Solution Structure of Human Calcitoninin Membrane-Mimetic Environment: TheRole of the Amphipathic HelixAndrea Motta,1* Giuseppina Andreotti,1 Pietro Amodeo,1 Giuseppe Strazzullo,1and Maria A. Castiglione Morelli2

1Istituto per la Chimica di Molecole di Interesse Biologico del CNR and Istituto Nazionale di Chimica dei SistemiBiologici, Napoli, Italy2Dipartimento di Chimica, Universita della Basilicata, Potenza, Italy

ABSTRACT The 32 amino acid hormonehuman calcitonin was studied at pH 3.7 and 7.4by multidimensional NMR spectroscopy in so-dium dodecyl sulfate micelles at 310K. Thesecondary structure was obtained from nuclearOverhauserenhancementspectroscopy(NOESY),3JHNa coupling constants, and slowly exchang-ing amide data. Three-dimensional structuresconsistent with NMR data were generated byusing distance geometry calculations. A set of265 interproton distances derived from NOESYexperiments, hydrogen-bond constraints ob-tained from amide exchange, and couplingconstants were used. From the initial randomconformations, 30 distance geometry struc-tures with minimal violations were selectedfor further refinement with restrained energyminimization. In micelles, at both pHs, thehormone assumes an amphipathic a-helix fromLeu9 to Phe16, followed by a type-I b-turnbetween residues Phe16 and Phe19. From His20onward the molecule is extended and no inter-action with the helix was observed. Therelevance of the amphipathic helix for thestructure–activity relationship, the possiblemechanisms of interaction with the receptor,as well as the formation of fibrillar aggregates,is discussed. Proteins 32:314–323, 1998.r 1998 Wiley-Liss, Inc.

Key words: AMBER; amphipathic helix; dis-tance geometry; DYANA; fibrilla-tion; NMR; sodium dodecyl sulfate

INTRODUCTION

Calcitonin (CT) is a peptide hormone produced bythe parafollicular cells of the thyroid gland in mam-mals and by the ultimobranchial glands of birds andfishes. Secreted in response to increased serumcalcium levels, it has a regulatory function in calcium-phosphorus metabolism and inhibits osteoclastic boneresorption and induces calcium uptake from bodyfluids.1 In addition, CT has been postulated to be aneuromodulator and/or neurotransmitter, as calcito-nin-like molecules have been found in the central

nervous system of many species, including the hu-man brain.2,3 Several mammalian and submamma-lian calcitonins have been isolated, sequenced, andsynthesized, displaying considerable differences inamino acid composition.4 In its common form, itconsists of 32 amino acids with an N-terminal disul-fide bridge between positions 1 and 7 and aC-terminal proline amide residue. Only nine resi-dues are common to all species studied so far, andthese are clustered at the two ends of the molecule(residues 3–7 and positions 1, 9, 28, and 32).

Circular dichroism (CD) and NMR studies haveshown that human CT (hCT) has little orderedsecondary structure in water. In structure-promot-ing solvents, different ordered structures are ob-served. In a cryoprotective mixture of dimethylsulfoxide/water, a short intramolecular antiparalleldouble-stranded b-sheet over residues 16–21 isformed, with two tight turns made by residues 3–6and 28–31.5 In a trifluoroethanol/water mixture, the9–22 residue region of hCT is seen to adopt a helicalconformation.6 In media with different concentra-tions of trifluoroethanol/water, CD has shown thatthe a-helix formation is a two-step process,7 while insodium dodecyl sulfate (SDS), only 15% of thyroidalcalcitonin residues are folded in an a-helical confor-mation.8

In general, fish calcitonins have been found to bemore potent biologically than mammalian ones. Threenative calcitonins, salmon (sCT), porcine CT andhCT, and an analog of eel CT are used therapeuti-cally,9 mainly for the treatment of osteoporosis,Paget’s disease, and hypercalcemia of malignancy.However, sCT, differing from hCT in 16 of the 32amino acids, is reported to produce antibodies in asignificant number of patients,10 while hCT easilyassociate and precipitate as insoluble fibrils uponstoring in aqueous solution, which is a defect intherapeutic use. Thus, it would be desirable to

