Structural Determinants of Salmon Calcitonin Bioactivity

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 α-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.

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 halflife 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 ␣-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 conforma-tional 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 ␣-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][23][24][25][26][27][28][29][30][31][32] of the hormone (23) (that is, residues located outside of the helix (12)), we designed an sCT mutant (referred to as Leu 23 ,Ala 24 -sCT) in which Pro 23 and Arg 24 were substituted for Leu 23 and Ala 24 , respectively (24). Insertion of two helixpromoting 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 ␣-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 ␣-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.
NMR Experiments-For acquisition of NMR spectra, the concentration of each sample in 90% 1 H 2 O, 10% 2 H 2 O (Cortec-Net, Paris, France), and 100% 2 H 2 O 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. 1 H 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)-(2,2,3,3-2 H 4 ) 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 doublepulsed 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 t 1 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 t 1 and t 2 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. 3 J HN␣ coupling constants of isolated resonances were measured from onedimensional experiments acquired with 131,072 points after application of strong Lorentian-Gaussian resolution enhancement.
Structure Calculations for Leu 23 ,Ala 24 -sCT; Simulated Annealing and Molecular Dynamics Refinement-Distance restraints were obtained from NOESY spectra of Leu 23 ,Ala 24 -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 Gln 14 NH i -␣CH i (0.27 nm) and the Pro 32 ␣CH i -␤CH i (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 Lys 11 -Leu 12 NH i -NH i ϩ 1 (0.28 nm), the Lys 11 -Leu 12 ␣CH i -NH i ϩ 1 (0.35 nm), and the Ser 13 -Leu 16 ␣CH i -␤CH i ϩ 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 ϩ10%, 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 3 J NH␣ 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 dihedral angles were restrained in the range of 170 -190 degrees. The ␤-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 Ϫ6 distance-weighted ambiguous restraints were used.
The Leu 23 ,Ala 24 -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 ϫ 10 3 kJ mol Ϫ1 nm Ϫ2 and 83.33 kJ mol Ϫ1 rad Ϫ2 . In all 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 Leu 23 ,Ala 24 -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 Leu 23 ,Ala 24 -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 ϭ 300 K and P ϭ 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 threedimensional structures of Leu 23 ,Ala 24 -sCT. As reference, the same calculation procedure was applied to sCT. The solution structures of sCT and Leu 23 ,Ala 24 -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 Ca 2ϩ levels in plasma were measured by Ca 2ϩ /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: The dose-response curve was made by linear regression, and 50% of effective dose (ED 50 ) 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 M insulin, and 0.1 M hydrocortisone in 5% CO 2 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/P i (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 l). Lyophilized 125 I-labeled sCT (5 Ci, specific activity 2000 Ci⅐mmol Ϫ1 ) was reconstituted in 100 mM HCl (200 l), separated into aliquots at 277 K in Eppendorf tubes (15 l each), and kept at 253 K, and for each 12-well plate one tube was thawed at room temperature, diluted with assay buffer (250 l), and used immediately. Twenty microliters of the 125 I-labeled sCT solution (15.2 pmol) were then added to each well, and the wells were mixed by gentle shaking. Thereafter, solutions (50 l) 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 (Ϸ500 M in 1 mM HCl) (43) in assay buffer. Binding was terminated by aspiration of the medium and washing of the cells with NaCl/P i three times. Cells were then removed from the wells by short treatment (1 min) with 0.5 M NaOH (2 ϫ 0.5 ml), and bound radioactivity was assessed by ␥-counting (counter efficiency Ϸ 75%). Nonspecific binding was determined as the binding of 100 nM sCT. This was assessed from 15 independent experiments to be 14.94% (Ϯ3.36). 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 (). Prediction of percentages of secondary structure from CD spectra was obtained using the k2d software, a Kohonen neural network with a twodimensional output layer (44).

Does the CT-Receptor Interaction Require a Helix of Definite Length?
Solution Structure of Leu 23 ,Ala 24 -sCT- Fig. 1 shows the CD spectra of Leu 23 ,Ala 24 -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% ␣-helix in water. In SDS a dominant ␣-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% ␣-helix (mutant 19, Table 1).
The detailed solution structure of Leu 23 ,Ala 24 -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 1 H 2 O and in 2 H 2 O allowed the identification of the amide ␣ and ␤ 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 Ser 5 to Asn 26 with only few interruptions due to crosspeaks overlap.
