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

详细信息    查看全文

• 作者：Stephen M. Condon ; Isabelle Morize ; Shelley Darnbrough ; Christopher J. Burns ; Bruce E. Miller ; Joanne Uhl ; Kathleen Burke ; Navinchandra Jariwala ; Kenneth Locke ; Paul H. Krolikowski ; N. Vasant Kumar ; a
• 刊名：Journal of the American Chemical Society
• 出版年：2000
• 出版时间：April 5, 2000
• 年：2000
• 卷：122
• 期：13
• 页码：3007 - 3014
• 全文大小：180K
• 年卷期：v.122,no.13(April 5, 2000)
• ISSN：1520-5126

文摘

Conformational restrictions in the form of [i, i + 4] lactam bridges were sequentially incorporatedinto 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 ofthe PTH/PTHrP receptor located on the surface of ROS 17/2.8 cells as measured by its ability to stimulateadenylyl cyclase activity (EC₅₀ = 0.29 nM). A second analogue, which constrains the entire C-terminal receptorbinding domain, bicyclo(Lys¹⁸-Asp²²,Lys²⁶-Asp³⁰)[Ala¹,Nle⁸,Lys¹⁸,Asp²²,Leu²⁷] hPTH(1-31)NH₂ (6), is alsoshown to be a potent agonist (EC₅₀ = 0.13 nM), thus providing further evidence for an extended helix as therelevant bioactive conformation in this region of the hormone. Adjacent lactam bridges were incorporated intothe analogue bicyclo(Lys¹³-Asp¹⁷,Lys¹⁸-Asp²²)[Ala¹,Nle⁸,Lys¹⁸,Asp^17,22,Leu²⁷]hPTH(1-31)NH₂ (7) to evaluatethe receptor's tolerance to conformational restriction in the midregion of the peptide. In fact, peptide 7 is alsoa highly potent agonist (EC₅₀ = 0.43 nM) in the cAMP-based assay, which suggests that at least one bioactiveform of the hormone requires a helical conformation extending from residue 13 to residue 22. Incorporationof all three lactam bridges afforded the most conformationally constrained PTH peptide agonist yet reported,tricyclo(Lys¹³-Asp¹⁷,Lys¹⁸-Asp²²,Lys²⁶-Asp³⁰)[Ala¹,Nle⁸,Lys¹⁸,Asp^17,22,Leu²⁷]hPTH(1-31)NH₂ (9). Peptide9 (EC₅₀ = 0.14 nM) forces residues 13-30 into an extended helical conformation and is a more potent PTHreceptor agonist than the parent linear hPTH(1-31)NH₂ (1, EC₅₀ = 4.7 nM). Comparative circular dichroismstudies indicate that peptide 9 is highly helical even in the absence of TFE, indicating that residues 1-12 arealso likely to be helical in the bioactive conformation. Taken together, these results provide strong structuralevidence that hPTH binds to its receptor in an extended helical conformation.