C-terminal analogues of parathyroid hormone: effect of C-terminus function on helical structure, stability, and bioactivity.
We have studied the effects of C-terminal group modifications (amide, methylamide, dimethylamide, aldehyde, and alcohol) on the conformation, adenylyl cyclase stimulation (AC), or binding of parathyroid hormone (hPTH) analogues, hPTH(1-28)NH(2) and hPTH(1-31)NH(2). hPTH(1-31)NH(2) has a C-terminal alpha-helix bounded by residues 17-29 [Chen, Z., et al. (2000) Biochemistry 39, 12766]. In both cases, relative to the natural analogue with a carboxyl C-terminus, the amide and methylamide had increased helix content whereas the dimethylamide forms had CD spectra more similar to the carboxyl one. Conformational effects were more pronounced with hPTH(1-28) than with hPTH(1-31), with increases in helix content of approximately 30% in contrast to 10%. Stabilization of the C-terminal helix of residues 1-28 seemed to correlate with an ability of the C-terminal function to H-bond appropriately. None of the analogues affected the AC stimulating activity significantly, but there was an up to 15-fold decrease in the level of apparent binding of the carboxyl hPTH(1-28) analogue compared to that of the methylamide and a 4-fold decrease in the level of binding of the aldehyde or dimethylamide. There was no significant change in binding activities for the 1-31 analogues. These observations are consistent with previous studies that imply the importance of a region of the hormone's C-terminal alpha-helix for tight binding to the receptor. They also show that modulation of helix stability does have an effect on the binding of the hormone, but only when the C-terminus is at the putative end of the helix. The similarity of AC stimulation even when binding changed 10-fold can be explained by assuming greater efficacy of the weaker binding PTH-receptor complexes in stimulating AC.
Potetinova
Zhanna
Z
Institute for Biological Sciences, National Research Council, Ottawa, Ontario, Canada K1A 0R6.
Barbier
Jean-René
JR
Suen
Tanya
T
Dean
Thomas
T
Gardella
Thomas J
TJ
Willick
Gordon E
GE
D000818:Animals; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D008253:Macaca mulatta; D008958:Models, Molecular; D010281:Parathyroid Hormone / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013816:Thermodynamics
D010281:Parathyroid Hormone; D010446:Peptide Fragments; C400002:parathyroid hormone (1-28); C101312:parathyroid hormone (1-31); C102836:parathyroid hormone (1-31)amide, human