This article is dedicated to the memory of Claudio Sellitti.*Correspondence to: Andrea Motta, Istituto per la Chimica di

Molecole di Interesse Biologico del CNR, via Toiano 6, I-80072Arco Felice (Napoli), Italy. E-mail: amot@nmr.icmib.na.cnr.it

Received 2 February 1998; Accepted 31 March 1998

PROTEINS: Structure, Function, and Genetics 32:314–323 (1998)

r 1998 WILEY-LISS, INC.

uncover the structure–activity relationship for hCTin order to prepare new high-potency moleculeswhich closely resemble hCT but are less antigenicthan sCT and prevent fibrillation. The molecularaspects of CT activity are not completely understoodand are still a matter of controversy, although thepresence of an amphipathic a-helix in the centralregion of CT has been reported to be important forthe interaction with lipids.11 Kaiser and cowork-ers12,13 provided evidence that the model of an amphi-pathic a-helical region in residues 8–22 is a usefulguide to designing potent sCT activity. However,other studies14–16 suggest that conformational flexibil-ity is also an important parameter.

The present study reports on the conformation ofhCT (Scheme I) in a membrane-like environment,such as that brought about by SDS micelles. Surfac-tant micelles can usefully be applied to simulate thecatalytic role of the membrane17 and to investigatethe conformational properties of hormones at theinterface. Since it is likely that the hormone willretain the conformation assumed at the interface,18

this investigation is expected to contribute informa-tion to the understanding of the receptor–hCT inter-action.

1 5 10Cys-Gly-Asn-Leu-Ser-Thr-Cys-Met-Leu-Gly-Thr-

15 20Tyr-Thr-Gln-Asp-Phe-Asn-Lys-Phe-His-Thr-Phe-

25 30Pro-Gln-Thr-Ala-Ile-Gly-Val-Gly-Ala-Pro-NH2

Scheme I: Amino Acid Sequence of hCT.

We have found that in the region Leu9-Phe16, hCTtakes up an amphipathic helix, with residues Phe16-Phe19 forming a type-I b-turn acting as end cappingto stabilize the C-terminus of the helix. From His20onward the molecule is extended and no interactionwith the helix was observed. According to theseresults, we discuss the importance of the amphi-pathic helix for the structure–activity relationship,its possible implication in the interaction with thereceptor, and, finally, the relevance of our structurefor hCT fibrillation.

MATERIALS AND METHODSPeptide Synthesis

hCT was prepared by classical methods of peptidesynthesis in solution and belonged to a batch previ-ously described.5

NMR Data Collection

For acquisition of NMR spectra, 5 3 10-3 M solu-tion of hCT in 95% 1H2O/5% 2H2O (v/v) (at pH 3.7 and7.4) or in 100% 2H2O (at pH* 3.3 and 7.0), both inphosphate buffer at 310K, were used. Isotopically

labeled solvents originated from Aldrich (Milwau-kee, WI). A solution of perdeuterated SDS was addedto a solution of the hormone, and the final mixtureremained transparent. The concentration of SDSwas maintained well above the critical micelle concen-tration, with a final hCT–SDS ratio of 1:120. Perdeu-terated SDS was obtained from Cambridge IsotopeLaboratories (Woburn, MA).

