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Originally published In Press as doi:10.1074/jbc.M603528200 on June 9, 2006

J. Biol. Chem., Vol. 281, Issue 34, 24193-24203, August 25, 2006
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Structural Determinants of Salmon Calcitonin Bioactivity

THE ROLE OF THE LEU-BASED AMPHIPATHIC {alpha}-HELIX*

Giuseppina Andreotti{ddagger}1, Blanca López Méndez{ddagger}12, Pietro Amodeo{ddagger}, Maria A. Castiglione Morelli§, Hiromichi Nakamuta, and Andrea Motta{ddagger}3

From the {ddagger}Istituto di Chimica Biomolecolare del Consiglio Nazionale delle Ricerche, Comprensorio Olivetti, Edificio A, 80078 Pozzuoli (Napoli), Italy, §Dipartimento di Chimica, Università della Basilicata, 85100 Potenza, Italy, and the Laboratory of Pharmacology, Department of Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences, Hiroshima International University, Hirokoshingai 5-1-1, Kure, Hiroshima 737-0112, Japan

Received for publication, April 12, 2006 , and in revised form, June 8, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Salmon calcitonin (sCT) forms an amphipathic helix in the region 9–19, with the C-terminal decapeptide interacting with the helix (Amodeo, P., Motta, A., Strazzullo, G., Castiglione Morelli, M. A. (1999) J. Biomol. NMR 13, 161–174). To uncover the structural requirements for the hormone bioactivity, we investigated several sCT analogs. They were designed so as to alter the length of the central helix by removal and/or replacement of flanking residues and by selectively mutating or deleting residues inside the helix. The helix content was assessed by circular dichroism and NMR spectroscopies; the receptor binding affinity in human breast cancer cell line T 47D and the in vivo hypocalcemic activity were also evaluated. In particular, by NMR spectroscopy and molecular dynamics calculations we studied Leu23,Ala24-sCT in which Pro23 and Arg24 were replaced by helix inducing residues. Compared with sCT, it assumes a longer amphipathic {alpha}-helix, with decreased binding affinity and one-fifth of the hypocalcemic activity, therefore supporting the idea of a relationship between a definite helix length and bioactivity. From the analysis of other sCT mutants, we inferred that the correct helix length is located in the 9–19 region and requires long range interactions and the presence of specific regions of residues within the sequence for high binding affinity and hypocalcemic activity. Taken together, the structural and biological data identify well defined structural parameters of the helix for sCT bioactivity.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The most recognized action of calcitonin (CT)4 is the inhibition of osteoclast-mediated bone resorption. This forms the basis for its primary clinical use in the treatment of bone-related disorders such as Paget disease, osteoporosis, and hypercalcemia of malignancy (1). CT activity also includes modulation of renal ion excretion, analgesia, inhibition of appetite, and gastric acid secretion as well as influence on reproduction via the effects on embryological implantation and sperm function (Ref. 1 and references therein). Recently, CT has been put forward as an ideal agent for treatment of osteoarthritis (2).

CT is a single-chain polypeptide hormone of 32 amino acids with an N-terminal disulfide bridge between positions 1 and 7 and a C-terminal amidated proline. CT species so far studied can be subdivided into three major classes: human/rodent, artiodactyl, and teleost/avian; of these, the members of the teleost/avian group are generally the most potent, although relative potency varies in a species- and isoform-specific manner (3). The higher potency combined with a longer in vivo half-life has led to fish-like CT, exemplified by salmon CT (sCT), as the standard form of CT used for the clinical treatment of bone disorders (4). However, the usefulness of CT is limited by the development of clinical resistance. This can be due to development of circulating antibodies against non-human CT (5) and/or loss of responsiveness to CT, presumably via receptor down-regulation and inhibition of new receptor synthesis (6). Furthermore, human CT (hCT) easily associates and precipitates as insoluble fibrils (7), which limits its usefulness as a therapeutic.

Structure-activity relationship of CT from various species has been studied extensively. In organic solvents (8, 9) sCT assumes an amphipathic {alpha}-helix in the region 8–22, whereas in SDS (10, 11) the helix spans the region 6–22, with the C-terminal tail folded back toward the helix. Molecular dynamics (MD) simulations (12) have indicated that the stable helix of sCT is confined in the region 9–19, with the flanking residues 6–8 and 20–22 acting as stabilizers and the C terminus interacting with the helix. Among the structural determinants suggested to explain CT bioactivity, a role for the central helix has been put forward for activity, aggregation, and fibril formation. Kaiser and coworkers (13, 14) provided evidence that the model of an amphipathic helix in the region 8–22 is a useful guide to designing potent sCT activity, although the primacy of conformational flexibility over amphipathicity has been invoked as an important parameter for activity (15). These apparently conflicting results could be explained by the existence of subtypes of CT receptor (CTR) showing variable affinity for helical and non-helical peptides (1, 16). Alternative RNA splicing yields multiple CTR mRNA isoforms. In man, at least six potential variants exist (3, 17, 18); however, the most common human CTR isoforms differ by the presence (hCTRb) or absence (hCTRa) of a 16-amino acid insert between amino acids 174 and 175, within the first intracellular loop of the receptor (19). Of these, the hCTRa is the major human receptor isoform and is expressed in essentially all tissues known to express the CTR. The {alpha}-helical central region of CT peptides has been reported to interact directly with the receptor N terminus (20), and a short segment of the hCTRa close to the transmembrane domain 1 is proximal to amino acid 19 of helix (21). Furthermore, a strict relationship between the binding receptor affinity and the helicity of the hormone has been observed, with a correlation between amphipathicity and potency (16, 22).

Two questions arise. Does the hormone-receptor interaction require a CT helix of definite length? Is the interaction simulated by any model amphipathic helix, as suggested by Kaiser and co-workers (13, 14)? Because the higher affinity of sCT in binding CTRs involves residues within the C terminus (amino acids 22–32) of the hormone (23) (that is, residues located outside of the helix (12)), we designed an sCT mutant (referred to as Leu23,Ala24-sCT) in which Pro23 and Arg24 were substituted for Leu23 and Ala24, respectively (24). Insertion of two helix-promoting residues should prolong the helical C terminus, so as to deal with the first question.

The second question was tackled by analyzing the conformational properties by circular dichroism (CD) and NMR spectroscopies of a number of sCT analogs showing variable helix length in SDS. Furthermore, we evaluated the receptor binding affinity and the in vivo hypocalcemic activity, which were linked to the helical length and chemical properties of the hydrophobic face. Our structural and biological data together with those of other CT analogs suggest a strict relationship between the amphipathic {alpha}-helix of definite length, located in the 9–19 region, and the biological activity of sCT. Because of variability of calcitonin sequences in that region (in particular, for sCT and hCT, 10 of the 16 amino acid substitutions are concentrated in the segment 8–22), it is suggested that the helix does not have to be a perfect amphipathic {alpha}-helix; it can be distorted or somewhat shortened but located in the 9–19 region. Also important is the Leu distribution within the helix; in fact, CT bioactivity is altered when Leu is substituted for Ala, showing that the chemical properties of the hydrophobic side chains within the helix are relevant for receptor binding affinity and the in vivo hypocalcemic activity.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Peptide Synthesis—sCT, Gly8-sCT, Met8-sCT, Ala9-sCT, Ala12-sCT, Ala16-sCT, Ala19-sCT, Ala12,Ala16-sCT, Ala12,Ala19-sCT, Ala16,Ala19-sCT, Ala12,Ala16,Ala19]-sCT, des-Leu9-sCT, des-Leu12-sCT, des-Leu16-sCT, des-Leu19-sCT, des-19Leu-Gly20-Thr21-sCT, des-L19,G20,T21,T22-sCT, Gly8-des-Leu19-sCT, Ala9-des-Leu19-sCT, Leu23,Ala24-sCT, MCT-I, MCT-III, sCT (1–23)-NH2, and hCT were synthesized on polyoxyethylenepolystyrene graft resin. Peptide chain assembly was performed using N-(9-fluorenyl)methoxycarbonyl chemistry (25) and in situ activation of amino acid building blocks by PyBOP (26). Peptide purity was confirmed by reversed phase high performance liquid chromatography and matrix-assisted laser desorption ionization time-of-flight mass spectrometry using a Bruker Biflex instrument (Bremen, Germany) in the linear mode at 19.5 kV.

