THE STRUCTURE OF HUMAN PARATHYROID-HORMONE FROM A STUDY OF FRAGMENTS IN SOLUTION USING H-1-NMR SPECTROSCOPY AND ITS BIOLOGICAL IMPLICATIONS

In order to gain insight into the structure of human parathyroid hormone (hPTH), four fragments [hPTH(1-34), hPTH(18-48), hPTH(28-48), and hPTH(53-84)], which cover all regions of the intact hormone, have been investigated by CD and NMR spectroscopy in combination with distance geometry, and restrained molecular dynamics and energy minimization calculations, under a variety of solution conditions. Significantly, all fragments showed little propensity to form stable structures in aqueous solution alone, and it was only on the addition of trifluoroethanol (TFE) that defined structural features were observed. In an extension of earlier work [Klaus et al. (1991) Biochemistry 30, 6936-6942], hPTH(1-34) in 70% trifluoroethanol (TFE) showed two helices that were longer than in 10% TFE, but essentially showed the same characteristics. Although overlap in the H-1 NMR spectra prevented the determination of quantitative NOE data for residues 26-30, the combination of the alpha-proton chemical shift data and quantitative NOE data indicated the helices extend from residues 3 to 13 and 15 to 29. No evidence was found for interaction of the two helical regions. The nature and extent of this second helix in the intact hormone were better defined from the data for hPTH(18-48). Under limiting solution conditions, where the fragment assumed its maximum helical content, a well-defined helix was observed between residues 21 and 38 with a possible discontinuity between Leu-28 and Gln-29. There was little evidence of any form of secondary structure between Gly-38 and the terminus of this fragment, Ser-48. In keeping with this result, the shorter fragment, hPTH(28-48), showed little evidence of stable secondary structure on addition of TFE. From the alpha-proton chemical shifts residues 23-27 appeared to sustain helical structure more readily than the rest of molecule under all solution regimes in both hPTH(1-34) and hPTH(18-48). In contrast to the other two longer fragments hPTH(53-84) showed little propensity for helical secondary structure even at the highest TFE concentrations. However, there was evidence that the molecule did adopt a defined three-dimensional structure. Various long-range NOE's were observed in 10% TFE that allowed the calculation of an open teriary structure consisting of an initial series of turns surrounded by a loop structure of several loose turns. Using these results, a model of the structural characteristics of the intact hormone, under conditions that simulate the membrane-like environment, was constructed and compared schematically with the large amount of in vitro and in vivo activity data that exists in the literature for the intact hormone and its fragments. The correlation between various structural features and activity is pointed out, and the apparent importance of the alpha-helical regions, in the N-terminal half of the molecule, for binding to the membrane-bound G-protein-coupled receptors is discussed.