Structure-function relationship studies of bovine parathyroid hormone [bPTH(1-34)] analogues containing α-amino-iso-butyric acid (Aib) residues

被引:7
作者
Peggion, E
Mammi, S
Schievano, E
Schiebler, L
Corich, M
Rosenblatt, M
Chorev, M
机构
[1] Univ Padua, Dept Organ Chem, CNR, Inst Biomol Chem, I-35131 Padua, Italy
[2] Beth Israel Deaconess Med Ctr, Dept Med, Charles A Dana Thorndike Labs, Bone & Mineral Metab Unit, Boston, MA 02215 USA
[3] Harvard Univ, Sch Med, Boston, MA 02215 USA
关键词
parathyroid hormone (PTH); conformation; 2D-NMR; molecular dynamics calculations;
D O I
10.1002/bip.10294
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The N-terminal 1-34 fragments of the parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) elicit the full spectrum of bone-related biological activities of the intact native sequences. It has been suggested that the structural elements essential for bioactivity are two helical segments located at the N-terminal and C-terminal sequences, connected by hinges or flexible points around positions 12 and 19. In order to assess the relevance of the local conformation around Gly 12 upon biological function, we synthesized and characterized the following PTH(1-34) analogues containing Aib residues: (I) A-V-S-E-I-Q-F-nL-H-N-Aib-G-K-H-L-S-Sn-L-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-Y-NH2 [[Nle(8,18) Aib(11), Nal(23),Tyr(34)]bPTH(1-34)-NH2]; (II) A-V-S-E-I-Q-F-nL-H-N-L-Aib-K-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-Y-NH2 [Nle(8,18), Aib(12),Nal(23),Tyr(34)]bPTH(1-34)-NH2]; (III) A-V-S-E-I-Q-F-nL-H-N-L-G-Aib-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-Y-NH2 {[Nle(8,18) Aib(13), Nal(23),Tyr(34)]bPTH(1-34)-NH2}; (IV) A-V-S-E-I-Q-F-nL-H-N- Aib-Aib-K-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-YNH2 {[Nle(8,18), Aib(11,12), Nal(23),Tyr(34)]bPTH(1-34)-NH2}; (V) A-V-S-E-I-Q-F-nL-H-N-L-Aib-Aib-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-Y-NH2 {[Nle(8,18), Aib(11,12), Nal(23),Tyr(34)]bpTH(1-34)-NH2}. (nL= Nle; Nal= L-(2-naphthyl)-alanine; Aib= alpha-amino-isobutyric acid.) The introduction of Aib residues at position 11 in analogue I or at positions 11 and 12 in analogue IV resulted in a 5-20-fold lower efficacy and a substantial loss of binding affinity compared to the parent compound [Nle(8,18), Nal(23),Tyr(34)]bPTH(1-34)-NH2. Both binding affinity and adenylyl cyclase stimulation activity are largely restored when the Aib residues are introduced at position 12 in analogue II, 13 in analogue III, and 12-13 in analogue V. The conformational properties of the analogues in aqueous solution containing dodecylphosphocholine micelles were studied by CD, two-dimensional (2D) NMR and computer simulations. The results indicated the presence of two helical segments in all analogues, located at the N-terminal and C-terminal sequences. Insertion of Aib residues at positions 12 and 13, or of Aib dyads at positions 11-12 and 12-13, enhances the stability of the N-terminal helix of all analogues. In all analogues the Aib residues are included in the helical segments. These results confirmed the importance of the helical structure in the N-terminal activation domain, as well as of the presence of the Leu(11) hydrophobic side chain in the native sequence, for PTH-like bioactivity. (C) 2003 Wiley Periodicals, Inc.
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页码:437 / 457
页数:21
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