NMR Experiments—For acquisition of NMR spectra, the concentration of each sample in 90% 1H2O, 10% 2H2O (Cortec-Net, Paris, France), and 100% 2H2O was 0.0012 mM. Solid perdeuterated SDS (Cambridge Isotope Laboratories, Woburn, MA) was added, and its concentration was maintained well above the critical micelle concentration, with a peptide-SDS molar ratio of about 1:120.

1H NMR spectra, acquired at the NMR Service of Istituto di Chimica Biomolecolare del CNR (Pozzuoli, Italy), were recorded at 600 MHz on a Bruker DRX-600 spectrometer using an inverse multinuclear probe head fitted with gradients along the x, y, and z axes. Spectra were referenced to internal sodium 3-(trimethylsilyl)-(2,2,3,3-2H4) propionate (Aldrich). Clean total correlation spectroscopy (TOCSY) (27, 28) and nuclear Overhauser enhancement spectroscopy (NOESY) (29) spectra were recorded by using the time-proportional phase incrementation of the first pulse and incorporating the excitation sculpting sequence (30) for water suppression. We used a double-pulsed field gradient echo with a soft square pulse of 4 ms at the water resonance frequency, with the gradient pulses of 1 ms each in duration. In general, 512 equally spaced evolution time period t1 values were acquired averaging 16 transients of 2048 points, with 6024 Hz of spectral width. Time-domain data matrices were all zero-filled to 4096 points in both dimensions, thus yielding a digital resolution of 2.94 Hz/point. Before Fourier transformation, a Lorentz-Gauss window with different parameters was applied for both t1 and t2 dimensions for all the experiments. NOESY spectra were obtained with different mixing times (0.10, 0.20, and 0.25 s); TOCSY experiments were recorded with a spin-lock period of 64 and 96 ms, achieved with the MLEV-17 pulse sequence. Both NOESY and TOCSY experiments were performed at 310 and 324 K. 3JHN{alpha} coupling constants of isolated resonances were measured from one-dimensional experiments acquired with 131,072 points after application of strong Lorentian-Gaussian resolution enhancement.

Structure Calculations for Leu23,Ala24-sCT; Simulated Annealing and Molecular Dynamics Refinement—Distance restraints were obtained from NOESY spectra of Leu23,Ala24-sCT recorded with 0.10- and 0.25-s mixing times in SDS micelles at 300, 310, and 324 K. Spectra taken at all temperatures show no significant differences in the distribution and intensity of NOE cross-peaks along the whole peptide sequence, thus indicating a similar solution structure. NOE volumes were integrated and calibrated with the TRIAD software (Tripos Inc., St. Louis, MO). In all, 304 NOE cross-peaks (97 intraresidual, 207 sequential and medium range) were converted into upper-limit distances and used together with van der Waals radii as distance restraints for structure calculations with the AMBER 6 package (31). As described for native hCT (32) and sCT (12), we used different NOE cross-peak reference for the calibration of intraresidual and interresidual NOEs. Furthermore, independent calibrations were performed for the NH-NH, the NH-aliphatic, and the aliphatic-aliphatic regions of NOESY spectra. Intraresidual NOEs were calibrated by using the Gln14 NHi-{alpha}CHi (0.27 nm) and the Pro32 {alpha}CHi-betaCHi (0.27 nm) NOEs for the NH-aliphatic and the aliphatic-aliphatic regions, respectively. Calibration of interresidual NOEs was achieved assigning regular intrahelical distances to isolated cross-peaks between residues for which an unambiguous experimental evidence of an helical conformation exists, i.e. the Lys11-Leu12 NHi-NHi + 1 (0.28 nm), the Lys11-Leu12 {alpha}CHi-NHi + 1 (0.35 nm), and the Ser13-Leu16 {alpha}CHi-betaCHi + 3 (0.29 nm) cross-peaks. Finally, all of the distances calculated from NOESY spectra acquired with 0.10-s mixing time were used with a tolerance of +10%, whereas a 20% tolerance was allowed for those coming from spectra recorded at longer mixing time. The self-consistency of the choice of reference distances was checked in the resulting structures. Additional conformational restraints were inferred from 3JNH{alpha} scalar couplings constraints. Only 11 scalar couplings were considered with values falling clearly below the 6–8-Hz region, a range that usually indicate rotationally averaged torsion angles (33). Restraints on the trans amide bonds were also imposed. The {omega} dihedral angles were restrained in the range of 170–190 degrees. The beta-methylene groups were stereospecifically assigned with the program HABAS (34). A total of 15 (4, 5, 7, 9, 11, 12, 14–19, 22, 23, 26) of 23 residues were stereospecifically assigned. When no stereospecific assignment was possible for methyl and methylene protons, the r–6 distance-weighted ambiguous restraints were used.

The Leu23,Ala24-sCT starting structure was generated with the AMBER 6 package using the special residue data base with reduced charges for amino acid side chains. We used the AMBER 91 all-atom forcefield (35) to compute the intrinsic strain energy and, additionally, a parabolic or linear penalty function for NOE distance bounds and torsion angles restraints. The force constants for distance and torsion angle restraints were, respectively, 8.33 x 103 kJ mol–1 nm–2 and 83.33 kJ mol–1 rad–2. Inall in vacuo calculations a distance-dependent dielectric function was used to mimic the presence of a high dielectric constant solvent, with a cutoff radius of 0.8 nm for nonbonded interactions and a residue-based pair-list routine. Structures were sampled and refined with a restrained simulated annealing (SA)/energy minimization approach previously described (36) giving a final set of 50 structures. Subsequently, MD calculations in methanol were performed on the lowest energy Leu23,Ala24-sCT structure using the particle mesh Ewald method (37) for calculating the full electrostatic energy of the unit cell. The AMBER-OPLS united atom force field (38) was employed for the peptide in conjunction with explicit methanol molecules and chloride counterions (39). The Leu23,Ala24-sCT initial structure was first neutralized with 3 chloride ions and subsequently solvated with 3211 methanol molecules in a periodic rectangular box (edge lengths of 6.94, 4.81, and 5.07 nm). Preparation and equilibration of the unit cell before the 1000-ps production run was performed as reported (36). Throughout the 1000-ps production run we collected coordinates, velocities, and energy every 10 ps to perform statistical analysis. Simulations were performed in the nTP ensemble (T = 300 K and P = 1 atm) with a constant temperature and constant pressure relaxation times, respectively, of 0.05 and 0.2 ps for the equilibration runs and 1 and 2 ps for the 1000-ps production run. The molecular graphics program MOLMOL (40) was used to analyze and represent the three-dimensional structures of Leu23,Ala24-sCT. As reference, the same calculation procedure was applied to sCT. The solution structures of sCT and Leu23,Ala24-sCT have been deposited with the Protein Data Bank (codes 2GLH and 2GLG, respectively).