1H NMR spectra were recorded at 500 MHz on aBruker DRX-500 spectrometer using an inverse mul-tinuclear probehead fitted with gradients along theX-, Y-, and Z-axes, and equipped with an SGI com-puter. Spectra were referenced to sodium 3-(trimeth-ylsilyl)-[2,2,3,3-2H4]propionate. Two-dimensionaldouble-quantum-filtered correlation spectroscopy(DQF-COSY),19 clean total correlation spectroscopy(TOCSY),20,21 and nuclear Overhauser enhancementspectroscopy (NOESY)22 spectra were recorded byusing the time-proportional phase incrementation ofthe first pulse,23 and incorporating the WATERGATEsequence24 for water suppression. The gradient pulseswere each 1 ms in duration and shaped to a sineenvelope. Usually, 512 equally spaced evolution timeperiod t1 values were acquired, averaging 16–64transients of 2,048 points, with 6,024 Hz of spectralwidth. Time-domain data matrices were all zero-filled to 4K in both dimensions, thus yielding adigital resolution of 2.94 Hz/pt. Prior to Fouriertransformation, a Lorentz-Gauss window with differ-ent parameters was applied for both t1 and t2 dimen-sions for all the experiments. NOESY spectra wereobtained with different mixing times (0.10, 0.15, and0.20 sec); TOCSY experiments were recorded with aspin-lock period of 0.064 and 0.096 sec, achievedwith the MLEV-17 pulse sequence.25

Slowly exchanging protons at pH* 7.0, were identi-fied by recording a NOESY spectrum (0.20-sec mix-ing time) of the peptide immediately after dissolu-tion in 2H2O. For isolated resonances, measurementsof 3JHNa coupling constants were obtained from one-dimensional experiments acquired with 128K pointsand application of strong Lorentz-Gauss resolutionenhancement. For overlapping lines, coupling con-stants were estimated from DQF-COSY spectra sothat they are reported as apparent values.26

Processing was performed using TRIAD (Tripos,Inc., St. Louis, MO) and AURELIA 2.027 (BrukerAnalytische) running on Silicon Graphics Indigo2

R4400 computers.

Distance Restraint Determination

An initial list of distances was obtained for hCTfrom a 0.1-sec mixing time NOESY spectrum re-corded at 310K. Volumes of nuclear Overhauserenhancements (NOEs) cross-peaks were integratedand calibrated with the AURELIA software. Theinterproton distances comprised 98 intraresidue dis-tances and 177 short- and medium-range interresi-due distances relative to amino acids less than five

315SOLUTION STRUCTURE OF HUMAN CALCITONIN

positions apart in the sequence. Analysis of the NOEdistance restraints revealed that intraresidue re-straints (namely, aCHi-NHi, bCHi-NHi, aCHi-bCHi)placed no restriction on the structure as the upperdistance limits became smaller than those imposedby the covalent geometry. This problem was circum-vented by calibrating differently intra- and interre-sidual NOEs by using the Leu9 aCHi-NHi andTyr12-Thr13 NHi-NHi11 distances, respectively, andthen scaling the intraresidual distances to fit thecorresponding allowed range. For each kind of intra-residual NOE, we identified the allowed distancerange from a dihedral systematic search using theAMBER standard covalent geometry. The details ofthis approach will be published later (manuscript inpreparation). A 60.01 nm tolerance was then al-lowed for each restraint. Restraints on the Cys1-Cys7 disulfide bond and on hydrogen bonds were alsoimposed. The disulfide bond was fixed by imposing arange of 0.20–0.21 nm on the S–S distance and arange of 0.30–0.35 nm on the Sg–Cb distances.Hydrogen bonds were imposed between pairs ofresidues for which standard intrahelical aCHi-NHi13

and aCHi-NHi14 NOEs were observed for a slow-exchanging amide proton.28 For each hydrogen bond,two upper limit restraints were used between theNH-O (0.25 nm) and the N-O (0.35 nm) atom pairs.

The b-methylene groups were stereospecificallyassigned with the program HABAS,29 which usesintraresidual and sequential NOEs and vicinal cou-pling constants 3JHNa and 3Jab. A total of 7 out of 20b-methylene groups were assigned (residues 7, 8, 9,12, 14, 19, and 24). The validity of stereospecificassignments was counterchecked by evaluating theirconsistency with the distance geometry (DG) struc-tures. When no stereospecific assignment was pos-sible for methyl and methylene protons, distanceconstraints were corrected for pseudoatom represen-tation.30 For methyl groups, an additional correctionof 0.05 nm was added to take into account themultiplicity.

