Addressing the Tertiary Structure of Human Parathyroid Hormone-(1-34)*

Maria PellegriniDagger , Miriam Royo§, Michael Rosenblatt§, Michael Chorev§, and Dale F. MierkeDagger par

From the Dagger  Gustaf H. Carlson School of Chemistry, Clark University, Worcester, Massachusetts 01610, the § Division of Bone and Mineral Metabolism, Harvard-Thorndike & Charles A. Dana Laboratories, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, and the  Department of Pharmacology and Molecular Toxicology, University of Massachusetts, Medical Center, Worcester, Massachusetts 01655

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Parathyroid hormone (PTH) regulates mineral metabolism and bone turnover by activating specific receptors located on osteoblastic and renal tubular cells and is fully functional as the N-terminal 1-34 fragment, PTH-(1-34). Previously, a "U-shaped" conformation with N- and C-terminal helices brought in close proximity by a turn has been postulated. The general acceptance of this hypothesis, despite limited experimental evidence, has altered the direction of the design of PTH-analogs. Examining the structure of human PTH-(1-34) under conditions that encompass the different environments the hormone may experience in the approach to and interaction with the G-protein-coupled receptor (including benign aqueous and saline solutions and in the presence of dodecylphosphocholine), we observe no evidence for a U-shape conformation or any tertiary structure. Instead, the N- and C-terminal helical domains, which vary in length and stability depending on the conditions, are separated by a highly flexible region of undefined conformation. These observations are in complete accord with recent conformational studies of PTH-related protein analogs containing lactams (Mierke, D. F., Maretto, S., Schievano, E., DeLuca, D., Bisello, A., Mammi, S., Rosenblatt, M., Peggion, E., and Chorev, M. (1997) Biochemistry 36, 10372-10383) or a model amphiphilic alpha -helix (Pellegrini, M., Bisello, A., Rosenblatt, M., Chorev, M., and Mierke, D. F. (1997) J. Med. Chem. 40, 3025-3031). Reliable structural data from different environmental conditions are absolutely requisite for the next step in the design of non-peptide PTH analogs.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Human parathyroid hormone (hPTH)1 has been the object of numerous studies for many years now, owing to its crucial role in the regulation of extracellular calcium homeostasis (1). More recently, we evidence a growing interest in PTH and PTH-derived agonists as anabolic agents in bone remodeling and their promise as new drugs for the treatment of osteoporosis. It has been established that the 34 residues at the N terminus of the 84-amino acid protein, hPTH-(1-34), are sufficient for high affinity binding to the PTH receptor (a G-protein-coupled receptor) and retain PTH-like bone-related activities (2, 3). Biological activity studies on hybrid peptides of PTH and the parathyroid hormone-related protein (PTHrP), which binds to and activates the same receptor, indicate a functional interaction between the 1-14 and 15-34 domains (4).

On the basis of theoretical methods for secondary structure prediction, both the N and C termini of PTH-(1-34) are expected to be helical. Furthermore, the amphipathic character of the two putative helices has suggested helix-helix interactions resulting in a "U-shaped" tertiary structure with a hydrophobic core and outwardly facing hydrophilic residues (5). Notably, only a low alpha -helical content could be identified for hPTH-(1-34) in aqueous solution ([theta ]r222 = -6800; average of literature values as reported by Willis (see Ref. 6) corresponding to approximately 8 residues in alpha -helix (see Ref. 7)). Only upon addition of the structure inducing solvent trifluoroethanol (TFE), a helix content consistent with the two predicted helical segments was observed. Unfortunately, the majority of the NMR-derived structures of hPTH-(1-34) in TFE (8-10) do not provide any evidence of long range interactions between the two helices. This has been attributed to the low dielectric constant (epsilon  = 26.7) of TFE, which could weaken hydrophobic interactions and therefore destabilize the alleged tertiary structure (11).

Recently, new evidence emerging from the NMR study of hPTH-(1-37) in aqueous solution in the presence of salts was interpreted in favor of the U-shape hypothesis (11). However, the reported long range NOEs are limited only to the turn portion of the U-shape and do not include residues further toward the termini. Therefore, the experimental data do not define the relative topological arrangement of the helices; any helix-loop-helix conformation is consistent with the data. In addition, the conformational consequences of the experimental conditions employed, namely the pH and the high salt concentration, were not investigated. Highly questionable U-shape conformations have been reported for PTHrP analogs in the presence of TFE (12-14).

