Backbone dynamics of human parathyroid hormone (1–34): Flexibility of the central region under different environmental conditions

Abstract

The presence of a stable tertiary structure in the bioactive N-terminal portion of parathyroid hormone (PTH), a major hormone in the maintenance of extracellular calcium homeostasis, is still debated. In this work, 15N relaxation parameters of the 33 backbone amides of human PTH(1-34) were determined in phosphate-buffered saline solution (PBS) and in the presence of dodecylphosphocholine (DPC) micelles. The relaxation parameters were analyzed using both the model-free formalism (G. Lipari and A. Szabo, Journal of the American Chemical Society, 1982, Vol. 104, pp. 4546-4549) and the reduced spectral density functions approach (J.-F. Lefevre, K. T. Dayie, J. W. Peng, and G. Wagner, Biochemistry, 1996, Vol. 35, pp. 2674-2686). In PBS, the region around Gly12 possesses a high degree of flexibility and the C-terminal helix is less flexible than the N-terminal one. In the presence of DPC micelles, the mobility of the entire molecule is reduced, but the stability of the N-terminal helix increases relative to the C-terminal one. A point of relatively higher mobility at residue Gly12 is still present and a new site of local mobility at residues 16-17 is generated. These results justify the lack of experimental nuclear Overhauser effect (NOE) restraints with lack of tertiary structure and support the hypothesis that, in the absence of the receptor, the relative spatial orientation of the two N- and C-terminal helices is undefined. The flexibility in the midregion of PTH(1-34), maintained in the presence of the membrane-mimetic environment, may enable the correct relative disposition of the two helices, favoring a productive interaction with the receptor.

Binding specificity of the ectodomain of the parathyroid hormone receptor

“…This is consistent with the results of an Ala scanning analysis described by Dean et al [31] and of the recently published crystal structure of a PTH (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34) peptide in complex with the PTH1R ectodomain [12]. Here, we extend these investigations by testing the whole PTH ligand for interactions.…”

“…For this, peptide libraries were generated in which every single amino acid position of PTH (20)(21)(22)(23)(24)(25)(26) was substituted against all 19 proteinogenic amino acids besides cysteine (Fig 1C). The densitometric analysis of the peptide library was quantified on two independent blots.…”

“…Pin point GPCR ECD-binding competent stretch of parathyroid hormone PTH (1-84) Narrow this stretch down to a core peptide region: PTH (20)(21)(22)(23)(24)(25)(26) Identify single residues crucial for binding: Arg20, Trp23…”

Design, conformational studies and analysis of structure–function relationships of PTH (1–11) analogues: the essential role of Val in position 2

“…In addition, a more stable C-terminal a-helical segment is present. These two helices are separated by two hinge motifs located around positions 12 and 19 (Scian et al 2006). According to the description, the interaction of PTH (1-34) with the receptor has been postulated to follow a ''two-domain'' model: the C-terminal portion interacts with the N-terminal extracellular domain of the receptor and then the N-terminal portion interacts with the PTH1R domain embedded in the membrane and elicits the cellular response (Hoare et al 2001;Gardella and Jüppner 2001;Shimizu et al 2002Shimizu et al , 2005Castro et al 2005;Gensure et al 2005;Wittelsberger et al 2006;Dean et al 2008;Pioszak et al 2009;Vilardaga et al 2011).…”

“…Our research approach (Scian et al 2006;Caporale et al 2009a) is aimed at the definition of the pharmacophoric moieties and tries to define the critical residues in the N-terminal PTH (1-11) segment. For this purpose, we decided to use both active and completely inactive analogues containing residues mimicking Val in position 2.…”