Crystal Structure of Human Parathyroid Hormone 1–34 at 0.9-Å Resolution (2000)

The N-terminal fragment 1–34 of parathyroid hormone (PTH), administered intermittently, results in increased bone formation in patients with osteoporosis. PTH and a related molecule, parathyroid hormone-related peptide (PTHrP), act on cells via a common PTH/PTHrP receptor. To define more precisely the ligand-receptor interactions, we have crystallized human PTH (hPTH)-(1–34) and determined the structure to 0.9-Å resolution. hPTH-(1–34) crystallizes as a slightly bent, long helical dimer. Analysis reveals that the extended helical conformation of hPTH-(1–34) is the likely bioactive conformation. We have developed molecular models for the interaction of hPTH-(1–34) and hPTHrP-(1–34) with the PTH/PTHrP receptor. A receptor binding pocket for the N terminus of hPTH-(1–34) and a hydrophobic interface with the receptor for the C terminus of hPTH-(1–34) are proposed. The N-terminal fragment 1–34 of parathyroid hormone (PTH), administered intermittently, results in increased bone formation in patients with osteoporosis. PTH and a related molecule, parathyroid hormone-related peptide (PTHrP), act on cells via a common PTH/PTHrP receptor. To define more precisely the ligand-receptor interactions, we have crystallized human PTH (hPTH)-(1–34) and determined the structure to 0.9-Å resolution. hPTH-(1–34) crystallizes as a slightly bent, long helical dimer. Analysis reveals that the extended helical conformation of hPTH-(1–34) is the likely bioactive conformation. We have developed molecular models for the interaction of hPTH-(1–34) and hPTHrP-(1–34) with the PTH/PTHrP receptor. A receptor binding pocket for the N terminus of hPTH-(1–34) and a hydrophobic interface with the receptor for the C terminus of hPTH-(1–34) are proposed. parathyroid hormone human PTH parathyroid hormone-related peptide trifluoroethanol transmembrane Parathyroid hormone (PTH)1 is an 84-amino acid polypeptide that regulates extracellular calcium homeostasis via actions directly on kidney and bone and indirectly on intestine by facilitating calcium absorption (1Potts Jr., J.T. Bringhurst F.R. Gardella T.J. Nussbaum S.R. Segre G.V. Kronenberg H.M. Endocrinology. 1995; 2: 920-966Google Scholar). Subcutaneous administration of hPTH-(1–34) once a day stimulates bone formation and increases bone mass in patients with osteoporosis (2Sone T. Fukunaga M. Ono S. Nishiyama T. Miner. Electrolyte Metab. 1995; 21: 232-235PubMed Google Scholar) and ovariectomized monkeys (3Brommage R. Hotchkiss C.E. Lees C.J. Stancill M.W. Hock J.M. Jerome C.P. J. Clin. Endocrinol. Metab. 1999; 84: 3757-3763Crossref PubMed Scopus (85) Google Scholar). Thus, hPTH-(1–34) has potential medical and pharmaceutical applications to the treatment of osteoporosis (4Lindsay R. Nieves J. Formica C. Henneman E. Woelfert L. Shen V. Dempster D. Cosman F. Lancet. 1997; 350: 550-555Abstract Full Text Full Text PDF PubMed Scopus (671) Google Scholar). PTH has both anabolic and catabolic effects on the skeleton. Persistent elevation of PTH causes increased bone resorption, whereas intermittently administered PTH results in enhanced bone formation (5Canalis E. Hock J.M. Raisz L.G. Bilezikian J.P. Marcus R. Levine M. The Parathyroids: Basic and Clinical Concepts. Raven Press, Ltd., New York1994: 65-82Google Scholar). The mechanism by which PTH exhibits its dual effects is not known. PTH interacts with a G protein-coupled, seven-transmembrane helix receptor (PTH/PTHrP or PTH1 receptor) to stimulate adenylyl cyclase (6Chase L.R. Aurbach G.D. J. Biol. Chem. 1970; 245: 1520-1526Abstract Full Text PDF PubMed Google Scholar) and phospholipase C (7Civitelli R. Reid I.R. Westbrook S. Avioli L.V. Hruska K.A. Am. J. Physiol. 