In Vivo Hypocalcemic Assay—Biological activity was estimated by in vivo hypocalcemic activity assay. Eight-week-old Wistar male rats (Japan SLC, Hamamatsu, Japan; Morini SAS, Polo D'Enza, Italy) whose body weights were in the range of 220–240 g were used. Rats were withdrawn food at least 12 h before experiments. All calcitonins were diluted with 1% gelatin in 0.9% NaCl solution at pH 3.2 and injected into subcutaneous space. Doses used were 7.5, 25, 75, 250, and 750 ng/kg of body weight for sCT and 25, 75, 250, and 750 ng/kg for different calcitonins. Blood (0.5 ml) was collected from the tail vein at 0, 1, and 3 h after administration, and Ca2+ levels in plasma were measured by Ca2+/pH analyzer (634, CIBA Corning Diagnostics).

The hypocalcemic response of each dose was normalized to that observed for 750 ng/kg of sCT, since it was found that a dose of 750 ng/kg of sCT was sufficient for the plateau to be reached. The following formula was used:

Formula(Eq. 1)
The dose-response curve was made by linear regression, and 50% of effective dose (ED50) value was calculated.

Cell Culture—Human breast cancer T 47D cells were cultured as described (20) in RPMI 1640 containing 10% heat inactivated fetal bovine serum, 1% streptomycin/penicillin, 0.1 µM insulin, and 0.1 µM hydrocortisone in 5% CO2 and 310 K. The latter hormones were omitted from the medium when subculturing cells to be used 1–3 days later for the receptor binding assay. Subculturing was performed with trypsin/EDTA as described (41, 42), and for the binding experiments cells were subcultured in 12-well dishes. Receptor binding experiments were performed when cells reached 90% confluence (1–3 days after subculture).

Receptor Binding Assay—The assay was performed according to established protocols (41–42). Briefly, cells in the 12-well dishes were washed with NaCl/Pi (1 ml) at room temperature, and then prewarmed (310 K) assay buffer (RPMI 1640 and 0.1% (w/v) bovine serum albumin) was added to the cells (950 µl). Lyophilized 125I-labeled sCT (5 µCi, specific activity 2000 µCi·mmol–1) was reconstituted in 100 mM HCl (200 µl), separated into aliquots at 277 K in Eppendorf tubes (15 µl each), and kept at 253 K, and for each 12-well plate one tube was thawed at room temperature, diluted with assay buffer (250 µl), and used immediately. Twenty microliters of the 125I-labeled sCT solution (15.2 pmol) were then added to each well, and the wells were mixed by gentle shaking. Thereafter, solutions (50 µl) of different concentrations of the peptides in assay buffer were added to the cells, and after gentle mixing cells were incubated for 1 h at room temperature. Peptide solutions were freshly made before each experiment by diluting peptide stocks ({approx}500 µM in 1 mM HCl) (43) in assay buffer. Binding was terminated by aspiration of the medium and washing of the cells with NaCl/Pi three times. Cells were then removed from the wells by short treatment (1 min) with 0.5 M NaOH (2 x 0.5 ml), and bound radioactivity was assessed by {gamma}-counting (counter efficiency {approx} 75%). Nonspecific binding was determined as the binding of 100 nM sCT. This was assessed from 15 independent experiments to be 14.94% (±3.36). Specific binding was the difference between total binding (tracer alone) and nonspecific binding.

CD Spectroscopy—Measurements were performed on a Jasco-J710 spectropolarimeter connected to a water bath used to control the temperature of the cell. Spectra were recorded in the far UV region (200–240 nm) at 300 K and pH 7.4 (20 mM phosphate, 100 mM NaCl) with a peptide concentration of 0.056 mM in a 1.0-cm path length cell. Spectra in the presence of 0.4 M SDS were acquired with a peptide concentration of 0.038 mM in a 0.1-cm path length cell. A spectral bandwidth of 2.0 nm and a scan speed of 10 nm/min were used. The precision of the data were improved by averaging five scans, and the results are reported as mean residue ellipticity ({theta}). Prediction of percentages of secondary structure from CD spectra was obtained using the k2d software, a Kohonen neural network with a two-dimensional output layer (44).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Does the CT-Receptor Interaction Require a Helix of Definite Length?
Solution Structure of Leu23,Ala24-sCT—Fig. 1 shows the CD spectra of Leu23,Ala24-sCT at pH 7.4 and 300 K in water (broken line) and in SDS (continuous line). Estimation of the secondary structure with the k2d neural network algorithm (44) suggests the presence of 20% {alpha}-helix in water. In SDS a dominant {alpha}-helix is clearly discernable, as two minima with high ellipticity values are observed at 220 and 208 nm, and we estimated the presence of 60% {alpha}-helix (mutant 19, Table 1).


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  		TABLE 1
CD and NMR structural characterization of CT peptides in the absence and presence of SDS

Results shown are with 0.056 mM peptide in the absence of SDS and 0.038 mM in the presence of 0.4 M SDS at 300 K in 20 mM phosphate and 100 mM NaCl, pH 7.4.

 
The detailed solution structure of Leu23,Ala24-sCT in SDS was obtained by NMR and SA and MD calculations. Assignment of proton spin systems was performed following the sequential methodology outlined by Wüthrich (33) using TOCSY and NOESY spectra recorded at 324 and 310 K to separate overlapping resonances. TOCSY experiments in 1H2O and in 2H2O allowed the identification of the amide {alpha} and beta protons of almost all of the amino acids. The complete assignment of long side-chain residues was accomplished by a combination of TOCSY and 0.25-s mixing-time NOESY experiments. Individual spin systems were used to identify characteristic short and medium range NOE connectivities. For example, in the NH-NH region of the 0.10-s mixing-time NOESY spectrum, it was possible to follow the connectivities from Ser5 to Asn26 with only few interruptions due to cross-peaks overlap.


Figure 1
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  		FIGURE 1.
Circular dichroism spectra of Leu23,Ala24-sCT. Spectra were recorded at 300 K in 20 mM phosphate, 100 mM NaCl, pH 7.4, at peptide concentration of 0.056 mM (dashed line) and in 0.4 M SDS at a peptide concentration of 0.038 mM (solid line). The results are reported as mean residue ellipticity.

 
The secondary structure of Leu23,Ala24-sCT was delineated from qualitative analysis of the sequential ({alpha}CHi-NHi + 1 and NHi-NHi + 1) and medium range ({alpha}CHi-NHi+n, 1 < n <4, and {alpha}CHi-betaCHi + 3) NOEs collected at 310 and 324 K, and from 3JHN{alpha} coupling constants. Fig. 2 summarizes the observed NOEs and the apparent 3JHN{alpha} coupling constants. In the region Val8-Ala24, strong NHi-NHi + 1 NOEs and weak {alpha}CHi-NHi + 1 cross-peaks suggest the presence of {alpha}-helical structure. This finding was supported by several unambiguous {alpha}CHi-NHi + 3, betaCHi-NHi + 1, {alpha}CHi-betaCHi + 3, and three {alpha}CHi-NHi + 4 cross-peaks. The absence of {alpha}CHi-NHi + 2 cross-peaks, suggestive of a 310 helix, together with 3JHN{alpha} < 6 Hz led to the conclusion that the Val8-Ala24 region forms an {alpha}-helix.

The presence of an NOE between {alpha}Leu4 and betaCys7 indicates that the helix could comprise residues up to Leu4. This behavior has also been observed for sCT, whereas in hCT no ring residue is part of the helix. Residues in the region 4–9 are important for the stabilization of the amphipathic {alpha}-helix; in fact, two long range NOEs (NHLeu4-betaLeu9, betaLeu4-NHLeu9), observed at all mixing times suggest close interaction between the ring and the helix.