Here, we report high resolution structures of hPTH-(1-34) in aqueous solution, investigating the effect of pH and salt concentration on secondary and tertiary structure (hPTH-(1-34) is equipotent to hPTH-(1-37) and displays identical affinity to the PTH/PTHrP receptor). In addition, we examined the peptide in the presence of dodecylphosphocholine (DPC) micelles. Micellar systems have often been used as an NMR-compatible model system for mimicking biological membranes (15-24). A membrane-ligand interaction and membrane-induced conformation have been proposed as an operational pathway for the interaction between peptide hormones and their receptors (25-28).

The structural study utilizes experimental data both from circular dichroism (CD) and NMR spectroscopies, providing, respectively, an estimate of the helix content and the location of the alpha -helix, possibly including long range interactions determining a tertiary fold. The NMR study was coupled with a protocol of structure calculation that could assure adequate sampling of conformational space (metric matrix distance geometry, DG), followed by further refinement with extensive molecular dynamics (MD) simulations incorporating explicit solvent. The high resolution conformational preferences of hPTH-(1-34) in aqueous and saline solutions and in the presence of a membrane mimetic is required to advance the structure-activity relationship of the hormone and provide a reliable template for rational design of non-peptidic PTH-mimetic agonists.

    EXPERIMENTAL PROCEDURES
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Introduction
Procedures
Results
Discussion
References

The synthesis and purification of the peptide followed published procedures (29).

Circular Dichroism--- Spectra were recorded using an Aviv CD spectrometer (model 62 DS), interfaced with a personal computer. Spectra were acquired on 30 µM solutions of the peptide in H2O, at different pH, buffer composition, and temperature and in the presence of DPC micelles. Each spectrum is the average of four scans from 255 to 195 nm. The ellipticity is reported as mean residue molar ellipticity ([theta ]r), in degrees cm2 dmol-1. Estimates of the helical fraction of the peptide were calculated using the value of [theta ]obs at 222 nm and the formula for percent helix = {([theta ]obs222 - [theta ]coil222)} - {([theta ]helix222 - [theta ]coil222)} × 100 (7). Estimations of the error associated with this formula has been discussed extensively in the literature (30, 31). Data manipulation and graphic representation were achieved with the program Kaleidograph.

NMR Methods-- hPTH-(1-34) was examined in benign water solution (1.4 mM, 9:1 H2O/2H2O, pH = 4.3, not corrected for deuterium), in the presence of buffer and salts (2 mM, 270 mM NaCl, 50 mM phosphate buffer, in 9:1 H2O/2H2O, pH = 6.0) and in the presence of micelles (1.4 mM, 9:1 H2O/2H2O, pH = 4.3, 200 mM DPC-d38 from CIL). All the experiments were recorded on a Varian Unity500 spectrometer (500 MHz) at temperatures varying between 285 and 318 K. Data processing utilized Varian (VNMR) or Felix (Biosym Technologies Inc., San Diego) software. Chemical shifts were referenced to the signal of tetramethyl silane (0.0 ppm).

The complete amino acid spin systems were identified from double quantum filtered correlation spectroscopy (32) and TOCSY (33, 34) spectra. NOESY (35, 36) and ROESY (37) experiments with mixing times of 125-350 ms were employed for the sequential assignment. All the experiments were recorded in the phase-sensitive mode according to the method detailed by States et al. (38). The TOCSY utilized a MLEV-17 sequence to realize mixing times of 40-70 ms with a spin-lock field of 10,000 Hz. In the ROESY experiment, a spin-lock field of 2500 Hz was realized by a continuous wave pulse. Suppression of the solvent signal was achieved by continuous wave presaturation at low power level during the relaxation delay (1.5-2 s), and for NOESY experiments also during the mixing time. The typical spectral width was 6500 Hz in both dimensions, with 4096 data points in t2 and 512-640 data points in t1 and with 32-128 scans at each increment. Forward linear prediction to 1024 points was applied to the incremented dimension; both dimensions were multiplied by Gaussian or shifted squared sine bell apodization functions, prior to Fourier transformation.