1988; 255: E660-E667PubMed Google Scholar) activities. Studies, both in vitro and in vivo, have shown that the N-terminal 1–34 fragment has the same biological activities as the intact hormone in eliciting cAMP responses and in stimulating bone formation (8Mosekilde L. Sogaard C.H. Danielsen C.C. Torring O. Endocrinology. 1991; 129: 421-428Crossref PubMed Scopus (171) Google Scholar). Truncation and mutagenesis studies on PTH-(1–34) have revealed that the N-terminal region is critical for activation of receptor signaling, whereas the N-terminal truncated peptide PTH-(3–34) is only a partial agonist, and the further shortened peptide PTH-(7–34) becomes a low affinity antagonist (9Tregear G.W. Van Rietschoten J. Greene E. Keutmann H.T. Niall H.D. Reit B. Parsons J.A. Potts Jr., J.T. Endocrinology. 1973; 93: 1349-1353Crossref PubMed Scopus (274) Google Scholar, 10Gardella T.J. Axelrod D. Rubin D. Keutmann H.T. Potts Jr., J.T. Kronenberg H.M. Nussbaum S.R. J. Biol. Chem. 1991; 266: 13141-13146Abstract Full Text PDF PubMed Google Scholar). Residues 17–31, near the C terminus of PTH-(1–34), are required for high affinity receptor binding (11Juppner H. Schipani E. Bringhurst F.R. McClure I. Keutmann H.T. Potts Jr., J.T. Kronenberg H.M. Abou-Samra A.B. Segre G.V. Gardella T.J. Endocrinology. 1994; 134: 879-884Crossref PubMed Scopus (0) Google Scholar). PTHrP is a polypeptide that is over-expressed in certain tumors and causes the syndrome of malignancy-associated humoral hypercalcemia (12Moseley J.M. Gillespie M.T. Crit. Rev. Clin. Lab. Sci. 1995; 32: 299-343Crossref PubMed Scopus (92) Google Scholar). Under physiological conditions, PTHrP is produced locally in a wide variety of tissues and is involved in cell growth, differentiation, and development of the fetal skeleton. There are 6 identical amino acids in the first 13 amino acids in the known PTH and PTHrP sequences (Fig. 1). Like PTH, PTHrP binds to the same G protein-coupled receptor, and its N-terminal fragment PTHrP-(1–34) has many functions that mimic those of full-length PTHrP-(1–141) as well as PTH-(1–34) and full-length PTH-(1–84) (12Moseley J.M. Gillespie M.T. Crit. Rev. Clin. Lab. Sci. 1995; 32: 299-343Crossref PubMed Scopus (92) Google Scholar, 13Blind E. Raue F. Knappe V. Schroth J. Ziegler R. Exp. Clin. Endocrinol. 1993; 101: 150-155Crossref PubMed Scopus (18) Google Scholar). In addition, hPTH-(1–34) and hPTHrP-(1–34) have similar three-dimensional structures based on NMR studies (14Gronwald W. Schomburg D. Tegge W. Wray V. Biol. Chem. 1997; 378: 1501-1508Crossref PubMed Scopus (16) Google Scholar, 15Weidler M. Marx U.C. Seidel G. Schafer W. Hoffmann E. Esswein A. Rosch P. FEBS Lett. 1999; 444: 239-244Crossref PubMed Scopus (25) Google Scholar). Various methods have been used to determine the structure of PTH, including dark-field electron microscopy, fluorescence spectroscopy, circular dichroism, and nuclear magnetic resonance (NMR) spectroscopy (14Gronwald W. Schomburg D. Tegge W. Wray V. Biol. Chem. 1997; 378: 1501-1508Crossref PubMed Scopus (16) Google Scholar, 15Weidler M. Marx U.C. Seidel G. Schafer W. Hoffmann E. Esswein A. Rosch P. FEBS Lett. 1999; 444: 239-244Crossref PubMed Scopus (25) Google Scholar, 16Fiskin A.M. Cohn D.V. Peterson G.S. J. Biol. Chem. 1997; 252: 8261-8268Google Scholar, 17Marx U.C. Austermann S. Bayer P. Adermann K. Ejchart A. Sticht H. Walter S. Schmid F.X. Jaenicke R. Forssmann W.G. Rosch P. J. Biol. Chem. 1995; 270: 15194-15202Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 18Barden J.A. Kemp B.E. Biochemistry. 