Cooperative interaction of arginine-19 and the N-terminal signaling domain in the affinity and potency of parathyroid hormone.
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.
Tsomaia
Natia
N
Department of Molecular Pharmacology, Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912, USA.
Shimizu
Masaru
M
Shimizu
Naoto
N
Gardella
Thomas J
TJ
Mierke
Dale F
DF
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001120:Arginine / Q000737:chemistry*; D019556:COS Cells; D002417:Cattle; D002522:Chlorocebus aethiops; D002942:Circular Dichroism; D018698:Glutamic Acid / Q000737:chemistry; D005998:Glycine / Q000737:chemistry; D006801:Humans; D018374:LLC-PK1 Cells; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010281:Parathyroid Hormone / Q000138:chemical synthesis / Q000737:chemistry* / Q000502:physiology; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry* / Q000502:physiology; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D044168:Receptor, Parathyroid Hormone, Type 1; D018016:Receptors, Parathyroid Hormone / Q000737:chemistry; D015398:Signal Transduction / Q000502:physiology; D013552:Swine
D008024:Ligands; C473449:PTH1R protein, human; D010281:Parathyroid Hormone; D010446:Peptide Fragments; D044168:Receptor, Parathyroid Hormone, Type 1; D018016:Receptors, Parathyroid Hormone; C102836:parathyroid hormone (1-31)amide, human; D018698:Glutamic Acid; D001120:Arginine; D005998:Glycine