©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Peptide Fragment of Human DNA Topoisomerase II Forms a Stable Coiled-coil Structure in Solution (*)

(Received for publication, April 11, 1995; and in revised form, May 11, 1995)

Valérie Frère (1), Frédéric Sourgen (1), Monique Monnot (1), Frédéric Troalen (2), Serge Fermandjian (1)(§)

From the  (1)Département de Biologie et Pharmacologie Structurales, URA 147 CNRS, Institut Gustave Roussy, Villejuif 94805 Cedex, France and (2)Service d'Immunologie Moléculaire, Institut Gustave Roussy, Villejuif 94805 Cedex, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
CONCLUSIONS
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Results are presented on a peptide fragment (1013-1056) from human DNA topoisomerase II . This was selected using the procedure of Lupas et al. (Lupas, A., Van Dyke, M., and Stock, J.(1991) Science 252, 1162-1164) for its potential to adopt a stable coiled-coil structure. The same theoretical treatment rejected the segment 994-1021 proposed by Zwelling and Perry (Zwelling, L. A., and Perry, W. M.(1989) Mol. Endocrinol. 3, 603-604) as a possible core for leucine-zipper formation. Our experimental studies combine cross-linking and CD analysis. Cross-linking establishes that the 1013-1056 fragment forms a stable homodimer in solution. Effects of increasing peptide concentration on CD spectra confirm that only the 1013-1056 fragment can undergo a coiled-coil stabilization from an isolated -helix. Unfolding experiments further show that the coiled-coil is more stable in guanidium chloride than in urea. Values of -6.8 and -7.4 kcal/mol for the dimerization free energy are determined by thermal and urea unfolding, respectively. These are strikingly similar to the value recently found for the dissociation/reassociation of the entire yeast topoisomerase II from sedimentation equilibrium experiments (Lamhasni, S., Larsen, A. K., Barray, M., Monnot, M., Delain, E., and Fermandjian, S.(1995) Biochemistry 34, 3632-3639), although their significance relatively to topoisomerase II undoubtedly requires further analysis.


INTRODUCTION

DNA topoisomerases II perform topological changes of DNA, such as catenation/decatenation, knotting/unknotting, and modification of the surpercoiling, by passing an intact double helix of DNA through a transient double-stranded break made in a second helix of DNA(1, 2) . These enzymes are implicated in DNA replication, recombination, and DNA transcription and are essential in mitosis and meiosis by intervening in condensation/decondensation and segregation of chromosomes. They further exhibit structural roles by probable associations to chromatin as they are a major component of the nuclear matrix. Topoisomerases II also present a crucial interest in the treatment of human cancers since they have been identified as the preferential cellular target for number of clinically important antitumoral drugs(3) .

The active form of topoisomerase II has been proposed to be dimeric (4, 5, 6) , although the motif of dimerization is not identified. Recently, Zwelling and Perry (7) have suggested on the basis of sequence examination that a leucine-zipper (994-1021 segment) could be at the origin of dimerization of human topoisomerase II , as also suggested by Caron and Wang(8) . This structure belongs to the widespread coiled-coil motif initially characterized by Crick in 1953 (9) and consisting of several amphipathic -helices wrapped around each other in a slightly left-handed supercoil(10, 11, 12, 13) . It is omnipresent in the b-ZIP family (for basic leucine-zipper)(14) , including DNA-binding proteins such as transcriptional factors (15) and protooncoproteins/oncoproteins as c-Fos and c-Jun, which all dimerize through a leucine-zipper motif(16) . An extensively analyzed leucine-zipper is given by the yeast transcriptional factor GCN4 (11, 12, 13, 17, 18) .

The coiling of -helices optimizes the interchain packing of apolar residues and also allows specific interchain ionic interactions in proteins as varied as fibrous, globular, or membrane-spanning molecules. The most striking feature of coiled-coil helices is the 4-3 or 3-4 hydrophobic repeat. This repeat, firstly identified by Hodges et al.(19) , consists of a repeating heptad sequence designated by the letters a-g, in which the positions a and d usually present hydrophobic residues. The a and d residues fall on the same side of the helix and pack in a regular ``knobs-into-holes'' pattern along the dimerization interface(9, 11) . This creates a hydrophobic core, which is critical for the dimerization process. Some charged residues in e and g positions of adjacent heptads could stabilize the dimer and contribute to the specificity of dimerization through electrostatic interactions(20, 21) .