Back-calculation of NOESY spectra utilized the matrix doubling method within the Felix program. The cutoff distance was set at 8 Å, the correlation time to 1 ns, and the smallest back-calculated intensity to be saved was set to 0.001, representing a 0.1% NOE enhancement.

Distance Geometry-- NOESY spectra acquired at 285 K with a mixing time of 200 ms were utilized to measure cross-peak volumes for the two water samples, while a mixing time of 150 ms at 308 K was employed for the DPC sample. The volumes were converted to distances using the two-spin approximation and the cross-peaks between couples of aromatic protons of Trp23 and between the two beta  protons of Trp23 (Ser1 for the DPC sample) as a reference (respectively: Trp23 4H/5H = 2.4 Å; Trp23 7H/6H = 2.4 Å; beta 1/beta 2 = 1.78 Å). Addition and subtraction of 10% to the calculated distances yielded upper and lower bounds utilized in the distance geometry calculations. A home-written program, based on the random metrization algorithm of Havel (39), was utilized to calculate an ensemble of structures fulfilling holonomic (constitutional) and experimental (proton-proton distances) restraints. The 100 starting structures were obtained by random sampling of the distances between the upper and lower bounds. They were first embedded in four dimensions and optimized versus the initial distance restraints (conjugate gradients) (40). This step was followed by distance-driven dynamics (DDD) (41, 42), carried out at 500 K for 100 ps, and by slow cooling to 1 K. A new metrization reduced the resulting structures to three dimensions for the next optimization and DDD. The DDD procedure applies a simple square well potential on holonomic and experimental distances and an additional penalty term to maintain chirality and planarity, to generate the violation "energy" and calculate the forces.

The ensemble calculations consist of the DDD method in which the penalty expression for the experimental restraints (distances) and the subsequent force applied are generated from an ensemble average, following published procedures (43, 44). The ensemble calculations utilized 10,000 steps of 20 fs at 500 K, followed by a slow reduction of the temperature to 1 K in 2500 steps.

Molecular Dynamics-- MD simulations were performed with GROMACS (45), and interactive modeling was performed using Insight II (Biosym Technologies Inc., San Diego). Only the structures resulting from the study in water and in the presence of salts were examined. The starting structures for the simulations were chosen from the low energy structures obtained from DG as described under "Results." All atoms and the solvent were simulated explicitly. The peptide structure was placed in a rectangular box of H2O (46, 47), allowing at least 10 Å between the walls and the peptide and using three-dimensional periodic boundary condition. The charges of ionizable groups correspond to the pH of the NMR solution; no counter ions were included. A time step of 2.0 fs was employed. Neighbor lists for calculation of nonbonded interactions were updated every 20 steps within a radius of 10 Å. No switching function was utilized. The system was energy-minimized for 100 steps using steepest descent. Following this, 10 ps of MD at 300 K (48) with the peptide restrained to its original position with a force constant of 1000 kJ mol-1 nm-1 were carried out. Experimental distance restraints were then introduced with a force constant of 6000 kJ mol-1 nm-1 for 20 ps, and subsequently raised to 10,000 kJ mol-1 nm-1 for the rest of the simulation (200-400 ps). Structures were sampled every 0.5 ps. One iteration took approximately 2.7 s on one processor of a SGI Challenger (R8000, 150 MHz). The resulting trajectories were examined with analysis programs in the GROMACS package and home-written Fortran programs.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

The helix content of hPTH-(1-34) based on CD spectra (given in Fig. 1) increases in the presence of phosphate buffer at pH >=  6 (from 24% in benign water to 28% in buffer) and reaches a maximum of 54% in the presence of DPC micelles. Spectra acquired in the presence of phosphate or acetate buffer but at acidic pH are almost identical to the spectrum in benign H2O (data not shown). The solution containing phosphate buffer at pH = 6 displays a small temperature dependence. A small decrease of helix content (indicated by the ellipticity at 222 nm) occurs upon changing the temperature from 5 to 45 °C.


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Fig. 1.   Circular dichroism spectra of hPTH-(1-34) in benign aqueous solution, saline solution, and in the presence of DPC micelles.