In the C terminus the presence of characteristic medium range NOEs such as {alpha}Leu23-betaAsn26 suggests that the helix may include residues 25 and 26. Furthermore, the presence of a capping (i + 5 -> i) {pi} turn can be inferred from calculations (see below). From Gly28 onward the molecule has a disordered conformation since only few weak sequential NOEs were detected. Moreover, different from sCT, the C-terminal region of Leu23,Ala24-sCT does not fold back toward the helix as no long range NOEs connecting residues far apart in the sequence were observed.


Figure 2
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  		FIGURE 2.
Amino acid sequence of Leu23,Ala24-sCT and diagrammatic representation of the short and medium range NOE connectivities observed in SDS micelles at 310 and 324 K. NOE intensities are indicated by the thickness of the bars. A filled triangle ({blacktriangleup}) below the residue name indicates a 3JHN{alpha} coupling constant of ~2–3 Hz measured for that residue.

 
Evidence was found for the presence of an isomer (22% of population) with cis peptide bond at Pro32. It was identified by an NOE between the H{alpha} protons of Thr31 and Pro32, whereas the trans form was identified by the presence of an {alpha}i-{delta}i + 1 NOE cross-peak. Usually, the relatively slow isomerization between the cis and trans forms of prolyl peptide bond induces additional cross-peaks for nearby residues. In CT spectra, two Thr31 cross-peaks were observed in the TOCSY fingerprint region.

Three-dimensional Structure of Leu23,Ala24-sCT—The initial 50 three-dimensional structures exhibit small root-mean square (r.m.s.) deviations from the experimental data and energy falling in the narrow range –1250 to –1378 kJ mol–1. The r.m.s. deviation values from interproton distances and dihedral angles are 0.0072 ± 0.0001 nm and 2.04 ± 0.30 degrees, respectively. The converged structures exhibit averaged backbone atomic r.m.s. deviation of 0.045 ± 0.011 nm for the Leu4-Gly28 helix. The best Leu23,Ala24-sCT structure in terms of potential energy was selected to perform a 1000-ps unrestrained MD (see "Experimental Procedures"). The backbone superposition of 100 structures of Leu23,Ala24-sCT in methanol generated every 10 ps of the whole unrestrained MD and the corresponding ribbon representation are reported in Fig. 3, A and B, respectively.


Figure 3
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  		FIGURE 3.
A, stereo view of the backbone superposition of the 100 Leu23,Ala24-sCT periodically sampled structures along the 1000-ps unrestrained MD. Structures were superimposed for pairwise minimum r.m.s. deviation of the N, C{alpha}, and C atoms of residues 4–28. B, stereo view of Leu23,Ala24-sCT structure showing the amphipathic property of the {alpha}-helix; hydrophobic residues, mainly leucine, are on the left side, whereas hydrophilic amino acids are on the right side. Hydrogen bonds along the backbone and in the N and C terminus helix cap motifs are represented as discontinuous lines.

 
As previously described, methanol was the solvent of choice for simulations (12). The {alpha}-helix (Fig. 3A) is well defined and includes 4LSTC7 of the N-terminal ring. The structure between Leu4 and Gly28 is characterized by an amphipathic {alpha}-helix, with Leu4, Val8, Leu9, Leu12, Leu16, Leu19, and Leu23 forming the hydrophobic face (Fig. 3B). The disulfide bridge between Cys7 (within the first turn of the helix) and Cys1 (external to the helix) straddles the helix N terminus, and it is associated with capping that stabilizes it. The NH groups of Thr6 and Cys7, which cannot form intrahelical hydrogen bonds, are hydrogen-bonded to the >CFormula O of Asn3 side chain and to >CFormula O of Asn3 backbone, respectively (Fig. 3B). Additional hydrogen bonds occur between the (acceptor) O{gamma} of Thr6 side chain and the (donor) backbone Asn3 NH (Thr6 -> Asn3) and between the side-chain groups of Ser2 and Thr6. This N-capping pattern recalls the beta-box motif (45) consisting of a hydrophobic interaction between N3' and N3 or N4 (N3' -> N3/N4) and a hydrogen-bonded beta turn between the NH at N3' and OFormula C< at Ncap. With regard to the C terminus stabilization of the helix, a {pi} {alpha}R-turn was identified. It is a generally occurring motif in {alpha}-helices as a distortion of the termini or in the middle (46) and consists of an i + 5 -> i hydrogen-bonded turn conformation ({pi} turn), with the (i + 4) th residue adopting an {alpha}R conformation. i + 5 -> i hydrogen bonds, as is outlined in Fig. 3B, occur between Gly30(HN) -> Thr25(O) and between Thr31(HN) -> Asn26(O), and an additional i + 4 -> i hydrogen bond is present between Ser29(HN) -> Thr25(O), known to stabilize {pi} {alpha}R-turns.

Time evolution of the intramolecular hydrogen bond pattern and of the backbone dihedral angles {phi} and {psi} gives an overall estimation of the stability of the secondary structure in the molecular dynamics time scale. The length of the current simulation permitted detailing fluctuations in secondary structure in the nanosecond time domain. The series of consecutive residues from Leu4 to Gly28 (adopting an {alpha}-helical conformation in the structures coming from SA/energy minimization calculations) have all backbone dihedral angles near the observed mean values for {alpha} helices ({phi} =–60 ± 15° and {psi} =–40 ± 15°) with no r.m.s. deviations greater than 12 degrees for any residue. Intrahelical NH(i) -> CO(i-4) hydrogen bonds are also well conserved along the whole set of structures with percentages ranged from 87% to 99%. Both the N terminus and the C terminus patterns of helix-terminating hydrogen bonding remained essentially invariant, thus supporting the stability of the overall peptide folding.

The time variation of {phi} and {psi} dihedral angles along the 1000-ps unrestrained MD trajectory (not shown) confirms the stability of the Leu23,Ala24-sCT helix in the region 4–28. The only local correlated variations are observed for {psi}29 and {phi}30, outside the helical region. The absence of any structural drift on the simulated time scale was also noted in a two-dimensional plot of the r.m.s. deviation values (not shown), calculated from each pair of superimposed molecules, of the 100 Leu23,Ala24-sCT conformations taken at regular intervals through the 1000-ps of the unrestrained MD simulations. We only observed local and reversible transitions.

Receptor Binding Affinity of Leu23,Ala24-sCT—Calcitonin exerts its biological activity upon binding a receptor that is a closely related member of the B family of G protein-coupled receptors with putative seven transmembrane domain (for review, see Ref. 1). Calcitonin receptors are widely distributed in the body but are most concentrated in the hypothalamus, bone, and kidney as well as in several cancer cell lines including the human breast cancer cell line T 47D (42). We have used the T 47D cell line to assess human receptor binding affinities of Leu23,Ala24-sCT as compared with sCT, the strongest known naturally occurring inhibitor, and hCT, a weak ligand. Binding affinities were evaluated via competitive inhibition of the specific binding of the radioligand 125I-labeled sCT that binds with high affinity and selectivity to the calcitonin receptors of this cell line (47). As shown in Fig. 4, Leu23,Ala24-sCT showed decreased binding affinity compared with sCT. Receptor binding affinity of Leu23,Ala24-sCT (IC50 = 1.8 nM) was ~2.5 lower than the affinity of sCT (IC50 = 0.69 nM), which in turn was ~6 times more potent than hCT (IC50 = 4.9 nM). Both sCT and hCT IC50 values are in perfect agreement with those reported (47). The lower binding affinity of Leu23,Ala24-sCT compared with sCT was also consistent with its decreased in vivo hypocalcemic potency with respect to sCT.


Figure 4
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  		FIGURE 4.
Human CT receptor binding of sCT, Leu23,Ala24-sCT and hCT to T47D cells assessed via displacement of bound 125I-labeled sCT. Specific radioligand binding is plotted versus the concentration of competing sCT ({diamondsuit}), Leu23,Ala24-sCT ({square}), and hCT ({blacktriangleup}). Data represent, respectively, the mean of 10, 13, and 12 assays ± S.E., indicated by the bars.

