Local Conformation around Position 12 of the (1-34) Fragment of Parathyroid Hormone Probed by Substitution with Aib Residues

The N-terminal 1-34 fragment of parathyroid hormone (PTH) is sufficient to elicit the physiological functions of the intact hormone which controls the level of calcium ions in the blood. The biologically relevant conformation of PTH(1-34) is thought to include two critical points of flexibility around residue 12 and residue 19. These two hinges are believed to connect two alpha-helical regions at either end of the molecule. In order to assess the relationship between biological activity and local conformation around Gly12, we prepared and studied several PTH(1-34) analogs containing Aib residues at position 11, 12, and 13. Here, we describe four such analogs, containing [Aib11], [Aib12], [Aib11,12] or [Aib12,13]. Compared to PTH(1-34), introduction of Aib11 reduced binding affinity by more than 40-fold and resulted in ~5-fold lower efficacy. The [Aib12] and [Aib]12,13 analogs were 2-4-fold more potent than PTH(1-34) but displayed a reduction of 2-4-fold in binding affinity. CD and NMR studies in dodecylphosphocholine micelles accompanied by MD simulations indicate that replacement of Gly12 with Aib alone or in conjunction with the substitution of Lys13 with Aib, increases the stability and the extent of the N-helical domain. Conversely, Aib11 tends to decrease slightly the amount of ordered structure, but more so in the doubly mutated analog [Aib11,12]. The results on the Aib11-containing analogs are in line with our previous observation on the functional relevance of the side chain in position 11. The possible presence of a type-I beta-turn around residues 12-13 in the [Aib12] and [Aib12,13] analogs may be responsible for their potent in vitro activity.