1993; 32: 7126-7132Crossref PubMed Scopus (62) Google Scholar, 19Klaus W. Dieckmann T. Wray V. Schomburg D. Wingender E. Mayer H. Biochemistry. 1991; 30: 6936-6942Crossref PubMed Scopus (81) Google Scholar, 20Strickland L.A. Bozzato R.P. Kronis K.A. Biochemistry. 1993; 32: 6050-6057Crossref PubMed Scopus (50) Google Scholar, 21Chorev M. Behar V. Yang Q. Rosenblatt M. Mammi S. Maretto S. Pellegrini M. Peggion E. Biopolymers. 1995; 36: 485-495Crossref PubMed Scopus (14) Google Scholar, 22Pellegrini M. Royo M. Rosenblatt M. Chorev M. Mierke D. J. Biol. Chem. 1998; 273: 10420-10427Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 23Neugebauer W. Surewicz W.K. Gordon H.L. Somorjai R.L. Sung W. Willick G.E. Biochemistry. 1992; 31: 2056-2063Crossref PubMed Scopus (57) Google Scholar). Results from these diverse approaches have not yet yielded a consistent structure for this peptide. In part, this uncertainty arises from the flexible nature of small peptides in solution as well as from different experimental conditions such as protein concentration, solvent conditions, pH, temperature, and different methods used for data interpretation. The general consensus is that PTH-(1–34) and PTHrP-(1–34) have an N-terminal helix and a C-terminal helix that vary in length and stability depending on the specific experimental conditions and are connected by a highly flexible mid-region. The C-terminal helix is more stable than the N-terminal helix. In aqueous solution, PTH-(1–34) and PTHrP-(1–34) form fewer and less stable secondary structural elements than under membrane-mimicking conditions, such as dodecylphosphocholine micelles (20Strickland L.A. Bozzato R.P. Kronis K.A. Biochemistry. 1993; 32: 6050-6057Crossref PubMed Scopus (50) Google Scholar) or palmitoyloleoylphosphatidylserine vesicles (23Neugebauer W. Surewicz W.K. Gordon H.L. Somorjai R.L. Sung W. Willick G.E. Biochemistry. 1992; 31: 2056-2063Crossref PubMed Scopus (57) Google Scholar), or in the presence of a secondary structure-inducing solvent such as trifluoroethanol (TFE) (18Barden J.A. Kemp B.E. Biochemistry. 1993; 32: 7126-7132Crossref PubMed Scopus (62) Google Scholar, 19Klaus W. Dieckmann T. Wray V. Schomburg D. Wingender E. Mayer H. Biochemistry. 1991; 30: 6936-6942Crossref PubMed Scopus (81) Google Scholar, 20Strickland L.A. Bozzato R.P. Kronis K.A. Biochemistry. 1993; 32: 6050-6057Crossref PubMed Scopus (50) Google Scholar, 22Pellegrini M. Royo M. Rosenblatt M. Chorev M. Mierke D. J. Biol. Chem. 1998; 273: 10420-10427Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 23Neugebauer W. Surewicz W.K. Gordon H.L. Somorjai R.L. Sung W. Willick G.E. Biochemistry. 1992; 31: 2056-2063Crossref PubMed Scopus (57) Google Scholar). Several of the NMR studies have been interpreted to show a “U-shaped” tertiary structure with the N- and C-terminal helices interacting with each other to form a hydrophobic core (17Marx U.C. Austermann S. Bayer P. Adermann K. Ejchart A. Sticht H. Walter S. Schmid F.X. Jaenicke R. Forssmann W.G. Rosch P. J. Biol. Chem. 1995; 270: 15194-15202Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 18Barden J.A. Kemp B.E. Biochemistry. 