Cooperative Interaction of Arginine-19 and the N-Terminal Signaling Domain in the Affinity and Potency of Parathyroid Hormone

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Abstract
Residue 19 of parathyroid hormone (PTH) plays a unique role in the interaction process with the PTH1 receptor. A Glu19 → Arg19 substitution, based on the Arg19 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 Arg19 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 Glu19 with Arg19 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, 1H NMR, and extensive ligand/receptor molecular dynamics simulations. The structural data clearly illustrate the helix-stabilizing effect of Arg19 substitution propagating N-terminally from position 19 to the pentaglycine linker. Overall, these studies suggest that an α-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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