NMR in Benign H2O-- The NMR study confirmed the presence of helical structure and localized the secondary structure elements to segments in the N- and C-terminal part of the peptide sequence. The analysis of the chemical shift values of the Halpha protons, compared with values for amino acids in a random-coil peptide (49), chemical shift index (CSI), provides a first estimate of secondary structure (50, 51). The results, shown in Fig. 2A, indicate a helical region (CSI <=  -0.1) extending from residue 17 to 30, separated by a zero point from a second "helical" domain with a small deviation from random coil values, centered at Leu7.


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Fig. 2.   The chemical shift index of the alpha -protons of hPTH-(1-34) in benign aqueous solution, pH 4.3 (A), in 50 mM phosphate buffer with 270 mM NaCl, pH 6.0 (B), and in water in the presence of DPC micelles (C).

The NOESY spectra are characteristic of a mostly disordered structure. A classical alpha -helix structure typically generates Halpha (i)-HN(i+3), Halpha (i)-HN(i+4), Halpha (i)-Hbeta (i+3), HN(i)-HN(i+2) and long stretches of continuous HN(i)-HN(i+1) NOEs. A certain degree of order is indicated by a series of HN(i)-HN(i+1) NOEs visible throughout all the sequence (4-5, 5-6, 8-9, 10-11, 12-13, 13-14, 14-15, 16-17, 17-18, 20-21, 21-22, 23-24, 24-25, 28-29, 29-30, 30-31, 31-32, and 33-34), and many of the missing NOEs are due to resonance overlapping. Typical alpha -helix NOEs of type Halpha (i)-HN(i+3) but of weak intensity are present for i = 6,7 consistent with one loop of helix in the N terminus and for i = 24,26, which together with a Trp23HN-Arg25HN NOE identify a probable C-terminal helix. A total of 95 structures were obtained from the DG calculations, all with comparable low penalty functions. Low order parameters for the phi  and psi  dihedral angles indicating disorder are observed, even for the 6-10 and 21-29 regions, consistent with the scarcity of NOEs and the low intensity of the cross-peaks indicative of alpha -helix (Fig. 3). The pairwise r.m.s. deviation values calculated as an average of the 95 structures are 1.9 Å for residues 6-10, 2.6 Å for residues 20-27, and 8.7 Å (using only the backbone atoms) for the entire peptide, indicating the predominance of flexible over ordered structures in benign aqueous solution. The structural features consist of an irregular helix between residues 21 and 26 and a loop centered on residues 7-8. Extensive MD simulations (200 ps with explicit solvent) confirm that the irregular alpha -helical structure in the identified regions is energetically stable and consistent with the experimental NOEs. The average phi ,psi dihedral angles from the simulation are reported in Table I.


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Fig. 3.   Backbone dihedral angle order parameters calculated for the ensemble of structures obtained from DG calculations of hPTH-(1-34) under the different conditions: benign aqueous, saline solution, and in the presence of DPC micelles. The phi  and psi  dihedral angles are indicated by filled and open bars, respectively.

                              
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Table I
Values of the dihedral angles phi ,psi (± standard deviation) for hPTH-(1-34) in H2O and in H2O in the presence of salts (U-shaped conformation)
The values are the average over the last 50 ps of the MD trajectories.

NMR in H2O and Salts-- The CSI values for alpha  protons in the presence of phosphate buffer and salts are similar to that observed in benign water solution (Fig. 2B). The extent of the negative deviations in this case is significantly larger for both the C- and N-terminal helix. This qualitatively indicates a larger content/population of alpha -helix, as do the greater dispersion of chemical shifts and the larger number of medium range NOEs (Fig. 4). The HN(i)-HN(i+1) NOEs are present along the entire peptide sequence. An N-terminal alpha -helix from residue 5 to 12 is indicated by 4 Halpha (i)-HN(i+3) (i = 5, 7, 9, 10) and 3 HN(i)-HN(i+2) (i = 3, 8, 12) cross-peaks. NOEs between Gly12HN and Ser14HN and Gly12Halpha -Leu15HN are of weak intensity and may result from a small population in which the helix extends to residue 15. In addition, Halpha (i)-Hbeta , delta (i+3) (i = 2, 3, 5, 6, 7) and Halpha (i)-Hbeta , delta (i+4) (i = 3, 6) NOEs are observed.


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Fig. 4.   The fingerprint region of spectra for hPTH-(1-34) in benign aqueous solution, pH 4.3, TOCSY (A) and NOESY (B), and in 50 mM phosphate buffer with 270 mM NaCl, pH 6.0, TOCSY (C) and NOESY (D).