Structure Calculations

Structures were generated by the DG programDYANA.31 A total of 60 structures were calculated bysimulated annealing, starting with a total of 4,000molecular dynamics (MD) steps and other defaultvalues of temperature and time steps. The best 30structures in terms of target function were refined byenergy minimization (EM) performed with theSANDER module of the AMBER 4.1 package,32,33

using the AMBER all-atom 1991 parametrization. Adistance-dependent dielectric constant e 5 r wasused to roughly take into account solvation effects. Acutoff radius of 0.8 nm for nonbonded interactions,with a residue-based pairlist routine, was used in allcalculations. The force constant for distance re-straints in EM were 500 kJ.mol-1·nm-2. EM wasperformed using a combination of steepest descent

and conjugate gradient algorithms with a gradientconvergence norm of less than 10-4 kJ· mol-1·nm-1.

Molecular structures have been drawn and ana-lyzed with the graphics program MOLMOL.34

RESULTSNMR Analysis and Secondary Structure

All experiments were run at pH 3.7 and at thephysiological pH 7.4, in order to separate overlap-ping resonances and check for possible conforma-tional variations. Assignment of proton spin systemswas obtained with the sequential methodology out-lined by Wuthrich.35 From the amide protons, TOCSYexperiments allowed identification of the a and the bprotons of almost all of the amino acids. Residueswith a long sidechain were identified by a combina-tion of TOCSY and NOESY experiments. Individualspin systems were placed in the primary structureby identification of characteristic short- and medium-range NOE connectivities. Figure 1 shows theNH–NH region of a NOESY spectrum at 0.15-secmixing time, 310K, pH 3.7. It is possible to follow theconnectivities from Leu4 to Val29, with only fewinterruptions due to cross-peak overlap and to thepresence of Pro23. The secondary structure of hCTwas delineated from qualitative analysis of the se-quential (aCHi-NHi11 and NHi-NHi11) and medium-range (aCHi-NHi1n, 1 , n , 4, and aCHi-bCHi13)NOEs,35 from slowly exchanging amide protons,35

and from 3JHNa coupling constants.36 Figure 2 summa-rizes the observed NOEs, the relative exchange ratesof amide protons and the apparent 3JHNa couplingconstants for hCT at 310K and pH 3.7. The fact thatin the central region the NHi-NHi11 NOEs are in-tense, while the aCHi-NHi11 NOEs are much weaker,implies a generally helical structure.37 The observa-tion of several unambiguous aCHi-NHi13, aCHi-bCHi13 and a single aCHi-NHi14 cross-peaks (Fig. 2)supports the presence of a helix. Further corrobora-tive data come from slowly exchanging amides:except for Thr13 and Gln14, all the amide protons inthe Leu9-Phe16 region are in slow exchange. Theslow exchange most likely indicates intramolecularhydrogen bonding; in fact, it is unlikely that a slowlyexchanging proton is buried in the interior of abiomolecule as small as hCT, although burial withinthe hydrophobic core of the micelle is also possible.3JHNa , 6 Hz in the Leu9-Phe16 region also supportthe presence of a helix.36 Furthermore, we observedaCHi-NHi12 cross-peaks, suggestive of a 310 helix,38

only in the N-terminal ring and not in the middleregion of the peptide, thus concluding that theLeu9-Phe16 region of hCT forms an a-helix. Severalintraresidual and sequential NOEs are observed forthe Cys1-Cys7 ring. However, the absence of medium-range NOEs at all mixing times indicates that noresidues of the ring are part of the helix, as insteadfound for sCT.39

