Molecular Characterization of the Receptor-Ligand Complex for Parathyroid Hormone † Christian Ro ¨lz, ‡,§ Maria Pellegrini, ‡ and Dale F. Mierke* ,‡,| Department of Molecular Pharmacology, Physiology, & Biotechnology, DiVision of Biology and Medicine, Box G-B4, and Department of Chemistry, Brown UniVersity, ProVidence, Rhode Island 02912 ReceiVed December 14, 1998; ReVised Manuscript ReceiVed March 24, 1999 ABSTRACT: Molecular models for the interaction of parathyroid hormone (PTH) with its G-protein-coupled receptors (PTH1 and PTH2) have been developed. The proposed ligand-receptor complex is based on experimental data from spectroscopic investigations of the hormone and receptor fragments as well as theoretical structure predictions based on homology analysis with proteins of known structure. From the insight afforded by the models, biochemical and pharmacological observations can be correlated with specific molecular or atomic interactions. The ligand selectivity of PTH2, specifically the lack of binding of His 5 -containing analogues, can be ascribed to unfavorable steric interactions (the binding pocket is markedly smaller in PTH2 than PTH1) as well as repulsive Coulombic forces between amino acids of like-charge (a positively charged H384 is located in the binding pocket in PTH2). The model of PTH1 suggests that the constitutive activity observed from the incorporation of a positively charged amino acid at position 223, found at the cytoplasmic end of TM2, is caused by a Coulombic attraction to E465, at the cytoplasmic end of TM7, leading to an association of TM2 and TM7 and thereby ligand-free activation. Additionally, a number of important interactions in the ligand-receptor complex are described along with predictions of the pharmacological profile which will result from specific modifications at these sites. In this regard, the models described here allow for atomic insight into the biochemical data currently available and allow targeting of future mutations to probe specific ligand/receptor interactions and thereby further our understanding of the functioning of this important hormone system. Parathyroid hormone (PTH) 1 plays a major role in the regulation of calcium and phosphate homeostasis. PTH is one of the few bone-active agents proven to be anabolic (1) and therefore has been the subject of intensive study (2-4). The hormone mediates its effects via a G-protein coupled receptor (PTH1) localized in bone and kidney (5-9). Parathyroid hormone related protein (PTHrP), evolutionary related to PTH, displays similar binding and pharmacological profiles at PTH1 (10). Truncation studies on the native hormones, have shown that the N-terminal peptides, PTH- (1-34) and PTHrP-(1-34), are sufficient for high-affinity binding to and activation of PTH1 (9, 11-13). The 15-34 region of these sequences, sharing only minimal sequence homology, see Table 1, contains the principal determinants of binding to PTH1 and can inhibit the binding of either natural ligand, suggesting a common location for interaction on the receptor surface (13-15). The amino-terminal portion of each hormone, which is highly conserved with 8 out of 13 residues being identical (Table 1), is essential for acti- vation of PTH1. On the basis of the similar pharmacological profiles of PTH-(1-34) and PTHrP-(1-34), it seems likely that both ligands will interact with PTH1 in a similar fashion. In 1995, a second receptor for parathyroid hormone (PTH2) was discovered (16). Although sequentially related to PTH1 (51% amino acid sequence identity), the ligand se- lectivity was markedly different: while PTH1 responds fully to both PTH and PTHrP, PTH2 responds only to PTH (16- 18). From mutation studies, residue 5 was found to be an important switch in the ligand selectivity observed for PTH2 (i.e., Ile 5 containing PTHrP activated PTH2) (17, 19) Furthermore, the Ile 5 -PTHrP analogue competed against PTH binding. These results suggest that the ligand selectivity of PTH2 is localized around the binding site for residue 5. To understand the underlying mechanism responsible for this ligand selectivity, we undertook the generation of molecular models of the ligand-receptor complexes for both PTH1 and PTH2. The molecular models were assembled using structural building blocks obtained by NMR spectroscopy and by computer modeling using structurally related proteins as templates (rhodopsin) and sequentially homologous proteins † This work was supported, in part, by Grant R29-GM54082 from the National Institutes of Health (D.F.M.) and Grant 98/02903 from the Deutscher Akademischer Austauschdienst (C.R.). * To whom correspondence should be addressed. Phone: (401) 863- 2139. Fax: (401) 863-1595. E-mail: Dale_Mierke@Brown.edu. ‡ Department of Molecular Pharmacology, Physiology, & Biotech- nology. § Permanent address: Department of Organic Chemistry and Bio- chemistry, Technical University of Munich, Lichtenbergstr. 4, D-85747 Garching, Germany. | Department of Chemistry. 1 Abbreviations: Bpa, benzoyl phenylalanine; DG, distance geom- etry; DPC, dodecylphosphocholine; EC, extracellular loop; G-Protein, guanine nucleotide-binding regulatory protein; GPCR, G-protein coupled receptor; MD, molecular dynamics; NOE, nuclear Overhauser enhance- ments; NOESY, nuclear Overhauser enhancement spectroscopy; PTH, human parathyroid hormone; PTHrP, parathyroid hormone-related pro- tein; PTH1, parathyroid hormone receptor 1; PTH2, parathyroid hor- mone receptor 2; PDB, Brookhaven protein data bank; TM, transmem- brane. 6397 Biochemistry 1999, 38, 6397-6405 10.1021/bi9829276 CCC: $18.00 © 1999 American Chemical Society Published on Web 04/30/1999