1993; 32: 7126-7132Crossref PubMed Scopus (62) Google Scholar). However, the majority of the NMR analyses of PTH and PTHrP do not provide evidence of long range interactions between the two helices. To create more potent and orally available analogs of PTH, detailed structural information on the peptide should aid in characterizing the molecular interactions between the ligand and receptor. Human PTH-(1–34) (LY333334, Lilly) was expressed in Escherichia coli cells. The inclusion bodies were solubilized in 7 m urea and captured by a reverse phase column. hPTH-(1–34) was purified through a cation exchange-column (FFSP, Amersham Pharmacia Biotech) with a gradient of 0.1–0.3 m sodium chloride at pH 2.5 in 7m urea followed by a reverse phase column with a gradient of 22–32% acetonitrile in 20 mm glycine at pH 9, refolded, and freeze-dried. Selenomethionine hPTH-(1–34) was synthesized on an ABI-430A peptide synthesizer usingt-butoxycarbonyl amino acids. Thet-butoxycarbonyl seleno-l-methionine was prepared from l-selenomethionine using di-t-butyldicarbonate. The selenomethionine hPTH-(1–34) was purified by a Vydac C18 column with a gradient of 10–50% acetonitrile in 0.1% trifluoroacetic acid at pH 2 and Phenomenex Primeshere 10 C18 column with a gradient of 15–35% acetonitrile in 0.05 mammonium bicarbonate at pH 8 on a fast protein liquid chromatography system (Amersham Pharmacia Biotech). Identified fractions were pooled, frozen, and lyophilized. Mass spectroscopy analysis showed complete incorporation of selenomethionine into the peptide. hPTH-(1–34) was crystallized at 20 °C by the hanging drop vapor diffusion method. Single crystals were obtained by mixing 20 mg ml−1hPTH-(1–34) in 20% glycerol, at a 1:1 ratio (v/v), with a solution containing 2.5 m ammonium sulfate, 5% isopropanol, and 0.1m sodium acetate buffer, pH 4.5. Crystals appeared overnight and continuously grew to 0.6 × 0.2 × 0.1 mm3 in a week. Crystals of selenomethionine hPTH-(1–34) were obtained by repeated seeding under the same conditions as described above with 10 mg ml−1 selenomethionine hPTH-(1–34) in 20% glycerol and 20 mm sodium citrate, pH 4.5. For cryogenic data collection, hPTH-(1–34) crystals were flash-frozen in liquid nitrogen. X-ray data were collected at −170 °C by a Mar CCD detector at the Industrial Macromolecule Crystallography Association beam line ID-17 at Advanced Photon Source in Argonne National Laboratories. Data were integrated and reduced using the program HKL2000 (24Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38526) Google Scholar). The crystals belong to hexagonal space group P65 with unit cell dimensions of 30.18 Å (a) and 110.44 Å (c). Three data sets were collected for a selenomethionine hPTH-(1–34) crystal at wavelengths of 0.9795, 0.97936, and 0.9840 Å for multiple wavelength anomalous dispersion phasing (see Table I).Table IData collection, phasing, and refinement statisticsDiffraction data (space group P65)Native dataMAD dataPeakInflection pointRemote pointWavelength (Å)1.000000.979360.979500.98400Unit cell dimensions a (Å)30.1830.1930.1430.16 c (Å)110.44111.04110.88110.96Resolution range14.0–0.920.0–2.020.0–2.020.0–2.0Total reflections111749419763862343184Unique reflections37765389438593865Completeness (%)1-aThe numbers in the parentheses are statistics for the highest resolution shell.90.5 (75.5)99.7 (99.5)99.7 (99.5)99.7 (99.5)R merge1-bRmerge = Σ‖I i − 〈I〉‖/Σ I i, where I i is the intensity of an individual measurement, and 〈I〉 is the mean intensity of all measurements of I.