Structural Basis for Parathyroid Hormone-related Protein Binding to the Parathyroid Hormone Receptor and Design of Conformation-selective Peptides

Parathyroid hormone (PTH) and PTH-related protein (PTHrP) are two related peptides that control calcium/phosphate homeostasis and bone development, respectively, through activation of the PTH/PTHrP receptor (PTH1R), a class B G protein-coupled receptor. Both peptides hold clinical interest for their capacities to stimulate bone formation. PTH and PTHrP display different selectivity for two distinct PTH1R conformations, but how their binding to the receptor differs is unclear. The high resolution crystal structure of PTHrP bound to the extracellular domain (ECD) of PTH1R reveals that PTHrP binds as an amphipathic α-helix to the same hydrophobic groove in the ECD as occupied by PTH, but in contrast to a straight, continuous PTH helix, the PTHrP helix is gently curved and C-terminally “unwound.” The receptor accommodates the altered binding modes by shifting the side chain conformations of two residues within the binding groove: Leu-41 and Ile-115, the former acting as a rotamer toggle switch to accommodate PTH/PTHrP sequence divergence, and the latter adapting to the PTHrP curvature. Binding studies performed with PTH/PTHrP hybrid ligands having reciprocal exchanges of residues involved in different contacts confirmed functional consequences for the altered interactions and enabled the design of altered PTH and PTHrP peptides that adopt the ECD-binding mode of the opposite peptide. Hybrid peptides that bound the ECD poorly were selective for the G protein-coupled PTH1R conformation. These results establish a molecular model for better understanding of how two biologically distinct ligands can act through a single receptor and provide a template for designing better PTH/PTHrP therapeutics.The parathyroid hormone receptor (PTH1R)³ is a class B G protein-coupled receptor (GPCR) that transduces signals from two related signaling molecules that have distinct functions in biology: parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) (Ref. 1; reviewed in Ref. 2). PTH is an 84-amino acid polypeptide endocrine hormone that is produced by the parathyroid glands and secreted into the circulation in response to low calcium levels (reviewed in Refs. 3–5), to act on bone and kidney cells and thus restore blood calcium to normal levels. In bone, PTH directly stimulates osteoblasts, resulting in bone formation (reviewed in Ref. 6), which in turn activate osteoclasts to induce bone resorption. In the kidney, PTH stimulates the reabsorption of filtered calcium, inhibits the reabsorption of phosphate, and stimulates the synthesis of 1,25-dihydroxyvitamin D3. The paradoxical anabolic/catabolic actions of PTH on bone can be modulated by exogenous PTH, and provide the molecular basis for the clinical use of PTH as an anabolic therapy for osteoporosis (7). Anabolic PTH therapy requires intermittent administration to minimize bone-resorptive effects, which predominate with sustained administration of PTH. PTHrP is a 141-amino acid polypeptide that was originally isolated as the factor responsible for humoral hypercalcemia of malignancy (8–11) and was subsequently shown to be a critical developmental paracrine factor that controls endochondral bone formation (Refs. 12, 13; reviewed in Ref. 14). PTHrP can also mediate bone-anabolic effects when administered to osteoporosis patients (15) and has been suggested to be more anabolic than PTH due to a differential effect on the coupled bone formation and resorptive responses (16).PTH and PTHrP are encoded by separate genes, each of which is found in vertebrate species ranging from fish to man. How PTH and PTHrP evolved to mediate distinct biological activities: calcium/phosphate homeostasis and tissue development, respectively, via actions upon a single receptor, remains unclear. Amino acid sequence homology is most apparent in the first 34-residue segments of the proteins, and N-terminal 34-residue peptide fragments of PTH and PTHrP are sufficient for high affinity binding to the PTH1R and are generally found to be equally potent for stimulating cAMP formation in PTH1R-expressing cells (1). The interaction of the (1–34)-length ligand with the PTH1R has been postulated to follow a “two-domain” model: residues within the approximate (1–14) segment interact with the 7-transmembrane (7-TM) helical domain embedded in the membrane, and residues within the approximate (15–34) segment interact with the N-terminal extracellular domain (ECD) of the receptor (17, 18). The 1–14 domains of PTH and PTHrP share eight amino acid sequence identities, reflecting a critical role in activating the receptor (18), while the 15–34 domains share only three amino acid identities, despite a critical role in imparting high affinity binding to the receptor.Recent studies suggest that PTH and PTHrP differ in their relative capacities to bind to two pharmacologically distinguishable high-affinity PTH1R conformations (19–22). One conformation, termed R⁰, is stable in the presence of GTPγS, but presumably in the absence of G protein coupling, correlates with prolonged signaling responses in vitro and in vivo, and is bound preferentially by PTH-(1–34). The other conformation, termed RG, is sensitive to GTPγS addition, promoted by the overexpression of a high affinity variant of Gα_s, and bound preferentially by PTHrP-(1–36). A mechanistic basis for the differing capacities of PTH and PTHrP ligands to bind to these altered PTH1R conformations is not clear at present, although, both the (1–14) and (15–34) portions of PTH contribute importantly to the capacity to bind stably to the proposed R⁰ conformation (19, 21, 22).We previously developed a method that allowed us to determine the high resolution crystal structure of recombinant PTH1R ECD in complex with the 15–34 synthetic fragment of PTH (23). The PTH1R ECD adopts a tertiary fold that is conserved among class B GPCR ECDs (24–26), and the PTH(15–34)NH₂ domain binds as a continuous and straight amphipathic α-helix to a hydrophobic groove in the ECD. Here we present the high resolution crystal structure of the PTHrP 12–34 fragment in complex with the PTH1R ECD, which reveals a distinct docking conformation toward the C terminus of the PTHrP peptide. Based on the structural differences, we designed hybrid PTH/PTHrP peptides exchanged for residues involved in altered ECD contacts; functional analyses of these peptides confirmed that the altered modes of binding indeed translate into functional consequences in terms of receptor affinity. These results provide critical insights into how PTH and PTHrP can act through a single receptor, and a structural model for designing better PTH/PTHrP analogs for treating osteoporosis.