NMR study of a 34‐residue N‐terminal fragment of the parathyroid‐hormone‐related protein secreted during humoral hypercalcemia of malignancy

Abstract

The proton resonances of the biologically active peptide parathyroid-hormone-related protein (residues 1 -34) were assigned using one-dimensional spin-decoupling techniques, two-dimensional correlated spectroscopy and by comparing the spectra of the peptides 1 -20, 1 -25, 1 -29, 7 -34 and 15 -34. The conformation of 1 -34 was determined using one-and two-dimensional nuclear Overhauser enhancement spectroscopy in the rotating frame. Amide proton temperature coefficients, vicinal coupling constants and circular dichroic spectra helped reveal a surprisingly compact structure with residues 3 -9 forming a-helix, type-I p-turns between residues I0 -13 and 16-19 and several interactions between the N-terminal residues and the C-terminal residues. Of these latter, the strongest appeared to be between Asp-10 and Phe-22. One peptide surface in the deduced model presents multiple positive charges, while the opposite surface has a hydrophobic character possibly functioning to exclude water from the binding interface and enhancing the binding constant.Humoral hypercalcemia of malignancy is a common complication of certain cancers caused by the secretion of a protein like human parathyroid hormone. Peptides secreted by human tumors associated with humoral hypercalcemia of malignancy [l] have recently been characterized [2 -41. Sequence studies have shown that there is significant homology between these hypercalcemia-associated peptides and human parathyroid hormone (PTH), particularly in the amino-terminal region where 8 of the first 13 residues are identical [l]. For this reason these peptides have been termed 'parathyroid-hormone-related protein', PTHrP. There is no similarity between PTHrP and PTH in the carboxyl-terminal region; like PTH, the biological activity of PTHrP appears to reside in the aminoterminal 34 residues [S, 61. Since the three-dimensional structure of PTH is not known, there are no clues to the possible structure of PTHrP. For this reason, we have studied the NMR spectra of the 34-residue synthetic PTHrP fragment in order to provide structural information about the biologically active fragment in solution. This work has revealed a surprisingly compact structure containing multiple interactions between the N-terminal and C-terminal residues. Furthermore, some structural basis is provided for understanding the large differences in agonist activity of the smaller peptide fragments. Abbreviations. AMX, AMPX, spin system of protons in amino acid residues where the sequence of letters in the alphabet matches the sequence of resonance frequencies, in order of increasing field strength; CPK, Corey-Pauling-Koltun space-filled molecular model; DQPSCOSY, double-quantum-filtered phase-sensi tive correlated spectroscopy; PSCOSY, single-quantum phase-sensitive correlated spectroscopy; PTH, parathyroid hormone; PTHrP, parathyroidhormone-related protein; ROESY, nuclear Overhauser enhancement spectroscopy in the rotating frame. MATERIALS AND METHODS Peptide synthesesA total of six peptides were synthesized for the final...

Solution Structure of Parathyroid Hormone Related Protein (Residues 1–34) Containing an Ala Substituted for an Ile in Position 15 (PTHrP[Ala15]-(1–34))

“…Thus, receptor binding must depend principally on the maintenance of the structure of the C-terminal helix 22-34 and the availability of Structure of PTHrP [Ala 15 ]- a binding interface within this segment. The structure of the C-terminal helix is maintained even without the hydrophobic interactions found in several other studies, in particular those between residues 15 and 23 in both PTH (16,21) and PTHrP (13,15,18,20). Other interactions, besides the formation of a hydrophobic core, act to stabilize the C-terminal helix.…”

“…Hence, the C terminus in the stabilized conformer is located close to the N terminus. The central helix (Lys 13 to Arg 19 ) and the turn formed at the Arg cluster serves to provide the connection between the N-and C-terminal helices. Interactions between the N-and C-terminal helices in most analogs appear transiently (13).…”

“…PTH and PTHrP both exhibit nascent structures in water, which are stabilized in dilute F 3 EtOH-d 2 . PTH is less well conserved, with the Nand C-terminal helices showing no evidence that they interact, unlike PTHrP-(1-34) in water (13). Thus PTHrP-(1-34) in dilute F 3 EtOH-d 2 more closely resembles PTH-(1-34) in water or salt buffer.…”

“…Both exhibit a hydrophobic core formed by the interaction of the side chains of residues 15 and 23 and similar C-terminal helices (16,21). Since PTH-(1-34) and PTHrP-(1-34) exhibited a flexible hinge at residues 13/14 (5, 14 -21), no particular interaction between the N-and C-domains could be detected except for weak interactions in water detected with a very long mixing time, such as those between residues 2 and 31, and 8 and 28 as examples (13). These investigations not only revealed the presence of separate N-and C-terminal helices, but with the exception of PTH in the presence of 10% F 3 EtOH-d 2 , a reverse turn was found in the intervening segment.…”

“…NMR studies have been carried out on both PTHrP-(1-34) and PTH- , in a range of solvents (5,(13)(14)(15)(16)(17)(18)(19)(20), as well as on the circulating fragment PTH-(1-37) (21). PTH and PTHrP both exhibit nascent structures in water, which are stabilized in dilute F 3 EtOH-d 2 .…”