The C-terminal helix is defined by a larger number of NOEs, including Halpha (i)-HN(i+3) (i = 18, 21, 22, 23, 24, 27, 28), HN(i)-HN(i+2) (i = 16, 19, 21), Halpha (i)-HN(i+2) (i = 23, 24), and Halpha (i)-HN(i+4) (i = 18, 21, 24), as well as a large number involving side chain protons. Medium range NOEs between the aromatic protons of Trp23 and Leu15 were also observed. Owing to overlapping of the beta  and gamma  protons of Leu15 with those protons of Leu24 and Leu28, residues closer in sequence to Trp23, only the cross-peaks involving the delta -CH3 protons of Leu15, characterized by a definite upfield shift, were considered non-ambiguous and utilized as experimental restraints in the following calculations. The NOE pattern observed, including the 15-23 connectivities, is similar to that reported by Marx et al. (11) for hPTH-(1-37) under analogous experimental conditions. Noteworthy is the presence of Halpha (i)-HN(i+1) cross-peaks of strong intensity, typical of an extended conformation and atypical for alpha -helix, indicating additional conformations present under these conditions.

A total of 229 informative distance restraints were used in the DG calculations, of which 149 from inter-residue NOEs. The phi ,psi values of the resulting 95 DG structures approach those of an ideal alpha -helix for residues 5-7 in the N terminus and 21-26 in the C terminus. The dihedral angle order parameters for phi ,psi show convergence of the structures (values > 0.8) about residues 4, 5, 6, 10, and 22-26 (Fig. 4). Pairwise r.m.s. deviation values are 0.92, 1.97, 0.76, and 6.4 Å for the heavy backbone atoms of residues 4-7, 17-27, 22-26, and 1-34, respectively. The average of the DG structures results in no NOE violation greater than 0.15 Å. However, single structures displayed violations of 0.35 Å to Halpha (i)-HN(i+1) sequential NOEs in different locations of the sequence. This is a clear indication that single structures cannot fulfill the experimental restraints produced by the coexistence of structured and unstructured peptide molecules equilibrating in solution. To address conformational averaging, ensemble calculations (44) were carried out.

A "minimum" ensemble of two structures, randomly chosen from the 95 DG structures, was utilized. In the ensemble approach (44), each structure must fulfill the constraints from the constitution (molecular connectivity), but only the ensemble average (average over the two structures) is required to fulfill the experimental distance restraints. Using the ensemble, the largest upper limit violation dropped to 0.06 Å. In Fig. 5, a ribbon representation of the two structures illustrates the coexistence of alpha -helix (in gray) and extended structure at the C terminus of the molecule. Therefore assuming an equilibrium between only two conformations is sufficient to generate agreement between the calculated structures and experimental data.


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Fig. 5.   Ribbon diagram of the two conformations resulting from the ensemble-based calculations. All of the experimental observations are fulfilled by averaging over this two-member ensemble. The different locations and extents of the alpha -helices are highlighted in gray; the side chain of Trp-23, used to align the conformations, is shown in ball-and-stick conformation.

To examine for the presence of tertiary structure, a plot of the distance between the N and C termini and the energy of the DG structures was carried out. The distances ranged between 10 to 45 Å, with no visible correlation between energy and distance; using the DG force field, based solely on connectivity and experimental distances, structures assuming a U-shape and "linear" conformation are equally favored. Representative structures of these extreme orientations of the two helical domains, linear and U-shaped (the angle between the axis of the two helices is approximately 55 degrees), were used as starting structures for extensive MD simulations. The aim of the MD simulations is to refine the local geometry in the presence of solvent, utilizing a full-atom representation force field, including Coulombic interactions and the Lennard-Jones attractive forces, which are not considered in the DG calculations.

Both structures were found to be stable in the time frame examined by the simulations (400 ps). The alpha -helical domains were preserved with the phi ,psi values adopting even more classical alpha -helix values. The carbonyl and HN groups align toward the axis of the helix producing hydrogen bonds that presumably stabilize the structure and act as a driving force toward the formation of the more classical alpha -helix. The average of phi ,psi dihedral angles from the MD trajectories is reported in Table I.