(%)1-aThe numbers in the parentheses are statistics for the highest resolution shell.5.4 (16.3)6.1 (11.1)6.1 (11.6)5.1 (8.9)Phasing by SOLVE (20.0–2.0 Å)Mean figure of merit0.86Refinement statistics (15.0–0.9 Å)F o > 0ςF o > 4ςR work1-cR = Σ‖F o c o is using of the whereas is using 5% of the = Σ‖F o c o is using of the whereas is using 5% of the from length The numbers in the parentheses are statistics for the highest resolution = Σ‖I i − 〈I〉‖/Σ I i, where I i is the intensity of an individual measurement, and 〈I〉 is the mean intensity of all measurements of = Σ‖F o c o is using of the whereas is using 5% of the in a The structure was by the program SOLVE J. Biol. 1999; PubMed Scopus Google Scholar) with the multiple wavelength anomalous dispersion The polypeptide was to the electron with program M. Methods Enzymol. 1997; PubMed Scopus Google Scholar). The was to resolution using the multiple wavelength anomalous dispersion data and to 0.9-Å resolution using the data in a for Crystallography and Press, New Scholar) by The was further in by the gradient with for all were and the from 20 to and from to were in 2 o c o c The for all data is is The structure protein and are in the conformation in was in using the protein The transmembrane of the PTH/PTHrP receptor were first determined by for transmembrane region by the and a consensus was The crystal structure of at Å H. P. A. C. J.P. E. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar) was used as a for the and of the transmembrane helices. of the transmembrane helices for the PTH/PTHrP receptor and were and was to create the helices of the PTH/PTHrP receptor. The of the and extracellular were in using the fragment data For the N-terminal receptor region the NMR structure determined in a M. A. Rosenblatt M. Chorev M. Mierke Biochemistry. 1998; PubMed Scopus Google Scholar) was into the was by the helix with the of The of the receptor of the hPTH-(1–34) from the crystal was into the receptor using two based on studies A. Mierke Pellegrini M. Rosenblatt M. Chorev M. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, A. Chorev M. Rosenblatt M. Endocrinol. 1998; PubMed Scopus Google Scholar). was to the of hPTH-(1–34) and of the receptor using the in were The hPTHrP-(1–34) was produced by using the crystal structure of hPTH-(1–34) as a The of hPTHrP-(1–34) binding to the PTH/PTHrP receptor was to the The structure of hPTH-(1–34) was determined by the multiple wavelength anomalous dispersion using selenomethionine hPTH-(1–34) with the program SOLVE J. Biol. 1999; PubMed Scopus Google Scholar). The structure was to a resolution of Å a data with the program Methods Enzymol. 1997; PubMed Scopus Google Scholar) of for the refinement the The structure of hPTH-(1–34) is a slightly helix (Fig. The is between and with a of between the N-terminal helix and the C-terminal helix between the of and and and a between and are hPTH-(1–34) is a and form two with the hydrophobic of these helices in different Thus, the hydrophobic of hPTH-(1–34) form a from the N terminus to the C terminus with the near (Fig. is a in all the known PTH and PTHrP (Fig. 1). the flexible nature of is in a helical conformation in the crystal of with a helix in was well whereas with a helix receptor binding affinity and adenylyl M. R.L. E. Rosenblatt M. Biochemistry. PubMed Scopus Google Scholar). these that the helical conformation is for biological structure of hPTH-(1–34) is a slightly helix as a in Residues and form two helices with hydrophobic in different The of the helices and are The of the two helices is to and are shown as and and and and hPTH-(1–34) is as and the the interface are The interface is the of the the from each form a shown as a and from the of from the hPTH-(1–34) in a the hPTH-(1–34) crystallizes as a in the hexagonal space group and from both