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The Bioactive Conformation of Human Parathyroid Hormone. Structural Evidence for the Extended Helix Postulate

2000; American Chemical Society; Volume: 122; Issue: 13 Linguagem: Inglês

10.1021/ja994033u

1943-2984

Stephen M. Condon, Isabelle Morize, Shelley Darnbrough, Christopher J. Burns, Bruce E. Miller, Joanne Uhl, Kathleen A. Burke, N. Jariwala, Kenneth T. Locke, Paul Krolikowski, N. Vasant Kumar, Richard Labaudinière,

Protein Kinase Regulation and GTPase Signaling

Conformational restrictions in the form of [i, i + 4] lactam bridges were sequentially incorporated into the shortest fragment of hPTH with recognized efficacy in the OVX rat model of osteoporosis, hPTH(1−31)NH2 (1). Cyclo(Lys18−Asp22)[Ala1,Nle8,Lys18,Asp22,Leu27]hPTH(1−31)NH2 (2) is a potent agonist of the PTH/PTHrP receptor located on the surface of ROS 17/2.8 cells as measured by its ability to stimulate adenylyl cyclase activity (EC50 = 0.29 nM). A second analogue, which constrains the entire C-terminal receptor binding domain, bicyclo(Lys18−Asp22,Lys26−Asp30)[Ala1,Nle8,Lys18,Asp22,Leu27] hPTH(1−31)NH2 (6), is also shown to be a potent agonist (EC50 = 0.13 nM), thus providing further evidence for an extended helix as the relevant bioactive conformation in this region of the hormone. Adjacent lactam bridges were incorporated into the analogue bicyclo(Lys13−Asp17,Lys18−Asp22)[Ala1,Nle8,Lys18,Asp17,22,Leu27]hPTH(1−31)NH2 (7) to evaluate the receptor's tolerance to conformational restriction in the midregion of the peptide. In fact, peptide 7 is also a highly potent agonist (EC50 = 0.43 nM) in the cAMP-based assay, which suggests that at least one bioactive form of the hormone requires a helical conformation extending from residue 13 to residue 22. Incorporation of all three lactam bridges afforded the most conformationally constrained PTH peptide agonist yet reported, tricyclo(Lys13−Asp17,Lys18−Asp22,Lys26−Asp30)[Ala1,Nle8,Lys18,Asp17,22,Leu27]hPTH(1−31)NH2 (9). Peptide 9 (EC50 = 0.14 nM) forces residues 13−30 into an extended helical conformation and is a more potent PTH receptor agonist than the parent linear hPTH(1−31)NH2 (1, EC50 = 4.7 nM). Comparative circular dichroism studies indicate that peptide 9 is highly helical even in the absence of TFE, indicating that residues 1−12 are also likely to be helical in the bioactive conformation. Taken together, these results provide strong structural evidence that hPTH binds to its receptor in an extended helical conformation.