Structures sampled every picosecond from the two MD simulations were analyzed with the program Procheck_NMR (52), to examine the "health" of the conformations with respect to allowed regions in the Ramachandran map, based on regular protein structures. For the U-shape and linear conformations, 90.6% and 89.8% of the residues, respectively, fall in regions of the Ramachandran map defined as "most favored" and "additionally favored." Only 4.5% and 2.9%, for the U-shape and linear, respectively, are in disallowed regions. This confirms that both folds are compatible with the NOEs between Leu15 and Trp23 without adopting unusual conformations.

NMR in H2O/DPC Micelles-- The pattern of two helices located at the N and C terminus of the molecule, very well defined by CSI and NOEs, is also observed in the membrane mimetic. The CSI, reported in Fig. 2C, show remarkably higher values in the two helical regions, compared with the values observed in the aqueous and saline conditions. The C-terminal helix extends less toward the N terminus, with residues 17-19 assuming values typical for a random coil structure. This is confirmed by NOEs; cross-peaks of type Halpha (i)-HN(i+3) (i = 2, 4, 5, 6, 10, 11, 12, 18, 20, 21, 22, 23, 24, 25, 26, 28, 29) and Halpha (i)-HN(i+4) (i = 5, 21, 22, 27) locate the two helices approximately from residues 2-4 to 15 and from 18-20 to 32. No long range NOEs were detected, even after increasing the mixing time of the NOESY experiments; no interactions between Trp23 and Leu15 were observed. A total of 249 informative distance restraints (144 are inter-residue) were utilized in the DG calculations. The 90 low energy structures fulfill the experimental distance restraints (the largest violation is 0.16 Å). The large number of NOEs typical of alpha -helix produces a converging ensemble of conformations, with values of the dihedral angles phi  and psi  characteristic for alpha -helix in the region 21-33 (pairwise r.m.s. deviation = 1.72 Å, average over 90 structures, backbone atoms). An irregular helix at the N terminus starts at residue 4 and extends to residue 15 and possibly 17, with strong deviations about Asp10 and Leu11 (r.m.s. deviation value of 2.85 Å for residues 4-14). The well ordered structure is reflected by high order parameters for the phi  and psi  dihedral angles (Fig. 3). Low values at residues 18 and 19 indicate flexibility in the central part of the molecule (importantly, there was no resonance overlap preventing the detection of NOEs in this region). A flexible domain in the center of the molecule allows a range of different orientations of the two well defined helices relative to each other. Consequently, the r.m.s. deviation value for the 34 amino acids of the peptide is very large (6.0 Å). The family of structures and the consequences of the flexible domain are indicated in Fig. 6. The average angle between the two helical domains is 115 ± 30°.


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Fig. 6.   A superposition of the structures from the DG calculations of hPTH-(1-34) in the presence of DPC micelles. To the left, the N-terminal helix, comprising residues 6-14, has been superimposed (heavy backbone atoms), while the C-terminal helix (residues 19-33), has been used in the superposition in the right panel. The conformational consequences of the flexible domain between the helices are clearly illustrated.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

The NMR studies and relative DG/MD calculations presented in this work indicate that the conformation of hPTH-(1-34) in aqueous solution depends largely on the experimental conditions. In addition, the presence of conformational averaging fast on the NMR time scale must be considered to produce accurate and high resolution structures consistent with the experimental observations.

In benign aqueous solution, the peptide is mainly disordered. The CD data account for about eight helical residues, in agreement with much of the literature data (6). The only NMR evidence for the presence of ordered structure (indeed, an equilibrium between ordered and random conformations) is provided by a few weak NOEs, typical of alpha -helix. These NOEs are in general agreement with the structure of Lee and Russell (53) and with our CD data, and provide additional evidence for one loop of helix at the N terminus. Our results are in stark contrast with the structure presented by Barden and Kemp (54). Despite very similar experimental conditions, their proposed conformation consists of two helical domains with a tertiary fold determined by a large number of long range interactions. These contrasting structures cannot be attributed to the difference at the C terminus (i.e. a free carboxylic acid used in this study versus a carboxamide used in their study). Importantly, the structure proposed by these authors is not consistent with the CD data. It is important to note that a significant overlap of NMR signals, particularly of the side chain resonances, are reported by these authors (54) that would make an unambiguous assignment of all of the NOEs listed in this publication particularly difficult. Additionally, the collapsing of the two helical domains to adopt a U-shape may arise from the utilization of in vacuo calculations for structure refinement. The observation of collapsed structures, particularly peptides, from the overemphasis of the non-bonded terms (i.e. Coulombic and van der Waals attraction) in the absence of explicit solvent has been addressed in the literature (55, 56).