 
In Vivo Hypocalcemic Activity of Leu23,Ala24-sCT—We also studied the in vivo hypocalcemic potency of Leu23,Ala24-sCT in rats. To evaluate the effect induced by the mutation introduced to prepare the analogs, native sCT was also tested. The 50% effective doses (ED50) were calculated (see "Experimental Procedures") from dose-response curves; 21.4 ng/kg was determined for sCT, whereas for Leu23,Ala24-sCT a value of 105.0 ng/kg was found. According to these results, the hypocalcemic activity of Leu23,Ala24-sCT is one-fifth that observed for sCT. All of the above results show that in Leu23,Ala24-sCT the longer helix (compared with the wild-type hormone) stabilized by the double mutation directly or indirectly reduces receptor binding affinity and in vivo hypocalcemic activity.

Is the Interaction Simulated by Any Amphipathic Helix?
CD and NMR Structural Characterization of CT Peptides in the Absence and Presence of SDS—The CD spectra of CT in the absence and in the presence of detergents have been previously described by several authors. Table 1 reports the mean residue ellipticities observed for sCT and analogs and hCT (as reference) in the absence and in the presence of SDS.

In SDS-free solution, except for MCT-I and MCT-III (Table 1, mutants 20 and 21), all reported calcitonins show no significant ordered structure. Prediction of secondary structure percentages from CD spectra with the k2d software (44) indicated an {alpha}-helix percentage of 30 and 35% for MCT-I and MCT-III, respectively. All of the other analogs show a helix percentage smaller than 20% (Table 1), with the lowest percentage (9%) for Ala16-sCT (mutant 6) and hCT (peptide 23). The addition of SDS affects all mutants, bringing about an increase in the mean residue ellipticities, with highest helical contents, as compared with sCT, for Ala16-sCT (mutant 6), des-Leu9-sCT (mutant 12), des-Leu19-sCT (mutant 15), des-19Leu-Gly20-Thr21-sCT (mutant 16), Leu23,Ala24-sCT (mutant 19), MCT-I (mutant 20), MCT-III (mutant 21), and sCT (1–23)-NH2 (mutant 22). In particular, the last four peptides take up 60, 66, 72 and 70% helix percentage, respectively (Table 1). On the contrary, hCT acquires much less helical structure than do sCT and mutants. Human CT is known to aggregate as a function of time (7); however, the concentrations used in our study are lower that those required for aggregation and no time or concentration dependence of the CD spectra were observed.

Localization of the {alpha}-helix by NMR was delineated from the qualitative pattern recognition approach of the sequential and medium range NOEs, as derived from two-dimensional NMR experiments (33). In water at pH 7, we observed strong {alpha}CHi-NHi + 1 connectivities along the whole peptide chain and weak NHi-NHi + 1 connectivities essentially concentrated in the central region. Furthermore, except for MCT-I (mutant 20) and MCT-III (mutant 21), we noticed scattered {alpha}CHi-NHi + 2 and betaCHi-NHi + 1 connectivities, which argue for the presence of a structure fluctuating between an extended chain and a sequence of turns located in the central region of CT (33). On the contrary, for MCT-I, and MCT-III, in the region Leu8-Leu19 we observed weak {alpha}CHi-NHi + 1 and strong NHi-NHi + 1 connectivities together with some medium range NHi-NHi + 2, {alpha}CHi-NHi + 3, {alpha}CHi-NHi + 4, and {alpha}CHi-betaCHi + 3 NOEs. They all hint at the presence of a well defined {alpha}-helix also in water, with a percentage of 38 and 40% for MCT-I, and MCT-III, respectively (data are not reported in Table 1).

Except for Ala12-sCT (mutant 5), the addition of SDS induces a helix in the central region of CT. We observed strong, non-ambiguous NHi-NHi + 1 and medium {alpha}CHi-NHi + 1 cross-peaks between all consecutive amino acids (as an example, see Fig. 2); the presence of such sequential NOEs is a first indication of a helical structure, which is confirmed by the observation of several medium-range NHi-NHi + 2, {alpha}CHi-NHi + 3, {alpha}CHi-NHi + 4, and {alpha}CHi-betaCHi + 3 NOEs. We found that the helix percentage obtained by NMR was always higher than that estimated from CD spectra. Except for Ala9-sCT (mutant 4), Ala12-sCT (mutant 5), Ala12,Ala16,Ala19]-sCT (mutant 11), des-Leu12-sCT (mutant 13), des-Leu16-sCT (mutant 14), and hCT (peptide 23), the helix always covers the 9–19 region (Fig. 5). The percentage and the helix location for each mutant are reported in Table 1.

In Vivo Hypocalcemic Activity and Receptor Binding Affinity of CT Mutants—We also studied the biological activity of CT mutants by investigating the in vivo hypocalcemic activity and receptor binding affinity, as described for Leu23,Ala24-sCT, and the results are reported in Table 2. To assess the effect induced by the mutation introduced to synthesize the analogs, native sCT was also tested, and the 50% effective doses (ED50) were calculated (see "Experimental Procedures") from dose-response curves. Receptor binding affinity of CT mutants was estimated on the T 47D cell line and compared with that of sCT and hCT (Table 2). For each analog, the affinity was evaluated via competitive inhibition of the specific binding of the radioligand 125I-labeled sCT to the calcitonin receptors of this cell line (47) and was referred to the affinity of sCT (IC50 = 0.69 nM).


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  		TABLE 2
Hypocalcemic activity and receptor binding potency of CT peptides

 


Figure 5
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  		FIGURE 5.
{alpha}-Helical content of CT mutants in SDS as obtained from the NMR qualitative pattern recognition approach (see "Results"). Horizontal bars symbolize the helix, a square represents a substitution, whereas a cross indicates a deletion within the sequence. Mutants are labeled as in Tables 1 and 2. The region corresponding to the putative biologically relevant helix is shaded.

 
Early reports indicate the presence in sCT of a stable {alpha}-helix in the region Leu9–Leu19 (12), with Leu19 interacting with the transmembrane domain 1 of receptor (21). Mutations or deletions at each side of the helix, as in Gly8-sCT (mutant 2), Met8-sCT (mutant 3) or des-Leu19-sCT (mutant 15), and des-19Leu-Gly20-Thr21-sCT (mutant 16) preserve the biological activity (Table 2) and the helix (Table 1), with an increase in the hypocalcemic activity. However, Ala9-sCT (mutant 4) shows a dramatic reduction of the biological potency of sCT, suggesting that this residue is crucial for sCT receptor binding. In fact, deletion of Leu9 in N{alpha}-Ac-sCT (8–32)-amide resulted in an 800-fold decrease in binding affinity (48). Mutations at both helical sides, as in [G8]-des-Leu19-sCT (mutant 17) and [A9]-des-Leu19-sCT (mutant 18), confirm the role of Leu9 in hypocalcemic activity as it increases in analog 17 (with a 9-fold decrease of the binding potency), but both activities are completely abolished in analog 18, in which the native Leu9 and Leu19 are substituted for or deleted. Taken together, these data suggest that for sCT the presence of Leu9 is fundamental for biological activity.