Here, we tested the reality of a coiled-coil motif in human topoisomerase II allowing its dimerization. Secondary structure predictions by the Garnier Osguthorpe Robson (GOR) method (22, 23) as well as circular dichroism experiments led us to discard the sequence 994-1021 proposed by Zwelling and Perry (7) and permitted instead the identification of a 44-amino acid segment(1013-1056) characterized by both a high -helix potential and five 4-3 hydrophobic repeats typical of coiled-coil motives. Glutaraldehyde cross-linking, detailed circular dichroism analysis, and derived thermodynamic results confirmed the dimeric coiled-coil structure of the 1013-1056 fragment in solution.


MATERIALS AND METHODS

Peptide Synthesis and Purification

The 1013-1056 sequence peptide from human topoisomerase II (NH-TVLDILRDLFELRLKYYGLRKEWLLGMLGAESAKLNNQARFILE-COOH) was prepared together with the 994-1021 fragment (NH-VFKLQTSLTCNSMVLFDHVGCLKKYDTVLDILRDLF-COOH) according to solid phase synthesis on an Applied Biosystems model 431A peptide synthesizer using Fmoc (9-fluorenylmethoxycarbonyl) chemistry with 4-hydroxymethyl-phenoxymethyl-copolystyrene, 1% divinylbenzene resin and 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate activation. The peptides were cleaved from the resin by a trifluoroacetic acid/1,2-ethanedithiol/thioanisole treatment for 2 h at room temperature. The crude peptide was purified by high performance liquid chromatography on a Beckman System Gold apparatus using two different columns successively: a reverse-phase (RP)() C Aquapore (22 1 cm; 20-µm particles; 330-Å porosity) with a linear gradient of 0-80% acetonitrile/water (with 0.1% trifluoroacetic acid); followed by a RP C Spherisorb (25 1 cm; 2-µm particles; 300-Å porosity) with a linear gradient of 0-100% acetonitrile/water (with 0.1% trifluoroacetic acid). In both cases, the flow rate was 4 ml/min. Peptide purity was estimated higher than 90% by amino acid analysis (6 N HCl; 1 h at 150 °C) on a Beckman model 6300 amino acid analyzer, by analytical high performance liquid chromatography using a RP C Nucleosyl column (25 0.46 cm; 5-µm particles; 300-Å porosity) with a linear gradient of 0-100% acetonitrile/water (with 0.1% trifluoroacetic acid) with a flow rate of 1 ml/min and by Edman degradation on an Applied Biosystems model 477A sequencing apparatus.

Glutaraldehyde Cross-linking

The glutaraldehyde cross-linking reaction was carried out at 20 °C. The reaction mixture contained 50 µM (260 µg/ml) of 1013-1056 peptide and 0.001% (v/v) glutaraldehyde. A control was achieved with 260 µg/ml lysozyme. This protein both behaves as a monomeric molecule at any concentration and presents an appropriate molecular weight (14,300) to follow the association intermediates of our fragment. Aliquots of the reaction mixture (10 µl) were taken at different times (0, 30, 60, 120, and 180 min) and were mixed with 2 µl of 1.5 M sodium borohydride. The reaction was stopped, each time, by boiling after addition of 38 µl of sample buffer (0.125 M Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, 100 mM DTT (dithiothreitol), and 0.01% bromphenol blue). 16 µl of cross-linking products were run on a sodium dodecyl sulfate-16.5% polyacrylamide gel electrophoresis (SDS-PAGE) according to the protocol of Schägger and von Jagow (24) . The gel resolution was increased by adding a 10% spacing gel between stacking and separating gels. Finally, the separated products were revealed by silver staining(25) .