Concerning hPTH-(1-34) in high salt concentration, our NMR data provide no evidence of tertiary structure or any long range interaction deriving from a hydrophobic collapse of residues 15 and 23, despite careful examination of NOESY spectra collected with differing mixing times and temperatures. The molecule is far too flexible to prefer any definite secondary or tertiary fold. Two regions of the sequence appear to have a tendency to fold into a alpha -helix and account for the CD spectra (namely residues 6-10 in the N terminus and residues 18-29 in the C terminus), but they do not constitute stable, long-lived alpha -helices.

The difference between the conformation in H2O at pH 4.3 and in saline solution does not arise exclusively from the different ionic strength of the solution or the salt utilized. CD spectra in aqueous solutions containing 50 mM phosphate buffer at pH 4.6 or 50 mM acetate buffer at pH 4.9 (results not shown) give similar values of [theta ]r222 to that observed for hPTH-(1-34) in benign H2O solution (-6800 versus -6300). The average ellipticity values measured for solutions containing 50 mM phosphate buffer at pH 6 or 9, in the absence or presence of NaCl, are [theta ]r222 of -8000, accounting for about 10 residues in alpha -helix.

The apparent discrepancy between the differences in the NMR spectra of the two solutions and the similarity of the CD spectra can be explained by averaging of the various conformational states in solution. Indeed, a good agreement between the NMR data and calculated structures for hPTH-(1-34) in water/salts was achieved only upon considering an equilibrium between helical and extended conformations. The ensemble of two structures utilized in the ensemble calculations produced two conformations, each of them representative of 50% of the population and displaying shorter alpha -helical segments, located in different, alternating regions of the peptide. If the combination of the helical segments observed in the two conformations is considered, it is consistent with the distribution of NOEs, indicative of 17-24 residues in an alpha -helix (residues 3-5 to 12 and 16-18 to 28-31), while the population of 50% of each conformation explains the 10 residues in alpha -helix calculated from the CD spectra.

Despite the similar medium range interactions observed here for hPTH-(1-34) and those reported for hPTH-(1-37) (11), the resulting conformations are drastically different. Indeed, the C-terminal helix is more stable in the presence of salt than in benign H2O and extends to include residues 16-18, placing the hydrophobic side chains of amino acids Trp23 and Leu15 at a short distance, separated by only two loops of helix. The close proximity may induce a hydrophobic interaction that will contribute to stabilize the helix. The interaction between amino acids containing aromatic rings with hydrophobic residues has been reported to significantly stabilize helices, although usually spaced i, i+4 residues apart (57). From the computational searching of conformational space, we find that the medium range NOEs between Leu15 and Trp23 are compatible with a large number of tertiary folds or topological arrangements of the two helical domains. This is a consequence of the flexibility of the central portion of the molecule producing a large variability of interhelical angles between the two helical domains. Two representative conformations, a linear and a U-shaped arrangement, representing the conformational extremes, were further examined by extensive MD simulations. During the MD simulations of the U-shaped conformation, the two helices (N- and C-terminal) are too far apart for interhelical contacts or justify a hydrophobic interaction as a stabilizing factor of the tertiary fold.

To further examine the putative U-shaped conformation, theoretical NOESY spectra were calculated using coordinates of a U-shaped conformation (as previously reported in Ref. 11) generated by molecular modeling. The back-calculation of NOEs from a structure is a powerful method for identification of conformations not consistent with the experimentally observed NOEs. One of the theoretical NOESY spectra is illustrated in Fig. 7; the experimental NOESY spectrum is shown for comparison in black. The agreement between the theoretical and experimental spectra is generally good. The higher regularity of the helices in the MD structure, discussed in the previous session, is reflected by an additional cross-peak, Ser3Halpha -Gln6HN, and by an increased intensity in the Ser3Halpha -Ile5HN and Asn10Halpha -Lys13HN cross-peaks. There are many additional theoretical NOEs produced by the U-shaped conformation; those giving rise to resolved, unambiguous cross-peaks are labeled in the figure. Clearly visible are NOEs between the aromatic protons of Trp23, which is buried in between the two helices, and backbone protons of Leu7 and Met8 belonging to the N-terminal helix. Additional correlations involve Leu24-Met8 and Arg20-Met8. The absence of these cross-peaks in the experimental NOESY indicates that under these experimental conditions, either such a conformation is not present or is not populated to an extent to generate observable NOEs.