are at the of the and The of from a with of from molecule, whereas from the of from the other (Fig. the hydrophobic interactions from the the N and C Residues and of the N-terminal helix from are in with and of the C-terminal helix from the other The conformation of each in the is similar for the conformation of the N-terminal that from its Thus, the mean is 0.05 Å the of from two are and Å all of 1–34 are The of different in the different Three for and two for and are in each Residues and have conformation in and two in the The of and are not between the two of these different and different structures the hPTH-(1–34) crystallizes as a in the unit in the space group P65 than as a in the space group results from studies with in a solution that was to conditions, not stable which is consistent with results obtained from (17Marx U.C. Austermann S. Bayer P. Adermann K. Ejchart A. Sticht H. Walter S. Schmid F.X. Jaenicke R. Forssmann W.G. Rosch P. J. Biol. Chem. 1995; 270: 15194-15202Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Thus, the is likely a in crystal than an of PTH-(1–34) in NMR studies have been on PTH and PTHrP in different solvent (14Gronwald W. Schomburg D. Tegge W. Wray V. Biol. Chem. 1997; 378: 1501-1508Crossref PubMed Scopus (16) Google Scholar). In NMR studies show that PTH-(1–34) and PTHrP-(1–34) form an N-terminal helix and a C-terminal helix connected by a highly flexible region in the of the crystal structure with the NMR structure of hPTH-(1–34) with U.C. Adermann K. Bayer P. Forssmann P. PubMed Scopus Google Scholar) by the of the C-terminal helices of the crystal structure with other NMR such as (17Marx U.C. Austermann S. Bayer P. Adermann K. Ejchart A. Sticht H. Walter S. Schmid F.X. Jaenicke R. Forssmann W.G. Rosch P. J. Biol. Chem. 1995; 270: 15194-15202Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar) and hPTHrP-(1–34) M. Marx U.C. Seidel G. Schafer W. Hoffmann E. Esswein A. Rosch P. FEBS Lett. 1999; 444: 239-244Crossref PubMed Scopus (25) Google Scholar) yielded similar as NMR structures were obtained under near physiological The highly flexible region in the NMR structures is to form a helix in crystal from NMR studies on PTH-(1–34) and PTHrP-(1–34) to the that the helical increases with or conditions that mimic the In the N- and C-terminal helices and of PTH-(1–34) were with a at NMR structures of PTHrP-(1–34) in showed two well helices and (14Gronwald W. Schomburg D. Tegge W. Wray V. Biol. Chem. 1997; 378: 1501-1508Crossref PubMed Scopus (16) Google Scholar). crystal structure is similar to the NMR structures determined in high of or under membrane-mimicking conditions (14Gronwald W. Schomburg D. Tegge W. Wray V. Biol. Chem. 1997; 378: 1501-1508Crossref PubMed Scopus (16) Google Scholar, V. T. W. Mayer H. Schomburg D. Tegge W. Wingender E. Biochemistry. 1994; PubMed Scopus Google Scholar). is not hPTH-(1–34) in the crystal has solvent The solvent of the hPTH-(1–34) crystal is less than with hydrophobic In the crystal structure the conformation of PTH-(1–34) is to its receptor as for the NMR structures under high of or membrane-mimicking conditions (23Neugebauer W. Surewicz W.K. Gordon H.L. Somorjai R.L. Sung W. Willick G.E. Biochemistry. 