Circular Dichroism

Circular dichroism spectra were recorded on a Jobin-Yvon Mark IV high sensitivity dichrograph linked to a PC microprocessor. Optical cells with path lengths of 10, 1, and 0.1 mm were placed in a thermostable cell holder. All spectra were recorded with a 1-nm step and were base-line corrected. Circular dichroism measurements are reported as mean residue molar ellipticity [] (degcmdmol). The relative content in -helix was deduced according to Zhong and Johnson(26) : % -helix = (-10), where is the circular dichroism per residue at 222 nm and is related to [] by: = []/3300. The concentration of the stock solution of 1013-1056 peptide was determined at pH > 12, 20 °C, from UV absorption in the aromatic region. Such a high basic medium was necessary in order to complete the ionization of the two tyrosine residues present in the peptide; however, all the dilutions were made at pH 10, that was enough to achieve dissolution. Obviously, neutral pH would be preferred to conduct association/reassociation experiments. However, inspection of our dimer model does not suggest any significant change within the side-chain groups participating in the coiled-coil stabilization between pH 7 and pH 10. The peptide ellipticities were measured 1 h after dilution at the chosen concentrations, these lying from 0.1 to 1200 µM, and 10 min after changes of temperature. These laps of time allow to reach the new equilibrium. The stock solutions of guanidine hydrochloride (GdnHCl) and urea used for the unfolding experiments were 7.3 M and 8.8 M, respectively. These concentrations were determined with refractive index measurements(27) . For GdnHCl and urea unfolding experiments, the samples were allowed to equilibrate in the presence of different concentrations of denaturant for a minimum of 4 h at room temperature before measuring the ellipticity.

Calculation of Thermodynamic Parameters from Thermal Unfolding

Supposing a transition between two states (folded dimer unfolded monomer), the molar fraction of dimer was calculated from thermal unfolding curves as: = ([] - [])/([] - []) where [] and [] are the ellipticity at 220 nm associated to monomer and dimer, respectively, and [] is the ellipticity at 220 nm at the considered temperature. The values of [] and [] in the transition region were obtained by extrapolating the pre- and post-transition regions. The equilibrium constant, K, is expressed as: K = /2C(1 - ) and, at T, K = 1/C [1], where C and T are the peptide concentration and the temperature at the half-transition, respectively(28) . For any process at equilibrium, -RT lnK = H - TS. Equation 1 provides an expression for K at T in terms of C. Plugging the equality given above, yields upon rearrangement: 1/T = R lnC/H + S/H. This equation corresponds to a straight line when the reciprocal of the melting temperature (1/T) is plotted against the natural logarithm of the peptide concentration (lnC). The slope and the intercept yield H and S, respectively.

Calculation of Thermodynamic Parameters from GdnHCl and Urea Unfoldings

The denaturation curves were analyzed by assuming that the transition between folding dimer/unfolding monomer is a two-state event (F 2U). An equilibrium constant, K, can be thus obtained at each individual GdnHCl and urea concentrations by: K = 2 C (1 - f)/f, where C is the peptide concentration and f is the folded fraction as determined from the ellipticity at 220 nm, namely: f = ([] - [])/([] - []) (27) . [] and [] are the ellipticities of the unfolded and native states, respectively, and [] is the observed ellipticity at any particular denaturant (GdnHCl or urea) concentration. The free energy of unfolding in the absence of denaturant (G HO) was estimated by a linear extrapolation of the unfolding free energy at each individual denaturant concentration to zero denaturant concentration, according to: G = G HO - m[denaturant], where m is the slope of the straight line, dG/d[denaturant].


RESULTS AND DISCUSSION

Identification of a Coiled-coil Motif in Human Topoisomerase II

Originally, the purpose of this work was to experimentally verify the reality of the leucine-zipper structure proposed on sequence examination grounds by Zwelling and Perry (7) for the 994-1021 segment of the human topoisomerase II . In this aim, we estimated the capacity of this sequence to adopt an -helical structure using the procedure of Biou et al.(23) based on the combination of two prediction methods: one by homology and the other by GOR III(22) . The results indicated that the 994-1021 segment was unfavorable to helical structurization. Instead, we found that the 1013-1056 segment located in the minimal region necessary to the activity of topoisomerase II (29) and which partially overlaps the 994-1021 segment exhibited a high probability to be structured in an -helix.

The method of Lupas et al.(30) was then applied to the 1013-1056 sequence as well as to the other -helical predicted segments of human topoisomerase II in order to evaluate their propensity for the coiled-coil structure. This method is based on the relative frequency of occurrence of amino acids at each position of the coiled-coil heptad repeat. The 1013-1056 segment presents the potentiality of having two possible 4-3 hydrophobic heptad repeats (noted as casesI and II in Fig. 1A). Almost all the residues found in positions a and d are of hydrophobic nature. Thus, they may form a hydrophobic interface that stabilizes the coiled-coil. In comparison, even if in the 994-1021 segment proposed as a possible leucine-zipper structure by Zwelling and Perry the leucine residues are found in the desirable d position, more than half of the residues in a position appear unfavorable to dimerization.