Analysis of Leu23,Ala24-sCT, MCT-I, MCT-III, sCT (1–23)-NH2 (mutants 19-22) indicates that the central helix alone (Table 1) does not warrant CT bioactivity. In fact, although all of the mutants 17-22 show helix percentage >40% and all cover the 9–19 region (Fig. 5, Table 1), the relative receptor binding potency varies from 0.001 (mutant 22) to 0.38 (mutant 19). Contrasting results are instead obtained for the hypocalcemic activity of Leu23,Ala24-sCT and MCT-I (low activity, Table 2) and [G8]-des-Leu19-sCT and MCT-III (high activity, Table 2). In particular, the inactivity of sCT (1–23)-NH2 (mutant 22) despite the helix suggests that other factors must be considered. We, therefore, substituted Leu with Ala at the sites 9, 12, 16, and 19 (mutants 4-11). Although the helix is preserved in all mutants (Table 1), only Ala16-sCT (mutant 6) and Ala19-sCT (mutant 7) show biological activity comparable with native sCT. Branched Leu residues at positions 9 and 12 seems to selectively favor the hypocalcemic activity because Ala16-sCT and Ala19-sCT (mutants 6 and 7) show 6200 and 5400 IU/mg, respectively, and 0.20 and 0.90 relative receptor binding potency (Table 2), whereas Ala9-sCT (mutant 4) and Ala12-sCT (mutant 5) are not active. It is suggested that the first half of the helix (including the 9–12 region) might be linked to the hypocalcemic activity. In fact, Ala16,Ala19-sCT (mutant 10) shows 7235 IU/mg (Table 2), whereas Ala12,Ala19-sCT (mutant 9) is inactive. Analysis of the 12–14 analogs shows that, except for Leu9 (mutant 12), whose biological activity is substantially unaffected (Val8 might be acting instead of Leu9), deletion of Leu12 or Leu16 (mutants 13 and 14) induces a reduction of the biological activation, most likely altering the structural periodic distribution of Leu within the 9–19 helix. Therefore, proper biological activity seems to require anchoring through Leu9 and Leu19 and the presence of Leu12 and Leu16 for the proper receptor interaction. This is in line with the 20-fold increase of hCT hypocalcemic activity, obtained by replacing aromatic amino acids by Leu residues (24, 49).

The inactive sCT (1–23)-NH2 (mutant 22) preserves all Leu residues at sites 9, 12, 16, and 19 but lacks the C-terminal region. It has been found for that CT in SDS takes up an amphipathic {alpha}-helix in the central region and an extended C-terminal tail (11, 12). However, although in hCT the two structural elements are independent, in sCT the tail and the helix interact through the contacts His17-Thr31, Lys18-Asn26, and Leu19-Asn26, (17). Such an interaction is absent in sCT (1–23)-NH2 (mutant 22), Leu23 ,Ala24-sCT, MCT-I, and MCT-III (mutants 19–21). Although the first two have structural limitations (mutant 22 lacks the 24–32 region, and mutant 19 takes up a 4–28 helix, Table 1), MCT-I, compared with sCT (footnotes of Table 1 or 2), presents Leu15 and Gln17 instead of Glu15 and His17, and MCT-III shows Gln17 instead of His17. This would suggest that charged residues within the helix play a role in the helix-tail interaction (17).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Although the molecular mechanism underlying CT bioactivity is not yet fully elucidated, it is well known today that the physiological effects of CT occur through receptor-mediated processes (1). The sCT sequence has been reported to contain two separate domains: an activation domain, which requires a minimum sequence of residues 3–6 and a binding region located between residues 9–32 (50). Furthermore, the C-terminal CT sequence (between residues 12 and 32) binds to the N-extracellular terminal domain of the receptor, which had previously been suggested to be responsible for ligand binding (20), whereas the N-terminal sequence (residues 1–11) interacts with the membrane-embedded domain and the associated loops, which had been suggested before to be the signal transduction domain (20). These results were confirmed by developing photolabile derivatives of sCT and hCT for photoaffinity labeling of CTR. It has been reported that the helical region 16–19 is in close contact with the receptor region Cys134–Lys141 (21, 51) and that hCT Met8 interacts with Leu368 in the third extracellular loop (52).

In general, receptor binding, signal transduction, and in vivo bioactivity of the calcitonins appear to be related to a variety of structural and conformational features that include the formation of an amphiphilic {alpha}-helix in the middle of the sequence (13, 14), conformational flexibility (15), long range interactions of the helix with the N-terminal ring as well as the C terminus (11, 12), the presence of specific regions and amino acid residues within the sequence, and also the aggregation propensity.

A comparison between the structures of sCT and hCT (12) indicated that sCT has a longer central helix with the C-terminal tail folded toward the helix, whereas in the human form the helix is shorter, and no interaction with the tail is present. Considering that sCT shows the highest bioactivity and hCT the lowest, a prominent role can be inferred for the length of the central amphipathic helix and/or the folding back of the C-terminal region. We have investigated here several sCT mutants with variable helix length and its effects on the hypocalcemic activity and receptor affinity. Furthermore, the analysis also confirmed a role for long range interactions as well as strict requirements for the hydrophobic residues within the helix and increased conformational flexibility. Mutants were selected to answer the following questions. Does the interaction with the receptor require an sCT helix of definite length? Is the interaction simulated by any amphipathic helix? We first studied the solution conformation, the receptor binding affinity, and the hypocalcemic potency for Leu23,Ala24-sCT (mutant 19), which was expected to take up a helix longer than sCT. Our results indicated that sCT and Leu23,Ala24-sCT both form an amphiphatic {alpha}-helix, but whereas in Leu23,Ala24-sCT it covers almost the whole sequence (Leu4-Gly28), in the native hormone a shorter region (Thr6–Phe22) is involved, with the region Leu9–Leu19 constantly found in helical conformation (12). Significantly, no helix-tail interaction was observed for Leu23,Ala24-sCT. This is due to the conformational constraints imposed by the longer helix that reduces the hinge region required to allow the folding back of the C terminus, as observed for sCT (Fig. 6). Therefore, the helix length and the folding back of the tail are strictly linked, giving sCT a wedge-shaped form that may be required for proper interaction with the receptor.

Compared with the native hormone, Leu23,Ala24-sCT exhibits less hypocalcemic activity and minor binding affinity (Table 2), supporting the suggestion that the helix length is related to sCT biological activity. Can we assess the helix borders defining the biologically relevant helix? The C terminus seems to be located at site 19, as des-Leu19-sCT and des-19Leu-Gly20-Thr21-sCT (mutants 15 and 16) are fully active (Table 2). A helix encompassing Leu19, as in Leu23,Ala24-sCT, MCT-I, and MC-III (mutants 19–21), shows low receptor binding potency and, except for MCT-III, low hypocalcemic activity. The N terminus appears to be located at Leu9, as Gly8-sCT (mutant 2) and Met8-sCT (mutant 3) show unaffected biological behavior.


Figure 6
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  		FIGURE 6.
Superposition of the Leu23,Ala24-sCT mean structure (black) on the bundle of sCT solution structures (gray). Structures were superimposed for pairwise minimum r.m.s. deviation of the N, C{alpha}, and C atoms of the common central helical region (residues 9–19).

 
By eliminating Leu from sites 9, 12, 16, and 19 (mutants 12–15), we altered the bioactivity when Leu12 or Leu16 were abolished, whereas deletion at sites 9 or 19 were irrelevant. These data suggest that at least one hydrophobic residue is important at each helical side and that the hydrophobic face is very responsive to Leu deletion. By changing Leu residues at sites 12, 16, and 19 into Ala (mutant 11), the biological activity is nullified, suggesting that the hydrophobic face of the conserved amphipathic helix requires a uniform distribution of branching, as Ala residues are not able to ensure interaction with the receptor. Selective substitution of Leu with Ala at sites 9, 12, 16, and 19 (mutants 4-7) suggests that Leu9 and Leu12 are more important for bioactivity than Leu16 and Leu19, as mutants 6 and 7 show biological activity comparable with sCT. According to this interpretation, Ala12,Ala16-sCT (mutant 8) and Ala12,Ala19-sCT (mutant 9) are completely devoid of activity, whereas A16,A19-sCT (mutant 10) shows reduction but compares with sCT.