The secondary structure of Leu 23 ,Ala 24 -sCT was delineated from qualitative analysis of the sequential (␣CH i -NH i ϩ 1 and NH i -NH i ϩ 1 ) and medium range (␣CH i -NH iϩn , 1 Ͻ n Ͻ4, and ␣CH i -␤CH i ϩ 3 ) NOEs collected at 310 and 324 K, and from 3 J HN␣ coupling constants. Fig. 2 summarizes the observed NOEs and the apparent 3 J HN␣ coupling constants. In the region Val 8 -Ala 24 , strong NH i -NH i ϩ 1 NOEs and weak ␣CH i -NH i ϩ 1 cross-peaks suggest the presence of ␣-helical structure. This finding was supported by several unambiguous ␣CH i -NH i ϩ 3 , ␤CH i -NH i ϩ 1 , ␣CH i -␤CH i ϩ 3 , and three ␣CH i -NH i ϩ 4 cross-peaks. The absence of ␣CH i -NH i ϩ 2 cross-peaks, suggestive of a 3 10 helix, together with 3 J HN␣ Ͻ 6 Hz led to the conclusion that the Val 8 -Ala 24 region forms an ␣-helix.
The presence of an NOE between ␣Leu 4 and ␤Cys 7 indicates that the helix could comprise residues up to Leu 4 . 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 ␣-helix; in fact, two long range NOEs (NHLeu 4 -␤Leu 9 , ␤Leu 4 -NHLeu 9 ), 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 ␣Leu 23 -␤Asn 26 suggests that the helix may include residues 25 and 26. Furthermore, the presence of a capping (i ϩ 5 3 i) turn can be inferred from calculations (see below). From Gly 28 onward the molecule has a disordered conformation since only few weak sequential NOEs were detected. Moreover, different from sCT, the C-terminal region of Leu 23 ,Ala 24 -sCT does not fold back toward the helix as no long range NOEs connecting residues far apart in the sequence were observed. Evidence was found for the presence of an isomer (22% of population) with cis peptide bond at Pro 32 . It was identified by an NOE between the H␣ protons of Thr 31 and Pro 32 , whereas the trans form was identified by the presence of an ␣ i -␦ i ϩ 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 Thr 31 cross-peaks were observed in the TOCSY fingerprint region.
Three-dimensional Structure of Leu 23 ,Ala 24 -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 Ϫ1250 to Ϫ1378 kJ mol Ϫ1 . The r.m.s. deviation values from interproton distances and  dihedral angles are 0.0072 Ϯ 0.0001 nm and 2.04 Ϯ 0.30 degrees, respectively. The converged structures exhibit averaged backbone atomic r.m.s. deviation of 0.045 Ϯ 0.011 nm for the Leu 4 -Gly 28 helix. The best Leu 23 ,Ala 24 -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 Leu 23 ,Ala 24 -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. As previously described, methanol was the solvent of choice for simulations (12). The ␣-helix (Fig. 3A) is well defined and includes 4 LSTC 7 of the N-terminal ring. The structure between Leu 4 and Gly 28 is characterized by an amphipathic ␣-helix, with Leu 4 , Val 8 , Leu 9 , Leu 12 , Leu 16 , Leu 19 , and Leu 23 forming the hydrophobic face (Fig. 3B). The disulfide bridge between Cys 7 (within the first turn of the helix) and Cys 1 (external to the helix) straddles the helix N terminus, and it is associated with capping that stabilizes it. The NH groups of Thr 6 and Cys 7 , which cannot form intrahelical hydrogen bonds, are hydrogenbonded to the ϾCAO of Asn 3 side chain and to ϾCAO of Asn 3 backbone, respectively (Fig. 3B). Additional hydrogen bonds occur between the (acceptor) O␥ of Thr 6 side chain and the (donor) backbone Asn 3 NH (Thr 6 3 Asn 3 ) and between the side-chain groups of Ser 2 and Thr 6 . This N-capping pattern recalls the ␤-box motif (45) consisting of a hydrophobic interaction between N 3Ј and N3 or N4 (N 3Ј 3 N3/N4) and a hydrogen-bonded ␤ turn between the NH at N 3Ј and OACϽ at Ncap. With regard to the C terminus stabilization of the helix, a ␣Rturn was identified. It is a generally occurring motif in ␣-helices as a distortion of the termini or in the middle (46)  Time evolution of the intramolecular hydrogen bond pattern and of the backbone dihedral angles and 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 Leu 4 to Gly 28 (adopting an ␣-helical conformation in the structures coming from SA/ energy minimization calculations) have all backbone dihedral angles near the observed mean values for ␣ helices ( ϭ Ϫ60 Ϯ 15°and ϭ Ϫ40 Ϯ 15°) with no r.m.s. deviations greater than 12 degrees for any residue. Intrahelical NH(i) 3 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 and dihedral angles along the 1000-ps unrestrained MD trajectory (not shown) confirms the stability of the Leu 23 ,Ala 24 -sCT helix in the region 4 -28. The only local correlated variations are observed for 29 and 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 Leu 23 ,Ala 24 -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 Leu 23 ,Ala 24 -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 Leu 23 ,Ala 24 -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 125 I-labeled sCT that binds with high affinity and selectivity to the calcitonin receptors of this cell line (47). As shown in Fig. 4, Leu 23 ,Ala 24 -sCT showed decreased binding affinity compared with sCT. Receptor binding affinity of Leu 23 ,Ala 24 -sCT (IC 50 ϭ 1.8 nM) was ϳ2.5 lower than the affinity of sCT (IC 50 ϭ 0.69 nM), which in turn was ϳ6 times more potent than hCT (IC 50 ϭ 4.9 nM). Both sCT and hCT IC 50 values are in perfect agreement with those reported (47). The lower binding affinity of Leu 23 ,Ala 24 -sCT compared with sCT was also consistent with its decreased in vivo hypocalcemic potency with respect to sCT.
In Vivo Hypocalcemic Activity of Leu 23 ,Ala 24 -sCT-We also studied the in vivo hypocalcemic potency of Leu 23 ,Ala 24 -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 (ED 50 ) were calculated (see "Experimental Procedures") from dose-response curves; 21.4 ng/kg was determined for sCT, whereas for Leu 23 ,Ala 24 -sCT a value of 105.0 ng/kg was found. According to these results, the hypocalcemic activity of Leu 23 ,Ala 24 -sCT is one-fifth that observed for sCT. All of the above results show that in Leu 23 ,Ala 24 -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.

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 ␣-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 Ala 16 -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 Ala 16 -sCT (mutant 6), des-Leu 9 -sCT (mutant 12), des-Leu 19 -sCT (mutant 15), des- 19 Leu-Gly 20 -Thr 21 -sCT (mutant 16), Leu 23 ,Ala 24 -sCT (mutant 19), MCT-I (mutant 20), MCT-III (mutant 21), and sCT (1-23)-NH 2 (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 ␣-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 ␣CH i -NH i ϩ 1 connectivities along the whole peptide chain and weak NH i -NH i ϩ 1 connectivities essentially concentrated in the central region. Furthermore, except for MCT-I (mutant 20) and MCT-III (mutant 21), we noticed scattered ␣CH i -NH i ϩ 2 and ␤CH i -NH i ϩ 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 Leu 8 -Leu 19 we observed weak ␣CH i -NH i ϩ 1 and strong NH i -NH i ϩ 1 connectivities together with some medium range NH i -NH i ϩ 2 , ␣CH i -NH i ϩ 3 , ␣CH i -NH i ϩ 4 , and ␣CH i -␤CH i ϩ 3 NOEs. They all hint at the presence of a well defined ␣-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 Ala 12 -sCT (mutant 5), the addition of SDS induces a helix in the central region of CT. We observed strong, nonambiguous NH i -NH i ϩ 1 and medium ␣CH i -NH i ϩ 1 crosspeaks 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 NH i -NH i ϩ 2 , ␣CH i -NH i ϩ 3 , ␣CH i -NH i ϩ 4 , and ␣CH i -␤CH i ϩ 3 NOEs. We found that the helix percentage obtained by NMR was always higher than that estimated from CD spectra. Except for Ala 9 -sCT (mutant 4), Ala 12 -sCT (mutant 5), Ala 12 ,Ala 16 ,Ala 19 ]-sCT (mutant 11), des-Leu 12 -sCT (mutant 13), des-Leu 16 -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 Leu 23 ,Ala 24 -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 (ED 50 ) were calculated (see "Experimental Procedures") from dose-re- sponse 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 125 I-labeled sCT to the calcitonin receptors of this cell line (47) and was referred to the affinity of sCT (IC 50 ϭ 0.69 nM).