Figure 1: A, the two possible coiled-coil structures (casesI and II) for the segment 1013-1056 of human DNA topoisomerase II determined according to the Lupas et al.(31) procedure. The characteristic 4-3 hydrophobic repeats in a and d positions are indicated. Figure also shows an alignment of several type II DNA topoisomerase sequences: HumA, HumB, Dm, and Sc, namely Homo sapiens (isoforms (1013-1056) and (1031-1056)), Drosophila melanogaster(9) , and Saccharomyces cerevisiae(3) , respectively. B, helical wheel representation of the coiled-coil (caseII) formed by the segment 1013-1056.



It has been established that the ratio of charged to apolar residues is indicative of the shape of protein containing a coiled-coil(31) . In the 1013-1056 segment, the positions b, c, e, f, and g are not really hydrophilic and the ratio of charged to apolar residues is found equal to 0.6. This could account for a coiled-coil integrated in a globular protein, which appears to be the case of topoisomerase II(32) , as it has been shown that the apolar groups can be more easily accommodated in the core of a globular unit than in a cylindrical one. Finally, case II, shown under the wheel representation in Fig. 1B, appears more appealing than case I as it displays a conservation of residues at a and d positions in DNA topoisomerases II of several species as diverse as the human and the yeast ones (Fig. 1A).

Glutaraldehyde Cross-linking

Fast fixation of protein association intermediates has been applied with success because of the accuracy and sensitivity of the method (reviewed in (33) ). We used the 1013-1056 peptide at 50 µM concentration, at which the peptide was shown to adopt a coiled-coil structure by circular dichroism (see below). The existence of cross-linked species was determined by SDS-PAGE (Fig. 2). Formation of a significant amount of dimer was detected immediately after mixing the peptide with glutaraldehyde, and the dimer remained the major oligomeric species from 0 to 3 h. After 3 h, we also detected small amounts of trimer, tetramer, and pentamer.


Figure 2: A polyacrylamide-SDS gel of the 1013-1056 human topoisomerase II peptide submitted to glutaraldehyde cross-linking (see ``Materials and Methods''). The products were revealed by silver staining analysis. The positions of molecular size standards (kilodaltons) are shown on the left, and those of the predicted oligomers formed by the 1013-1056 peptide are indicated on the right. T and MW, respectively, correspond to a control without cross-linker and to the molecular size standards.



The present cross-linking results can be considered as significant for the following reasons. First, low concentrations of cross-linker and peptide prevented nonspecific reactions. Second, no complex larger than the pentamer was detected even after 3 h, with a relative amount remaining insignificant compared to cross-linking of the dimer. This accounts for the fact that the dimer is the most stable complex and that formation of higher order oligomers is barely due to interactions of the dimer with peptide molecules in its surrounding. Third, no cross-linking of the monomeric molecule lysozyme used as control was detected under similar conditions. Thus, the glutaraldehyde cross-linking and subsequent SDS-PAGE experiments confirmed the autoassociative properties of the 1013-1056 peptide and pointed out the predominance of dimer over the largest oligomeric species.

Circular Dichroism Spectroscopy

The ability of the 1013-1056 peptide to form a two-stranded -helical coiled-coil was monitored by circular dichroism. Two criteria were used in the analysis: the helical content and the degree of coiling. The first was calculated from the intensity of the molar ellipticity at 220 nm, while the second was estimated from the ratio between the intensities of the bands at 220 and 208 nm. The ratio []/[] is about 0.8 for a single-stranded -helix and about 1.0 for a two-stranded -helical coiled-coil(34) . Changes of this ratio have been explained as follows. The amide n* transition (220-nm CD band) is essentially responsive to the -helical content(26) . On the other hand, the amide * excitation band at 208 nm polarizes parallel to the helix axis and is sensitive to whether the -helix is single-stranded or an interacting helix as in the case of the two-stranded coiled-coil. The decrease in the parallel band intensity, together, in fact, with a red shift of its maximum(35) , must reflect the conversion of a rigid single-stranded -helix to an -helical coiled-coil structure. Furthermore, clear-cut thermodynamic data reflecting the dissociation/reassociation may be obtained from CD experiments using as variables polypeptide concentration, temperature, or chemical denaturants.