316 A. MOTTA ET AL.

For the C-terminal decapeptide Pro23-Pro32, weessentially found sequential NOEs, meaning thatthe conformation is largely extended. However, thepresence of NHi-NHi11 and bCHi-NHi11 NOE connec-tivities (Fig. 2) is indicative of local structures andshort-range order. The absence of long-range NOEsbetween the C-terminal region and the helix is alsonoted. This rules out the possibility that theC-terminal tail of hCT is folded toward the helix, aswas found for sCT.39 Evidence was found for thepresence of isomers with cis peptide bonds at sites 23and 32 (16% cis at 310K). For Pro23, it was revealedby a small NOE connectivity35 between the a protonsof Phe22 and Pro23. An a–aNOE cross-peak betweenAla31 and Pro32 confirmed the cis isomer for Pro32.For both prolyl residues, the trans forms were identi-fied by NHi-di11 NOE cross-peaks. As previouslyreported for hCT5 and sCT,39,40 conformational hetero-geneity due to the relatively slow isomerization of cisand trans forms of proline residues brings aboutadditional cross-peaks for nearby residues. Morespecifically, we could follow two separate sequence-specific NOE pathways in the region Lys18-Pro32due to trans23-trans32 and cis23-trans32 for aminoacids around Pro23, and to trans23-trans32 andtrans23-cis32 for residues preceding Pro32. The ef-fects of the other possible isomer (cis23-cis32) oncross-peaks of residues around Ile27-Gly28 were notdetected because of its very low concentration.

It is well known that helix formation under theinfluence of SDS is enhanced by a decrease in pH;41

however, examination of the NOE patterns at pH 7.4

(not shown) indicates that this structure is pre-served at physiological pH.

Three-Dimensional Structure

The distribution of the observed NOEs along theamino acidic sequence of hCT is shown in Figure 3.The average number of constraints per residue is sixfor the Cys1-Thr6 region, 15 for the Cys7-His20region and nine for the Thr21-Pro32. Two notableexceptions are Leu4 and Gln24, having 15 and 18NOEs, respectively. Three-dimensional structures ofhCT were generated consistently with the NMR databy combined use of DG and restrained EM. From theinitial 60 structures as obtained from DYANA calcu-lations, 30 were selected for further refinement.They had no violations of the upper and lowerbounds of the NMR distance restraints greater than0.10 nm and 0.12 nm, respectively, and did notpredict any additional short interproton distancewith no observable NOEs. They were subjected torestrained EM using the AMBER package (see Mate-rials and Methods). A correlation was observed be-tween the values of the DYANA target function andthe energy of the structures after EM: the beststructures in terms of target function were also themost stable. The energy of these refined structureswere all in the narrow range from -3,225 to -3,775kJ·mol-1. Refinement produced a decrease of theoverall energy but the NOE restraint energies under-went a small increase upon minimization. The over-all agreement among individual conformers can beseen by global root-mean square (RMS) deviation.

Fig. 1. Amide region of a 0.15-sec mix-ing time NOESY spectrum of SDS-boundhCT in 95% 1H2O/5% 2H2O, pH 3.7, 310K.Where space permits, sequence numbersplaced horizontally and vertically to eachcross-peak label the v2 and v1 dimensionsof the spectrum, respectively.

317SOLUTION STRUCTURE OF HUMAN CALCITONIN

The average RMS deviation between the 12 beststructure pairs is 0.074 6 0.016 nm, for the backboneatoms in the 9–16 region, and 0.128 6 0.036 nmincluding all heavy atoms of the helical region. Theaverage sum of all violations for these structures is0.76 nm, while the average distance restraint viola-tion is 0.0067 nm.

Figure 4 presents a superposition of the polypep-tide backbone for the first 12 best structures of hCTobtained with the program DYANA and locally im-proved with restrained EM. A unique backbone foldis obtained for residues 9–16, which contains fourout of seven residues stereospecifically assigned inhCT. Lack of convergence is instead observed in thesecond half of the hormone (Asn17-Pro32) and, to alesser degree, in the N-terminal Cys1-Met8 region.Although the presence of the Cys1-Cys7 ring cer-tainly limits the accessible conformational space, atleast in comparison with the C-terminal part, thelack of convergence reflects the absence of long-range NOEs between ring and helix, and helix andtail. The effect of energy minimization was to regular-ize the structures and to reduce the spread of thebackbone dihedral angles. The distribution of the f