1992; 31: 2056-2063Crossref PubMed Scopus (57) Google Scholar). studies that have involved for the bioactive of PTH and PTHrP have used to secondary structural elements to for the presence of these were at different the peptide to the at i and i in an to a helical conformation. and studies have that helical by such biological this is highly to the M. A. Rosenblatt M. Chorev M. Mierke J. Chem. 1997; PubMed Scopus Google Scholar, I. S. C.J. B.E. J. K. K. J. Am. Chem. Scopus Google Scholar) that adenylyl in cells was increased a was between and or and of the was between 10 and In was between 13 and adenylyl was increased Maretto S. E. D. A. Mammi S. Rosenblatt M. Peggion E. Chorev M. Biochemistry. 1997; 36: PubMed Scopus Google Scholar). However, a between and in binding affinity and adenylyl Maretto S. E. D. A. Mammi S. Rosenblatt M. Peggion E. Chorev M. Biochemistry. 1997; 36: PubMed Scopus Google Scholar). the structures of I. S. C.J. B.E. J. K. K. J. Am. Chem. Scopus Google Scholar) and hPTHrP-(1–34) Maretto S. E. D. A. Mammi S. Rosenblatt M. Peggion E. Chorev M. Biochemistry. 1997; 36: PubMed Scopus Google Scholar) were both in extended helical similar to crystal structure of In the crystal structure of the well and are on the or of the by the slightly helix and are in the of the Thus, that helical structure in this flexible region of the peptide increases the biological of PTH and The and are on the of the hPTH-(1–34) helical (Fig. In these the biological activities by the helical or with the ligand-receptor interaction were at those Thus, in the region of hPTH-(1–34) as well as the of the helix to have the extended helical conformation in the crystal structure well the receptor binding conformation of hPTH-(1–34) in a flexible conformation in solution as in the extracellular a helical conformation is likely to the peptide approaches the hydrophobic receptor are further by the molecular studies on or interactions have that the region of the helices and extracellular the of the PTH/PTHrP receptor with the N terminus of PTH or PTHrP to M. Rosenblatt M. Bilezikian J.P. Raisz L.G. of Press, the N-terminal extracellular region of the receptor interacts with the C-terminal region of PTH or PTHrP ligand binding (11Juppner H. Schipani E. Bringhurst F.R. McClure I. Keutmann H.T. Potts Jr., J.T. Kronenberg H.M. Abou-Samra A.B. Segre G.V. Gardella T.J. Endocrinology. 1994; 134: 879-884Crossref PubMed Scopus (0) Google Scholar). Results from and mutagenesis two in the receptor of hPTH-(1–34) to of the receptor A. Mierke Pellegrini M. Rosenblatt M. Chorev M. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar) and of hPTH-(1–34) to of the receptor A. Chorev M. Rosenblatt M. Endocrinol. 1998; PubMed Scopus Google Scholar). A of the hPTH-(1–34) to the PTH/PTHrP receptor was by these of the PTH/PTHrP receptor was by using the resolution crystal structure of H. P. A. C. J.P. E. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar) as a for the transmembrane helices. The of the and extracellular were by a fragment data The NMR structure of the of the receptor M. A. Rosenblatt M. Chorev M. Mierke Biochemistry. 1998; PubMed Scopus Google Scholar), was hPTH-(1–34) was to the receptor the of the ligand-receptor interactions by and studies A. Mierke Pellegrini M. Rosenblatt M. Chorev M. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google A. Chorev M. Rosenblatt M. Endocrinol. 1998; PubMed Scopus Google Scholar). In (Fig. 6 the N-terminal region of for its binds to a pocket of the extracellular of and and the and extracellular of the receptor. The region of hPTH-(1–34) is between the first extracellular and the N-terminal extracellular region of the receptor to The C-terminal region of hPTH-(1–34) interactions with the binding of the PTH/PTHrP receptor (Fig. 6 interface of the hydrophobic interactions and of hPTH-(1–34) and and of the receptor) and interactions between of hPTH-(1–34) and and of the receptor as well as between of hPTH-(1–34) and of the receptor. Several models the NMR structure of hPTH-(1–34) with N- and C-terminal helices connected by a flexible have been in the for the binding of hPTH-(1–34) to the PTH/PTHrP receptor. A. Mierke Pellegrini M. Rosenblatt M. Chorev M. