Toward parathyroid hormone minimization: conformational studies of cyclic PTH(1-14) analogues.
The N-terminal fragment of PTH(1-34) is critical for PTH1 receptor activation. Various modifications of PTH(1-14) have been shown to result in a considerable increase in signaling potency [Shimizu et al. (2000) J. Biol. Chem. 275, 21836-21843]. Our structural investigations revealed an unusually stable helical structure of the signaling domain (1-14), where residues 6 (Gln) and 10 (Gln or Asn) were located on the same face of the alpha-helix. To test whether a stable N-terminal alpha-helix is required for productive interaction with PTH1 receptor, we designed two conformationally restricted PTH(1-14) analogues, each containing a lactam bridge at positions 6 and 10. Specifically, substitutions Gln(6)-->Glu(6) and Asn(10)-->Lys(10) were introduced into the most potent [Ala(1,3,12),Gln(10),Har(11),Trp(14)]PTH(1-14)NH2 agonist. Both the Glu(6)-Lys(10) and Lys(6)-Glu(10) lactam-bridged analogues were characterized to examine the importance of orientation of the lactam. According to biological studies [Shimizu et al. (2003) Biochemistry 42, 2282-2290], none of the 6/10 substituted analogues (linear or cyclic) remained as active as the parent peptide. However, relative to their corresponding linear peptides, lactam-bridged analogues either maintained potency or showed 6-fold improvement. High-resolution structures as determined by 1H NMR and NOE-restrained molecular dynamics simulations clearly illustrate the structural differences between the linear and cyclic PTH(1-14) fragments, supporting the hypothesis that an alpha-helix is the preferred bioactive conformation of the N-terminal fragment of PTH. In addition, our results demonstrate that the structural order of the very first residues (1-4) of the signaling domain plays a significant role in PTH action.
Tsomaia
Natia
N
Department of Molecular Pharmacology, Division of Biology & Medicine, Brown University, Providence, Rhode Island 02912, USA.
Pellegrini
Maria
M
Hyde
Kimberly
K
Gardella
Thomas J
TJ
Mierke
Dale F
DF
D019943:Amino Acid Substitution / Q000235:genetics; D003198:Computer Simulation; D006801:Humans; D007769:Lactams / Q000737:chemistry; D008024:Ligands; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D010281:Parathyroid Hormone / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010456:Peptides, Cyclic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D017433:Protein Structure, Secondary / Q000235:genetics; D044168:Receptor, Parathyroid Hormone, Type 1; D018016:Receptors, Parathyroid Hormone / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics; D012997:Solvents; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D007769:Lactams; D008024:Ligands; C473449:PTH1R protein, human; D010281:Parathyroid Hormone; D010446:Peptide Fragments; D010456:Peptides, Cyclic; D044168:Receptor, Parathyroid Hormone, Type 1; D018016:Receptors, Parathyroid Hormone; D012997:Solvents; C430893:parathyroid hormone (1-14)amide