Structure-Activity Relation of NH2-terminal Human Parathyroid Hormone Fragments

Abstract

Human parathyroid hormone (hPTH) is involved in the regulation of the calcium level in blood. This hormone function is located in the NH 2 -terminal 34 amino acids of the 84-amino acid peptide hormone and is transduced via the adenylate cyclase and the phosphatidylinositol signaling pathways. It is well known that truncation of the two NH 2 -terminal amino acids of the hormone leads to complete loss of in vivo normocalcemic function. To correlate loss of calcium level regulatory activity after stepwise NH 2 -terminal truncation and solution structure, we studied the conformations of fragments hPTH-(2-37), hPTH-(3-37), and hPTH-(4 -37) in comparison to hPTH-(1-37) in aqueous buffer solution under near physiological conditions by circular dichroism spectroscopy, two-dimensional nuclear magnetic resonance spectroscopy, and restrained molecular dynamics calculations. All peptides show helical structures and hydrophobic interactions between Leu-15 and Trp-23 that lead to a defined loop region from His-14 to Ser-17. A COOH-terminal helix from Met-18 to at least Leu-28 was found for all peptides. The helical structure in the NH 2 -terminal part of the peptides was lost in parallel with the NH 2 -terminal truncation and can be correlated with the loss of calcium regulatory activity.

International Union of Basic and Clinical Pharmacology. XCIII. The Parathyroid Hormone Receptors—Family B G Protein–Coupled Receptors

“…Although a good deal of variation is seen in the structures obtained from these studies, depending on the specific experimental conditions, e.g., peptide sequence, solvent systems used, a generally consistent finding is the presence of a relatively stable a-helical segment that extend for several turns within the approximate (15-34) region, with the N-terminal portion tending to be more random or to contain a short segment of weak a-helix Kemp, 1994, 1996;Marx et al, 1998Marx et al, , 2000Chen et al, 2000). TIP39 has also revealed a bihelical domain structure (Piserchio et al, 2000b) (Fig.…”

“…The presence of backbone flexibility or even a b-turn motif between the amino and C-terminal domain has raised the possibility that the bioactive PTH(1-34) peptide ligand adopts a folded or U-shaped structure (Marx et al, 1998(Marx et al, , 2000Chen et al, 2000). One of the NMR studies, however, specifically addressed this question for PTH(1-34) and failed to find evidence for interdomain tertiary interactions and thus argued against such a U-shaped structure .…”

“…First, the deletion of residues 1 and 2 in PTH peptides has been seen in solution phase NMR studies to result in a marked loss in the N-terminal a-helix (Marx et al, 1998). Valine-2, one of the most conserved residues in PTH and PTHrP ligands and a critical determinant of receptor activation, may thus not only form direct contacts with the receptor but help stabilize the bioactive ligand conformation.…”

Purification and Characterization of a Receptor for Human Parathyroid Hormone and Parathyroid Hormone-related Peptide

“…Understanding hormone-PTH1R and PTH1R-G protein interactions has relied largely on determination of functional consequences resulting from mutations in either the hormone or the receptor (13,(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36), analysis of receptor fragments after cross-linking to radioiodinated, p-benzoyl-L-phenylalaninemodified ligands (13,(37)(38)(39)(40), crystallographic resolution of PTH (41), and NMR of PTH (42)(43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53)(54)(55)(56)(57)(58), PTHrP (59 -68), and short segments of the PTH1R (see Refs. 69 and 70 and for review see Refs.…”

In situ growth of a PEG-like polymer from the C terminus of an intein fusion protein improves pharmacokinetics and tumor accumulation

“…Although this strategy is useful for a subset of proteins and peptide drugs to improve their pharmacological profiles, many proteins and peptide drugs -interferon alpha, glucagon-like peptide-1 (GLP-1), and parathyroid hormone are three examples-cannot be modified at their N terminus because their active site is at or near the N terminus of the molecule (22)(23)(24). Given these constraints on the modification of the N terminus of many pharmacologically relevant protein and peptide drugs, and the need for a toolbox of complementary and widely applicable methods for site-specific and stoichiometric polymer conjugation, we turned our attention to the C terminus, because it is the only other ubiquitous and universal site on a proteins' scaffold.…”