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar) have that the N terminus of hPTH-(1–34) to the extracellular of In a the N-terminal helix of PTH a space by the extracellular of the transmembrane helices and extracellular whereas the C-terminal helix of hPTH-(1–34) has two to the N-terminal extracellular region of the receptor C. Pellegrini M. Mierke Biochemistry. 1999; PubMed Scopus Google Scholar). the crystal structure of hPTH-(1–34) with its extended helical only of hPTH-(1–34) in all the known ligand-receptor mutagenesis studies in the C-terminal region of hPTH-(1–34) have that and are to T.J. Keutmann H.T. R. Potts Jr., J.T. Kronenberg M. Nussbaum S.R. Endocrinology. 1993; PubMed Scopus Google Scholar). and are by the receptor binding and A less of receptor binding affinity is is by In of by has on receptor In (Fig. 6 and of hPTH-(1–34) are at the of the hydrophobic whereas is at the of the hydrophobic is to the at this not receptor binding The interaction between hPTH-(1–34) and of the PTH/PTHrP receptor less for binding than other interactions a variety of were at T.J. Keutmann H.T. R. Potts Jr., J.T. Kronenberg M. Nussbaum S.R. Endocrinology. 1993; PubMed Scopus Google Scholar). To this mutagenesis studies on at the interface has been that PTHrP-(1–34) binds to the PTH/PTHrP receptor in the same as PTH-(1–34) C. Pellegrini M. Mierke Biochemistry. 1999; PubMed Scopus Google Scholar). We have a of hPTHrP-(1–34) using the crystal structure of hPTH-(1–34) and hPTHrP-(1–34) to the PTH/PTHrP receptor with the same as was followed by Residues and are all the known PTH and PTHrP whereas and are all hydrophobic that are (Fig. 1). Residues and of hPTHrP-(1–34) form similar interactions with receptor as the of hPTH-(1–34) in in which is in is in the hydrophobic and hPTHrP-(1–34) identical amino acids in the region only identical amino acids in the region However, the C of both peptides form similar helices that are to for high affinity receptor binding M. Royo M. Rosenblatt M. Chorev M. Mierke D. J. Biol. Chem. 1998; 273: 10420-10427Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 23Neugebauer W. Surewicz W.K. Gordon H.L. Somorjai R.L. Sung W. Willick G.E. Biochemistry. 1992; 31: 2056-2063Crossref PubMed Scopus (57) Google Scholar). were with a in the PTHrP bone anabolic was Z. D. J.P. T. J. Miner. PubMed Scopus Google Scholar). and NMR studies that in a helical conformation from to models for the interaction of PTH and PTHrP to the common PTH/PTHrP receptor the that the helices at the C-terminal of PTH and PTHrP-(1–34) are for the M. Royo M. Rosenblatt M. Chorev M. Mierke D. J. Biol. Chem. 1998; 273: 10420-10427Abstract Full Text Full Text PDF PubMed Scopus (92) Google W. Surewicz W.K. Gordon H.L. Somorjai R.L. Sung W. Willick G.E. Biochemistry. 1992; 31: 2056-2063Crossref PubMed Scopus (57) Google Scholar). A detailed structural analysis of PTH or PTHrP to its common PTH/PTHrP receptor required to this ligand-receptor binding and The structure of hPTH-(1–34) with the NMR structures and has of and interacting with the PTH/PTHrP receptor. has a for the ligand-receptor mechanism and to of PTH analogs and We H. J. F. W. W. R. D. C. A. M. F. J. M. A. H. V. J. J. B. H. J. D. and for