Early reports indicate the presence in sCT of a stable ␣-helix in the region Leu 9 -Leu 19 (12), with Leu 19 interacting with the transmembrane domain 1 of receptor (21). Mutations or deletions at each side of the helix, as in Gly 8 -sCT (mutant 2), Met 8 -sCT (mutant 3) or des-Leu 19 -sCT (mutant 15), and des- 19 Leu-Gly 20 -Thr 21 -sCT (mutant 16) preserve the biological activity ( Table 2) and the helix (Table 1), with an increase in the hypocalcemic activity. However, Ala 9 -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 Leu 9 in N ␣ -Ac-sCT (8 -32)-amide resulted in an 800-fold decrease in binding affinity (48). Mutations at both helical sides, as in [G 8 ]-des-Leu 19 -sCT (mutant 17) and [A 9 ]des-Leu 19 -sCT (mutant 18), confirm the role of Leu 9 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 Leu 9 and Leu 19 are substituted for or deleted. Taken together, these data suggest that for sCT the presence of Leu 9 is fundamental for biological activity.

TABLE 2
Hypocalcemic activity and receptor binding potency of CT peptides activity of Leu 23 ,Ala 24 -sCT and MCT-I (low activity, Table 2) and [G 8 ]-des-Leu 19 -sCT and MCT-III (high activity, Table 2). In particular, the inactivity of sCT (1-23)-NH 2 (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 Ala 16 -sCT (mutant 6) and Ala 19 -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 Ala 16 -sCT and Ala 19 -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 Ala 9 -sCT (mutant 4) and Ala 12 -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, Ala 16 ,Ala 19 -sCT (mutant 10) shows 7235 IU/mg (Table 2), whereas Ala 12 ,Ala 19 -sCT (mutant 9) is inactive. Analysis of the 12-14 analogs shows that, except for Leu 9 (mutant 12), whose biological activity is substantially unaffected (Val 8 might be acting instead of Leu 9 ), deletion of Leu 12 or Leu 16 (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 Leu 9 and Leu 19 and the presence of Leu 12 and Leu 16 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)-NH 2 (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 ␣-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 His 17 -Thr 31 , Lys 18 -Asn 26 , and Leu 19 -Asn 26 , (17). Such an interaction is absent in sCT (1-23)-NH 2 (mutant 22), Leu 23 ,Ala 24 -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 Leu 15 and Gln 17 instead of Glu 15 and His 17 , and MCT-III shows Gln 17 instead of His 17 . This would suggest that charged residues within the helix play a role in the helix-tail interaction (17).

DISCUSSION
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 Cys 134 -Lys 141 (21,51) and that hCT Met 8 interacts with Leu 368 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 ␣-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 Leu 23 ,Ala 24 -sCT (mutant 19), which was expected to take up a helix longer than sCT. Our results indicated that sCT and Leu 23 ,Ala 24 -sCT both form an amphiphatic ␣-helix, but whereas in Leu 23 ,Ala 24 -sCT it covers almost the whole sequence (Leu 4 -Gly 28 ), in the native hormone a shorter region (Thr 6 -Phe 22 ) is involved, with the region Leu 9 -Leu 19 constantly found in helical conformation (12). Significantly, no helix-tail interaction was observed for Leu 23 ,Ala 24 -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 wedgeshaped form that may be required for proper interaction with the receptor.
Compared with the native hormone, Leu 23 ,Ala 24 -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-Leu 19 -sCT and des- 19 Leu-Gly 20 -Thr 21 -sCT (mutants 15 and 16) are fully active (Table 2). A helix encompassing Leu 19 , as in Leu 23 ,Ala 24 -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 Leu 9 , as Gly 8 -sCT (mutant 2) and Met 8 -sCT (mutant 3) show unaffected biological behavior.
By eliminating Leu from sites 9, 12, 16, and 19 (mutants 12-15), we altered the bioactivity when Leu 12 or Leu 16 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 Leu 9 and Leu 12 are more important for bioactivity than Leu 16 and Leu 19 , as mutants 6 and 7 show biological activity comparable with sCT. According to this interpretation, Ala 12 ,Ala 16 -sCT (mutant 8) and Ala 12 ,Ala 19 -sCT (mutant 9) are completely devoid of activity, whereas A 16 ,A 19 -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, Gly 8 -des-Leu 19 -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 Ala 16 -sCT (mutant 6) and Ala 16 ,Ala 19 -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 Lys 14 instead of Gln (see the primary structure in footnote of Table 1). Furthermore, sCT (1-23)-NH 2 , which is fully helical (Table 1), is completely inactive ( Table 2). Taken together, data on MCT-III and sCT (1-23)-NH 2 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 ␣-helix, and indeed, the variability of the calcitonin sequences from different species also argues against such a conclusion. A stable ␣-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 ␣-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.