and c angles (Fig. 5) of the 12 lowest energy struc-tures reflects the extent to which the conformationhas been defined by NOE restraints. Good agree-ment was observed among the structures for thevalues of f and c of the helical region (Leu9-Phe16),which shows a reduced dihedral dispersion. Resi-dues Cys1-Met8 do not converge to a unique struc-ture. This is most likely due to the fact that thespreading observed for Met8 angles does not allowits inclusion into the helix, even though nine out oftwelve structures present values expected for ahelix. This is evident from Figure 6, which shows theRamachandran plot for Met8. While structure 8 hasa c angle not far from the expected helical value,structures 5 and 7 take up values close to theleft-handed helix. As a consequence, the ring ofstructure 8 is in the envelope of the nine helicalstructure set, while the rings of structures 5 and 7span a completely different region of space (labels 5and 7 in Fig. 4). Asn17 presents average f and cvalues similar to those expected for a helix. However,the progressive angular spreading (Fig. 5) and theNOE pattern (Fig. 2) suggest that the region Asn17-Lys18-Phe19 cannot be strictly included in the helix.

Fig. 2. Diagrammatic representation of sequential and second-ary structural interresidue NOEs observed for hCT in aqueousSDS. NOE intensities are indicated by the height of the bars.Effects to the Hd protons of Pro23 and Pro32, though observed,are not drawn. Circles indicate coupling constants data (solid

circles, apparent 3JHNa , 6.0 Hz; large open circles, measured3JHNa . 7.0 Hz; small open circles, 6.0 Hz # 3JHNa # 7.0 Hz). Solidcircles also label residues for which slow exchange of NH protonswith deuterons was observed.

318 A. MOTTA ET AL.

The presence of an NHi-NHi12 connectivity betweenAsn17 and Phe19, the 3JHNa coupling constants , 6Hz for Asn17 and . 7 Hz for Lys18, the slowlyexchanging amide of Phe19, suggest the possibleformation of a type-I turn in the region Phe16-Phe19. From His20 onward, the description emerg-ing from DG calculations points out the coexistenceof different flexible extended structures, which areresponsible for the lack of convergence and theabsence of structurally significant NOEs. This is

fully supported by unrestrained MD calculations onhCT with solvent, which indicate that the absence oflong-range NOEs stems from a conformational pref-erence of the molecule (manuscript in preparation).

DISCUSSION

The solution structure of hCT in SDS micelles hasbeen obtained from NMR data. Its identificationrelied on the observed NOE patterns, 3JNHa couplingconstants, and the amide proton exchange data.

Fig. 3. Distribution of distance restraintsalong the primary structure of hCT. Differenttype of NOEs are labeled with the followingcolor code: intraresidual, white; sequential,light gray; medium-range, dark gray; andlong-range, black.

Fig. 4. Stereoview of the Ca atoms of the 12 lowest energy structures of SDS-bound hCT.Structures were superimposed for pairwise minimum RMS deviation of the Ca atoms of residues9–16. The N- and C-termini of structures 5 and 7 are labeled.

319SOLUTION STRUCTURE OF HUMAN CALCITONIN

NOESY cross-peaks were carefully calibrated andemployed as restraints for structure calculationswith the DG program DYANA. Selected structureswere subsequently energy-refined with the AMBERprogram. The predominant conformational featureof hCT is an amphipathic a-helix between residues 9and 16. The exclusion of the region Asn17-Lys18-Phe19 from the helix is supported by the interre-sidual NOEs (Fig. 2) and the backbone torsionalangles (Fig. 5), although Asn17 has angular valuescompatible with a helix for some of the calculatedstructures. Both the NOE pattern and the distribu-tion of f and c angles suggest that the Phe16-Phe19region forms a type-I b-turn, which may act as endcapping to stabilize the C-terminus of the helix.Indeed, Asn and Lys, found at sites 17 and 18, havebeen shown to have good ability in stabilizing theC-terminus of an a-helix through hydrogen bond-ing.42 Our helix is shorter than that described in the

trifluoroethanol/water mixture,6 where the 9–22 resi-due region of hCT adopts a helical conformation.From His20 onward, the hormone takes up anextended structure which spans a large conical re-gion of space with no interaction with the helix. Thisis in contrast with sCT in SDS, in which theC-terminal decapeptide is in close association withthe helix.39,43

