Structure of Recombinant Human Parathyroid Hormone in Solution Using Multidimensional NMR Spectroscopy

Abstract

The solution structure of human parathyroid hormone, in the form of recombinant prolyl-hPTH(1-84), has been investigated by multidimensional NMR spectroscopy under conditions (aqueous trifluoroethanol) which favour the structured-state of the protein. Spin systems were identified from 3D 1H DQF (double-quantum filtered)-COSY and TOCSY spectra and sequence-specific assignments were from 2D 1H phase-sensitive NOESY spectra. Signal overlap was resolved in a 3D-NOESY-TOCSY spectrum and assignments were confirmed with 2D NOESY-15N-HMQC (heteronuclear multiple-quantum coherence) spectra taken of a sample universally labeled with 15N. A satisfactory set of final structures was calculated from the quantitative NOE data using restrained molecular dynamics and energy minimization calculations. The N-terminus is dominated by three, well defined helices between Ser-3 to Asn-10, Ser-17 to Lys-27 and Asp-30 to Leu-37, while the most significant structural features in the C-terminus are a short, less-well defined helix between Asn-57 to Ser-62 and a series of loose turns. These two terminal units are joined by an unstructured mid-region. The molecule shows a tendency towards tertiary structure, defined by a number of long-range NOEs. A detailed RMS deviation analysis allowed the final refined structures to be classified into a limited ensemble of stable conformations that reflect the inherent flexibility of the hormone in solution.

Comparative Study of Chicken and Human Parathyroid Hormone‐(1–34)‐Peptides in Solution with SDS

“…Recently, the structure analysis of the whole PTH was reported (Gronwald et al, 1996). With increasing interest in designing analogs of PTH for therapeutic use, the elucidated structure obtained in the present study may prove helpful in probing appropriate directions for future PTH analog design.…”

“…Such a conformational change was not observed for hPTH-(1-34). It takes directly an a-helical structure with SDS micelle as reported previously (Gronwald et al, 1996). The primary structure of cPTH-(1-34) differs from those of the other PTH fragments in the lack of the tribasic region (25)(26)(27).…”

Parathyroid Hormone (PTH)-(1–14) and -(1–11) Analogs Conformationally Constrained by α-Aminoisobutyric Acid Mediate Full Agonist Responses via the Juxtamembrane Region of the PTH-1 Receptor

“…The structure of PTH as it is bound to the P1R is not known, but NMR analyses of PTH analogs in a variety of polar and non-polar solvents suggest that the N-terminal portion of PTH (within residues Ser 3 to Lys 13 ) contains a short segment of ␣-helix. This segment is connected by a flexible bend or turn segment to a longer ␣-helix in the C-terminal portion of the molecule (within residues Ser 17 to Val 31 (12)(13)(14)(15)(16)(17)(18)). A recent crystallographic study of PTH(1-34) reveals a continuous helical structure that extends nearly the full length of the molecule (Ser 3 to Asn 33 ) with only a slight (15°) bend at the midsection (19).…”

“…In a recent series of studies on the PTH (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) scaffold peptide, we showed that certain substitutions at several sites in the peptide resulted in enhanced potency (23)(24)(25). The most potent PTH (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) analog thus identified was [Ala 3,12 ,Gln 10 (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)), which is 1,600-fold more potent than native PTH (1)(2)(3)…”

Purification and Characterization of a Receptor for Human Parathyroid Hormone and Parathyroid Hormone-related Peptide

“…Understanding hormone-PTH1R and PTH1R-G protein interactions has relied largely on determination of functional consequences resulting from mutations in either the hormone or the receptor (13,(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36), analysis of receptor fragments after cross-linking to radioiodinated, p-benzoyl-L-phenylalaninemodified ligands (13,(37)(38)(39)(40), crystallographic resolution of PTH (41), and NMR of PTH (42)(43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53)(54)(55)(56)(57)(58), PTHrP (59 -68), and short segments of the PTH1R (see Refs. 69 and 70 and for review see Refs.…”