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Fig. 7.   A superposition of the theoretical NOESY spectrum (red) from back-calculation using the U-shaped conformation and the experimental NOESY spectrum (black) of hPTH-(1-34) in 50 mM phosphate buffer with 270 mM NaCl, pH 6.0. Many of the cross-peaks derived from the U-shaped conformation not observed in the experimental spectrum are labeled (see "Results").

The NMR study of hPTH-(1-34) in the presence of DPC micelles indicates that indeed the membrane environment can induce a higher degree of conformational order, extending and stabilizing both the N-terminal and C-terminal alpha -helix. If the two helices, and the consequent amphipathic exposure of the amino acids, are relevant elements in the bioactive conformation, the interaction with the cellular membrane could present to the receptor a ligand folded into a preferred conformation (25, 58). Similar to the results obtained in water and saline solution the two helical domains are separated by a region of flexibility, in this case centered about residues 18 and 19 (i.e. shifted more toward the C terminus than was observed in the other two environments). This allows the two "halves" of the molecule to adopt a range of relative orientations. However, no evidence for helix-helix interaction in a tertiary structure was observed in the presence of DPC. Likewise, we could not find any support for a turn conformation in the connecting loop separating the two helices.

In conclusion, under the experimental conditions examined in this study, the formation of a hydrophobic core between the two amphiphilic helices, resulting in a U-shaped conformation is not detectable either in aqueous solution or in the presence of micelles. A conformation in which the N and C termini of hPTH are in close proximity may indeed be preferred upon binding to the receptor, as suggested by biological activities and receptor binding affinities of chimeric PTH/PTHrP (4). However, our results clearly indicate that, even if the U-shape fold is energetically accessible, the peptide does not assume this conformation in solution. Instead it is possible that the flexibility of the molecule, allowing different relative spatial arrangements of the two helical domains, is a requirement to adopt the proper conformation for interacting with the receptor. Results from a series of lactam containing PTHrP-derived analogs have shown that flexibility around residue 19 is required for high biological activity of these analogs (59). The PTHrP analog, RS-66271, containing a model amphiphilic alpha -helix, likewise displayed a flexible domain immediately N-terminal of the C-terminal helix (60). The magnitude of the conformational changes induced by the receptor will only be obtained from analysis of the receptor bound hormone. We are approaching this by photoaffinity cross-linking and mapping out contact points between the hormone and receptor (61). These investigations, coupled with high resolution structures of the hormone in the different environments that the hormone may experience upon interacting with the receptor, will provide the structural insight required for the rational design of PTH-derived agonists.

    ACKNOWLEDGEMENT

We gratefully acknowledge the assistance of Dr. Robert Talanian of BASF (Worcester, MA) with the CD measurements.

    FOOTNOTES

* This work was supported in part by Grants R01-DK47940 (to M. R.) and GM54082 (to D. F. M.) from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

par To whom correspondence should be addressed. Present address: Dept. of Molecular Pharmacology, Box G-B4, Brown University, Providence, RI 02912. Tel.: 401-863-2139; Fax: 401-863-1595; E-mail: dale_mierke{at}brown.edu.

1 The abbreviations used are: hPTH, human parathyroid hormone; CSI, chemical shift index; DDD, distance-driven dynamics; DG, distance geometry; DPC, dodecylphosphocholine; G-protein, guanine nucleotide-binding regulatory protein; MD, molecular dynamics; NOE, nuclear Overhauser enhancements; NOESY, nuclear Overhauser enhancement spectroscopy; PTH, parathyroid hormone; PTHrP, parathyroid hormone-related protein; r.m.s., root-mean-square; ROESY, rotational-Overhauser enhancement spectroscopy; TFE, trifluoroethanol; TOCSY, total-correlation spectroscopy.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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