Structure–activity studies of several peptide hor-mones have generally been indicative of the strongcorrelation between amphipathic a-helical struc-tures and pharmacological activity.44 In the case ofCT, high helical contents in solution or bound toamphiphiles correlate well with receptor-bindingand hypocalcemic potencies,11 and minimally homolo-gous sCT analogs that retain the physicochemicalproperties characteristic of the amphipathic a-helixalso retain high potency in pharmacological as-says.12,13 Furthermore, deletion of the helical residue

Fig. 5. Plot of the f and c angles for each residue of the best 12 structures of hCT. The numbersmark corresponding structures.

320 A. MOTTA ET AL.

16 from sCT or hCT results in substantial loss ofpharmacological potency.45 However, analogs basedon des-Leu19-sCT which have lost the phospholipid-binding properties associated with the sCT amphi-pathic a-helical structure, nevertheless retain mostof the in vivo hypocalcemic potency of native sCT.46

In addition, the high hypocalcemic activity reportedfor [Gly8]-sCT does not fit with the simple expecta-tions of a high amphipathic a-helix content, andanalogs of sCT with multiple deletions in residuepositions 19–22 also retain high potency in vivo,indicating that the C-terminal end of the proposedhelix is not essential either.47

In agreement with Kaiser and coworkers,12,13 webelieve that the presence of an amphipathic helix inthe central regions of hCT and sCT is relevant for thebiological activity of CT. A comparison between MDcalculations on sCT and hCT in methanol (ourunpublished results) indicate that the helix spansthe regions Val8-Lys18 and Leu9-Phe16, respec-tively. The fact that deletion of residue 16 from hCTor sCT results in substantial loss of pharmacologicalpotency can be explained by the destabilization ofthe C-terminal end-capping of the helix. NOESYspectra obtained at 0.10, 0.15, and 0.20 sec mixingtimes of des-Phe16-hCT and des-Leu16-sCT (notshown) indicate almost complete unwinding of thehelix for hCT, while sCT breaks in a helical segment(Val8-Gln14) and a turn (Leu19-Tyr22). This sug-gests that there exist a minimum requirement forthe CT helix of about ten residues necessary for thepharmacological activity, and which corresponds tothe length of the hydrophobic regions in known

signal sequences.48 This requirement can also justifythe high hypocalcemic activity reported for des-Leu19-sCT, [Gly8]-sCT, and for analogs with mul-tiple deletions at sites 19–22. MD simulations showthat the region Gly8-Leu-Gly-Lys11 in [Gly8]-sCTtakes up a type-I b-turn and precedes a helicalsegment terminating at Lys18. For turns of type I(and type I’), sequential and medium-range interpro-ton distances coincide closely with those in the 310

helix;35 thus, the type-I turn could actually initiatethe helix. In fact, MD calculations have shown thatfor 22 out of 30 structures the turn is included in thehelix forming a helix in the region Gly8-Lys18. Beingresidues 19–22 outside of the helical region, in thecorresponding des-analogs the amphipathic helix isunperturbed, thus justifying the retention of highpotency in vivo.

