Residue 19 of the Parathyroid Hormone: Structural Consequences

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Abstract
Residue 19 of the parathyroid hormone (PTH) has been shown to play an important role in both binding to and activation of the PTH receptor; specifically, Arg19-containing analogues have improved biological function over similar Glu19 peptides {Shimizu et al. (2002) Biochemistry41, 13224−13233}. Additionally the juxtamembrane portion of the receptor is involved in the different biological responses. Here, we determine the conformational preferences of PTH analogues to provide a structural basis for their biological actions. On the basis of circular dichroism results, the Arg19 → Glu19 mutations within the context of both PTH(1−20) and PTH(1−34) analogues lead to increases in helix content, ranging from a 8−15% increase. High-resolution structures as determined by 1H NMR and NOE-restrained molecular dynamics simulations clearly illustrate the difference between Arg19 and Glu19−PTH(1−20), particularly with the extent and stability of the C-terminal helix. The Arg19-containing analogue has a well defined, stable α-helix from Ser4−Arg19, while the Glu19 analogue is less ordered at the C-terminus. On the basis of these observations, we propose that position 19 of PTH(1−20) must be α-helical for optimal interaction with the juxtamembrane portion of the receptor. This mode of binding extends the current view of PTH binding (indeed ligand binding for all class B GPCRs), which invokes a bihelical ligand with the C-terminus of the ligand interacting with the N-terminus of the receptor (responsible for binding) and the N-terminus of the ligand interacting with the seven-helical bundle (leading to receptor activation).