH1. The binding and signaling capacities of the PG5 analogues were diminished relative to those of PTH(1-34), indicating that the residue 10-14 region of PTH provides more than just a simple linker function. To probe this further, the structural consequences of the glycine linker and its interaction with PTH1 were examined by circular dichroism, (1)H NMR, and extensive ligand/receptor molecular dynamics simulations. The structural data clearly illustrate the helix-stabilizing effect of Arg(19) substitution propagating N-terminally from position 19 to the pentaglycine linker. Overall, these studies suggest that an alpha-helix is the preferred conformation for the residue 15-20 region of PTH and that residues 10-14 are also required for full affinity and potency of the hormone. |
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