It has been reported that the types of interactionsthat may be operative in the SDS micellar systemrecall those important for protein-lipid association inmembrane.49 This suggests that the amphipathica-helix found in hCT and sCT could be involved inthe mechanism(s) of interaction with the receptor.Nakamuta et al.50 have inferred two independentbinding sites which are able to discriminate differentconformational features of CT molecules. StructuralNMR solution studies of peptides in micellar sys-tems and solid-state NMR experiments on orientedbilayer samples suggest that the structure can bevery similar in both media, but information on theorientation of the molecule is only achieved in solid-state experiments.51 This suggests that peptidesshowing otherwise similar secondary structure inmicelles, such as hCT and sCT, may have differentorientations in bilayer and consequently differentmechanisms for interacting with the membrane. Wesuggest that the two independent binding sitesproposed by Nakamuta et al.50 are actually able todiscriminate the orientation of the interacting amphi-pathic helix according to the following mechanisms:1) tangential interaction of the hCT amphipathichelix with the membrane surface,52 followed by theinteraction with the receptor; and 2) perpendicularinsertion into the membrane of the wedge-like helix-tail structure, similar to that of sCT,43 with a directinteraction with the receptor. Both mechanisms relyon the presence of the amphipathic helix, and recall,respectively, the proposed tangential attachment ofthe colicin E1 channel-forming domain to membranesurface by electrostatic interactions,53 and the laststep of the model proposed by Gierasch and col-leagues for signal- or leader-peptide function indetermining protein translocation across the periplas-mic membrane in bacteria.54 Indirect support for thishypothesis comes from the observation that in SDSthe helix-C terminus interaction is present in sCT43

but not in hCT, and that the C-terminal region ofsCT contributes to receptor binding.15

Fig. 6. Ramachandran plot for the Met8 residue in the best 12structures of hCT. Isolines enclose normally allowed regions forstandard (i.e., different from Gly, Val, Ile, and Thr) residues. Thenumbers label structures 5, 7, and 8.

321SOLUTION STRUCTURE OF HUMAN CALCITONIN

It is very well known that hCT easily associatesand precipitates as insoluble fibrils upon storage inaqueous solution, which is a defect in therapeuticuse. Arvinte et al.55 have reported that hCT mol-ecules form a-helical and intermolecular b-sheetsecondary structure approximately 24 hr after dis-solving the peptide in aqueous solution, while sCT ishighly stable in aqueous solution, with the fibrilla-tion process occurring much slower than that of hCT.Aggregation of hCT leads to a large degree of struc-tural polymorphism, starting from protofibrils mostlikely containing three or four hCT monomers percross-section.55,56 Kanaori and Nosaka57 have shownthat NMR peaks of residues in the N-terminal andcentral regions broaden and disappear faster thanthose in the C-terminal region, suggesting that themolecular association of hCT is initiated by intermo-lecular hydrophobic interaction in the N-terminaland central regions and that the C-terminal region issubsequently involved in the fibrillation. We haveobserved the formation of a gel in a 4-week-oldsample of hCT in aqueous SDS stored at roomtemperature, while no evidence of aggregation wasobserved for a 12-month-old sample of sCT. This canbe related to the different behavior in the fibrillationof the two calcitonins. Comparison of the amino acidsequence of hCT and sCT shows only small differ-ences in the respective hydrophobicity for each resi-due in the hydrophobic side of the amphipathic helix.We believe, instead, that the differences in the‘‘tertiary’’ structure can explain the different behav-ior. In SDS, both calcitonins take up an amphipathica-helix in the central region and an extendedC-terminal tail. However, while in hCT the twostructural elements are independent, in sCT a hydro-phobic interaction between the tail and the helix ispresent.39,43 This would suggest that the stability ofsCT in solution is linked to the presence of thisintramolecular interaction, which prevents molecu-lar association through hydrophobic helical interac-tions. On the contrary, in hCT the availability of thehydrophobic side of the helix could favor the forma-tion of a helical bundle in the critical nucleus;subsequently, the C-terminus in an extended confor-mation could provide a template for the a-helicalrods in the fibril formation. This is in agreementwith the data reported by Arvinte et al.55

We are currently simulating the formation of thehCT fibrils by using the reported SDS-bound struc-ture and the supramolecular structural polymor-phism model proposed by Bauer et al.56 This isexpected to contribute to further improvement of thelong time stable aqueous therapeutic formulations ofhCT.58

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323SOLUTION STRUCTURE OF HUMAN CALCITONIN