Mutants 2, 3, 7, 12, 15, and 16 show hypocalcemic activity higher than sCT (Table 2), but receptor binding potency is identical or only slightly lower than sCT (Table 2). It is conceivable that mutations bring about local arrangements within the helix, which favor a better accommodation onto the receptor, in agreement with the increased conformational flexibility required for CT bioactivity (15). On the contrary, Gly8-des-Leu19-sCT (mutant 17) and MCT-III (mutant 21) have hypocalcemic activity more than two times that of sCT, with no correlation with the receptor binding affinity (Table 2). To a lesser extent, a similar behavior is observed for Ala16-sCT (mutant 6) and Ala16,Ala19-sCT (mutant 10). Such behavior might be linked to some unknown step in the receptor-mediated processes and/or to the mechanistic effect brought about by mutations. It is possible that sliding of the helix on the receptor surface locks the hormone in an active conformation similar, but not identical, to that of the native conformation. Although MCT-III preserves the amphipathic helix (19, 20), it abrogates positively charged residues at sites 11 and 17, bearing Lys and His, respectively, in native sCT and inserts Lys14 instead of Gln (see the primary structure in footnote of Table 1). Furthermore, sCT (1–23)-NH2, which is fully helical (Table 1), is completely inactive (Table 2). Taken together, data on MCT-III and sCT (1–23)-NH2 suggest that the native charge distribution with the helix-tail interaction and the right helical length are sufficient to regulate sCT bioactivity.

The data reported confirm the crucial role of the helix for CT bioactivity. However, the central region does not have to be postulated as a perfect amphipathic {alpha}-helix, and indeed, the variability of the calcitonin sequences from different species also argues against such a conclusion. A stable {alpha}-helical region from residues 9–19, although exhibiting amphipathicity, is amenable to sequence variation, and at least some properties of the hormone benefit from imperfections in the segregation of hydrophobic and hydrophilic residues. Our results also indicated that an ideal amphipathic {alpha}-helix might not necessarily correlate with high CT bioactivity. Furthermore, the bioactivity is associated with specific local conformational features of the backbone, side chains, and the shape of the molecule. It is conceivable that the more compact structure brought about by such conformational constraints also affects the rate of metabolic degradation and clearance, which is reduced by the presence of strong peptide secondary structure.


   FOOTNOTES
 
* This work was supported in part by Consiglio Nazionale delle Ricerche/Ministero dell'Istruzione, Università e Ricerca Legge 449/97 DM 30/10/2000 (to AM). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and structure factors (codes 2GLH and 2GLG) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

This paper is dedicated to the memory of Gianfranco Borin. Back

1 Contributed equally to this work. Back

2 Supported in part by European Community Grant ERBFMRXCT960069. Present address: Centro de Investigaciones Biológicas, CSIC, C/Ramiro de Maetzu 9, 28040 Madrid, Spain. Back

3 To whom correspondence should be addressed: Istituto di Chimica Biomolecolare del CNR, Comprensorio Olivetti, Edificio A, Via Campi Flegrei 34, I-80078 Pozzuoli (Napoli), Italy. Tel.: 39-081-8675-228; Fax: 39-081-8041-770; E-mail: amotta{at}icmib.na.cnr.it.

4 The abbreviations used are: CT, calcitonin; CTR, CT receptor; sCT, salmon CT; hCT, human CT; MD, molecular dynamics; hCTRa, human calcitonin receptor isoform differing by the absence of 16-amino acid insert; Leu23,Ala24-sCT, salmon calcitonin that substitutes Pro23-Ala24 for Leu-Ala; TOCSY, two-dimensional total correlation spectroscopy; NOE, nuclear Overhauser effect; NOESY, two-dimensional NOE spectroscopy; SA, simulated annealing; r.m.s., root-mean square. Back


