Structure-Function Studies of Analogues of Parathyroid Hormone (PTH)-1-34 Containing beta-Amino Acid Residues in Positions 11-13

The 1-34 N-terminal fragments of human parathyroid hormone (PTH) and PTH-related protein (PTHrP) elicit the full spectrum of bone relevant activities characteristic of the intact hormones. The structural elements believed to be required for receptor binding and biological activity are two helical segments, one N-terminal and one C-terminal, connected by hinges or flexible points located around positions 12 and 19. To test this hypothesis, we synthesized and characterized the following analogs of PTH-(1-34), each containing single or double substitutions with beta- amino acid residues around the putative hinge located at position 12: I. [Nle8,18,beta-Ala11,12,Nal23,Tyr34]bPTH-(1-34)NH2; II. [Nle8,18,beta-Ala12,13,Nal23,Tyr34]bPTH-(1-34)NH2; III. [Nle8,18,beta-Ala11,Nal23,Tyr34]bPTH-(1-34)NH2; IV. [Nle8,18,beta-hLeu11,Nal23,Tyr34]bPTH-(1-34)NH2; V. [Nle8,18,beta-Ala12, Nal23,Tyr34]bPTH-(1-34)NH2; VI. [Nle8,18,beta-Ala13, Nal23,Tyr34]bPTH-(1-34)NH2. (beta-hLeu= beta-homo-leucine; beta-Ala= beta-alanine; Nal= L-2-naphthyl-alanine; Nle=norleucine) Analogs I and III exhibit very low binding affinity and are devoid of adenylyl cyclase activity. Analog II, in spite of its very low binding capacity is an agonist. Biological activity and binding capacity are partially restored in analog IV, and completely restored in analogs V and VI. The conformational properties of the analogs were investigated in aqueous solution containing dodecylphosphocholine (DPC) micelles as a membrane-mimetic environment using CD, 2D-NMR and molecular dynamics calculations. All peptides fold partially into the alpha-helical conformation in the presence of DPC micelles, with a maximum helix content in the range of 30-35%. NMR analysis reveals the presence of two helical segments, one N-terminal and one C-terminal, as a common structural motif in all analogs. Incorporation of beta-Ala dyads at positions 11,12 and 12,13 in analogs I and II, respectively, enhances the conformational disorder in this portion of the sequence but also destabilizes the N-terminal helix. This could be one of the possible reasons for the lack of biological activity in these analogs. The partial recovery of binding affinity and biological activity in analog IV, compared to the structurally similar analog III, is clearly the consequence of the re-introduction of Leu side-chain of the native sequence. In the fully active analogs V and VI the helix stability at the N-terminus is further increased. Taken together, these results stress the functional importance of the conformational stability of the helical activation domain in PTH-(1-34). Contrary to expectation, insertion of a single beta-amino acid residue in positions 11, 12 or 13 in analogs III-VI does not favor a disordered structure in this portion of the sequence.