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

Julian A Barden,Ruth M Cuthbertson,Wu Jia-Zhen,Jane M Moseley,Bruce E Kemp

doi:10.1074/jbc.272.47.29572

Julian A Barden, Ruth M Cuthbertson + Show 3 more

Open Access

https://doi.org/10.1074/jbc.272.47.29572

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Abstract

The structure of human parathyroid hormone (PTH) related protein (residues 1-34) containing an Ala substituted for an Ile in position 15 was studied by two-dimensional proton nuclear magnetic resonance spectroscopy. This mutant retains quite high levels of adenylate cyclase activity based on slightly reduced PTH receptor binding capacity. Three segments of helix were revealed extending from His5 to Lys11, Lys13 to Arg19, and from Phe22 to Thr33/Ala34, with a decided kink between the first two helices around Gly12. N- and C-terminal helices were stabilized by charged and hydrophobic side chain interactions between His5 and Glu30, Asp17 and both His9 and His25, and between Leu8 and Ala29, resulting in a globular molecule occupying a single conformation. While the structure of the entire mid-molecule region differed greatly from the structure of the native peptide, the structure of both N- and C-terminal regions remains essentially unaltered. The residues responsible for initiating signal transduction in the mutant are located in the vicinity of the residues responsible for receptor binding. The C-terminal amphipathic helix forming the receptor binding site exhibits reduced binding as a result of the closely applied N-terminal signal transduction-activating region. Although not contributing directly to receptor binding, the N-terminal region can sterically affect hormone binding through modifications to certain N-terminal side chains.

Highlights

Parathyroid hormone related protein (PTHrP)1 was discovered because of its expression by cancers, commonly of squamous origin, giving rise to the syndrome of humoral hypercalcemia of malignancy (HHM)
PTHrP acts largely as a paracrine regulator in several tissues [1, 2], including smooth muscle, at many locations where it acts via PTH/PTHrP receptors to relax smooth muscle
PTHrP is an example of a peptide possessing discrete structural domains for receptor binding and agonist activity

Summary

Introduction

Parathyroid hormone related protein (PTHrP) was discovered because of its expression by cancers, commonly of squamous origin, giving rise to the syndrome of humoral hypercalcemia of malignancy (HHM) (for reviews, see Refs. 1 and 2). PTHrP-(1–34) in dilute F3EtOH-d2 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 revealed the presence of separate N- and C-terminal helices, but with the exception of PTH in the presence of 10% F3EtOH-d2, a reverse turn was found in the intervening segment. Any interaction between the N- and C-terminal helices in PTH/PTHrP-(1–34) would be expected to be weakened under these conditions

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