   ACKNOWLEDGMENTS
 
We thank Dominique Melck and Emilio P. Castelluccio (Istituto di Chimica Biomolecolare del Consiglio Nazionale delle Ricerche) for excellent technical assistance and computer system maintenance, respectively.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Purdue, B. W., Tilakaratne, N., and Sexton, P. M. (2002) Receptors Channels 8, 243–255[CrossRef][Medline] [Order article via Infotrieve]
  2. Manicourt, D. H., Devogelaer, J. P., Azria, and Silverman, S. (2005) J. Musculoskelet. Neuronal Interact. 5, 285–293[Medline] [Order article via Infotrieve]
  3. Gorn, A. H., Lin, H. Y., Yamin, M., Auron, P. E., Flannery, M. R., Tapp, D. R., Manning, C. A., Lodish, H. F., Krane, S. M., and Goldring, S. R. (1992) J. Clin. Investig. 90, 1726–1735[Medline] [Order article via Infotrieve]
  4. Zaidi, M., Inzerillo, A. M., Troen, B., Moonga, B., Abe, E., and Burckhardt, P. (2002) in Principles of Bone Biology (Bilezikian, J. P., Raisz, L. G., Rodan, G. A., eds) Vol. 2, pp. 1423–1440, Academic Press, San Diego, CA
  5. Singer, F. R., Aldred, J. P., Neer, R. M., Krane, S. M., Potts, J. T., Jr., and Bloch, K. J. (1972) J. Clin. Investig. 51, 2331–2338[Medline] [Order article via Infotrieve]
  6. Wada, S., Martin, T. J., and Findlay, D. M. (1995) Endocrinology 136, 2611–2621[Abstract]
  7. Arvinte, T., Cudd, A., and Drake, A. F. (1993) J. Biol. Chem. 268, 6415–6422[Abstract/Free Full Text]
  8. Meyer, J.-P., Pelton, J. T., Hoflack, J., and Saudek, V. (1991) Biopolymers 31, 233–241[CrossRef][Medline] [Order article via Infotrieve]
  9. Meadows, R. P., Nikonowicz, E. P., Jones, C. R., Bastian, J. W., and Gorenstein, D. G. (1991) Biochemistry 30, 1247–1254[CrossRef][Medline] [Order article via Infotrieve]
  10. Motta, A., Pastore, A., Goud, N. A., and Castiglione Morelli, M. A. (1991) Biochemistry 30, 10444–10450[CrossRef][Medline] [Order article via Infotrieve]
  11. Castiglione Morelli, M. A., Pastore, A., and Motta, A. (1992) J. Biomol. NMR 2, 335–348[CrossRef][Medline] [Order article via Infotrieve]
  12. Amodeo, P., Motta, A., Strazzullo, G., and Castiglione Morelli, M. A. (1999) J. Biomol. NMR 13, 161–174[CrossRef][Medline] [Order article via Infotrieve]
  13. Moe, G. R., and Kaiser, E. T. (1985) Biochemistry 24, 1971–1975[CrossRef][Medline] [Order article via Infotrieve]
  14. Green, F. R., III, Lynch, B., and Kaiser, E. T. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 8340–8344[Abstract/Free Full Text]
  15. Epand, R. M., Epand, R. F., Orlowski, R. C., Seyler, J. K., and Colescott, R. L. (1986) Biochemistry 25, 1964–1968[CrossRef][Medline] [Order article via Infotrieve]
  16. Nakamuta, H., Orlowsky, R. C., and Epand, R. M. (1990) Endocrinology 127, 163–169[Abstract/Free Full Text]
  17. Moore, E. E., Kuestner, R. E., Stroop, S. D., Grant, F. J., Matthewes, S. L., Brady, C. L., Sexton, P. M., and Findlay, D. M. (1995) Mol. Endocrinol. 9, 959–968[Abstract/Free Full Text]
  18. Chen, W. J., Armour, S., Way, J., Chen, G., Watson, C., Irving, P., Cobb, J., Kadwell, S., Beaumont, K., Rimele, T., and Kenakin, T. (1997) Mol. Pharmacol. 52, 1164–1175[Abstract/Free Full Text]
  19. Gorn, A. H., Rudolph, S. M., Flannery, M. R., Morton, C. C., Weremowicz, S., Wang, T. Z., Krane, S. M., and Goldring, S. R. (1995) J. Clin. Investig. 95, 2680–2691[Medline] [Order article via Infotrieve]
  20. Stroop, S. D., Nakamuta, H., Küstner, R. E., Moore, E. E., and Epand, R. E. (1996) Endocrinology 137, 4752–4756[Abstract]
  21. Pham, V., Wade, J. D., Purdue, B. W., and Sexton, P. M. (2004) J. Biol. Chem. 279, 6720–6729[Abstract/Free Full Text]
  22. Epand, R. F., Orlowski, R. C., and Epand, R. M. (2004) Biopolymers 76, 258–265[CrossRef][Medline] [Order article via Infotrieve]
  23. Houssami, S., Findlay, D. M., Brady, C. L., Martin, T. J., Epand, R. M., Moore, E. E., Murayama, E., Tamura, T., Orlowski, R. C., and Sexton, P. M. (1995) Mol. Pharmacol. 47, 798–809[Abstract]
  24. Andreotti, G., and Motta, A. (2004) J. Biol. Chem. 279, 6364–6370[Abstract/Free Full Text]
  25. Carpino, L. A., and Han, G. Y. (1972) J. Org. Chem. 37, 3404–3409[CrossRef]
  26. Coste, J., Le-Nguyen, D., and Castro, B. (1990) Tetrahedron Lett. 31, 205–209[CrossRef]
  27. Braunschweiler, L., and Ernst, R. R. (1983) J. Magn. Reson. 53, 521–528
  28. Griesinger, C., Otting, G., Wüthrich, K., and Ernst, R. R. (1988) J. Am. Chem. Soc. 110, 7870–7872[CrossRef]
  29. Jeener, J., Meier, B. H., Bachmann, P., and Ernst, R. R. (1979) J. Chem. Phys. 71, 4546–4553[CrossRef]
  30. Hwang, T.-L., and Shaka, A. J. (1995) J. Magn. Reson. 112, 275–279[CrossRef]
  31. Pearlman, D. A., Case, D. A., Caldwell, J. W., Ross, W. S., Cheatham, T. E., III, DeBolt, S., Ferguson, D., Seibel, G., and Kollman, P. (1995) Comput. Phys. Commun. 91, 1–41
  32. Motta, A., Andreotti, G., Amodeo, P., Strazzullo, G., and Castiglione Morelli, M. A. (1998) Proteins 32, 314–323[CrossRef][Medline] [Order article via Infotrieve]
  33. Wüthrich, K. (1986) NMR of Proteins and Nucleic Acids, pp. 162–175, John Wiley & Sons, Inc., New York
  34. Güntert, P., Braun, W., Billeter, M., and Wüthrich, K. (1989) J. Am. Chem. Soc. 110, 3997–4004[CrossRef]
  35. Weiner, S. J., Kollman, P. A., Nguyen, D. T., and Case, D. A. (1986) J. Comput. Chem. 7, 230–252[CrossRef]
  36. Jourdan, F., Lazzaroni, S., López Méndez, B., de Julio, M., Lo Cantore, P., Amodeo, P., Iacobellis, N. S., Evidente, A., and Motta, A. (2003) Proteins 52, 534–543[CrossRef][Medline] [Order article via Infotrieve]
  37. Darden, T., York, D., and Pedersen, L. (1993) J. Chem. Phys. 98, 10089–10092[CrossRef]
  38. Jorgensen, W. L., and Tirado-Rives, J. (1988) J. Am. Chem. Soc. 110, 1657–1664[CrossRef]
  39. Lybrand, T. P., McCammonn, J. A., and Wipff, G. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 833–835[Abstract/Free Full Text]
  40. Koradi, R., Billeter, M., and Wuthrich, K. (1996) J. Mol. Graph. 14, 51–55[CrossRef][Medline] [Order article via Infotrieve]
  41. Kazantzis, A., Waldner, M., Taylor, J. W., and Kapurniotu, A. (2002) Eur. J. Biochem. 269, 780–791[Medline] [Order article via Infotrieve]
  42. Lamp, S. J., Findlay, D. M., Moseley, J. M., and Martin, T. J. (1981) J. Biol. Chem. 256, 12269–12274[Free Full Text]
  43. Kapurniotu, A., and Taylor, J. W. (1995) J. Med. Chem. 38, 836–847[CrossRef][Medline] [Order article via Infotrieve]
  44. Andrade, M. A., Chacón, P., and Morán, F. (1993) Protein Eng. 6, 383–390[Abstract/Free Full Text]
  45. Seale, J. W., Srinivasan, R., and Rose, G. D. (1994) Protein Sci. 3, 1741–1745[Medline] [Order article via Infotrieve]
  46. Rajashakar, K. R., and Ramakumar, S. (1996) Protein Sci. 5, 932–946[Medline] [Order article via Infotrieve]
  47. Findlay, D. M., Michelangeli, V. P., Orlowski, R. C., and Martin, T. J. (1983) Endocrinology 112, 1288–1291[Abstract/Free Full Text]
  48. Feyen, J. H. M., Cardinaux, F., Gamse, R., Bruns, C., Azria, M., and Trechsel, U. (1992) Biochem. Biophys. Res. Commun. 187, 8–13[CrossRef][Medline] [Order article via Infotrieve]
  49. Maier, R., Kamber, B., Riniker, B., and Rittel, W. (1976) Clin. Endocrinol. 5, (suppl.) 327–332
  50. Bergwitz, C., Gardella, T. J., Flannery, M. R., Potts, J. T., Jr., Kronenberg, H. M., Goldring, S. R., and Juppner, H. (1996) J. Biol. Chem. 271, 26469–26472[Abstract/Free Full Text]
  51. Dong, M., Pinon, D. I., Cox, R. F., and Miller, L. J. (2004) J. Biol. Chem. 279, 1167–1175[Abstract/Free Full Text]
  52. Dong, M., Pinon, D. I., Cox, R. F., and Miller, L. J. (2004) J. Biol. Chem. 279, 31177–31182[Abstract/Free Full Text]
  53. Epand, R. M., Epand, R. F., and Orlowski, R. C. (1985) Int. J. Pept. Protein Res. 25, 105–111[Medline] [Order article via Infotrieve]
  54. Epand, R. M., Epand, R. F., and Orlowski, R. C. (1990) Eur. J. Biochem. 188, 633–635[Medline] [Order article via Infotrieve]
  55. Epand, R. M., Seyler, J. K., and Orlowski, R. C. (1986) Eur. J. Biochem. 159, 125–127[Medline] [Order article via Infotrieve]
  56. Epand, R. M., Epand, R. F., Stafford, A. R., and Orlowski, R. C. (1988) J. Med. Chem. 31, 1595–1598[CrossRef][Medline] [Order article via Infotrieve]
  57. Epand, R. M., Epand, R. F., and Orlowski, R. C. (1985) Biochem. Biophys. Res. Commun. 152, 203–207[CrossRef]
  58. Moe, G. R., Miller, R. J., and Kaiser, E. T. (1983) J. Am. Chem. Soc. 105, 4100–4102[CrossRef]
  59. Epand, R. M., Stahl, G. L., and Orlowski, R. C. (1986) Int. J. Pept. Protein Res. 26, 501–507

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