Peptide Concentration Effects

The 1013-1056 peptide CD spectra show two minima at 208-210 and 222 nm indicative of a significant proportion of the peptide residues in the -helical conformation(36) . The molar ellipticity value at 220 nm, [], became more negative with increasing peptide concentration (Fig. 3A). The concentration value at the half-transition increased with the temperature (5, 9, and 24 µM peptide at 6, 20, and 40 °C, respectively). An isolated -helix appears marginally stable in aqueous solution, and its stabilization requires interactions provided by tertiary and quaternary structures. Thus, the [] concentration dependence shown by the peptide results from the stabilization of the -helix through its oligomerization. As the concentration of peptide is increased, the monomer-oligomer equilibrium is shifted toward the formation of oligomer which increases the -helical content. At 200 µM peptide concentration, CD spectral analysis with the method of Zhong et al.(28) provides 50% of -helix content. However, above 200 µM, the ellipticity at 220 nm becomes concentration-independent, suggesting the complete association of the fragment. We may note that, in the same conditions, the CD spectra of the 994-1021 fragment reflected an -helix content of approximately 15%, which does not vary with the peptide concentration between 166 µM and 5.2 mM. This confirms the weak potential for the corresponding segment in topoisomerase II to adopt a stable coiled-coil structure.


Figure 3: A, dependence curves of the mean residue molar ellipticity at 220 nm versus the 1013-1056 peptide concentration (logarithmic scale) in water, pH 10 at: 5 °C (), 20 °C (), and 40 °C (). B, dependence curve of []/[]versus the 1013-1056 peptide concentration in water at pH 10, 5 °C.



Certainly more revealing for the self-association of the 1013-1056 peptide in solution, the ratio []/[] in CD spectra at 6 °C was found to increase from about 0.85 at low peptide concentration (<2 µM) to approximately 1.08 at higher peptide concentration (>10 µM) (Fig. 3B). Moreover, the peptide concentration corresponding to the half-transition was found to be the same (6 µM), whether the transition was monitored from the ellipticity at 220 nm or the ratio []/[]. We also observed the expected red shift in the maximum of the parallel band (208 to 210 nm), which, when added to the other effects, accounts for the stabilization of a coiled-coil structure from an isolated -helix.

Thermal Unfolding

Occurrence of an isodichroic point at 203 nm during the thermal denaturation of the 1013-1056 peptide (Fig. 4A) provides support for the existence of a predominant two-state structural transition, which according to the above results is presumed to correspond to the conversion of an -helical coiled-coil into an unfolded monomer. [] decreased with increasing temperature, but the phenomenon was found quantitatively reversible with the decrease of temperature. The thermal curves at different peptide concentrations showed discernible cooperative transitions. A clear dependence of the melting temperature, T, on peptide concentration could be detected (Fig. 4B) with T values of 33, 53, 67, and 77 °C for peptide concentrations of 16, 50, 100, and 160 µM, respectively. This concentration dependence of Talso characterizes an oligomeric structure. The thermodynamic parameters were determined from the thermal unfolding curves according to the procedure described under ``Materials and Methods'' (Fig. 4B, inset). The relative magnitude of H (-11.3 kcal/mol) and TS (-4.5 kcal/mol) indicate that the coiled-coil formation is enthalpically driven. The free energy G was found equal to -6.8 kcal/mol. This free energy is similar to those values determined for -helical coiled-coil models (37) and allows assessment of the stability of the coiled-coil formed by the 1013-1056 peptide.


Figure 4: A, The CD spectra of the 1013-1056 peptide recorded in water at pH 10 from 5 °C (bottomspectrum) to 70 °C (upperspectrum) by 5° increments. The peptide was used at 50 µM concentration. B, the thermal melting profiles for peptide concentrations of 16 µM (), 50 µM ([), 100 µM (), and 160 µM (). The denatured fraction, f, was calculated as f = 1 - , where is the molar fraction of dimer (see ``Materials and Methods''). Inset, the plots of 1/T against the natural logarithm of the peptide 1013-1056 concentration yield the H and S values (H = -11.2 kcal/mol and S = -15 cal/molK).



Trifluoroethanol Effects

TFE can increase the helicity of single-stranded peptides(37) . However, TFE is also a denaturant of tertiary and quaternary structure stabilized by hydrophobic interactions. For instance, it has been shown previously that 50% TFE is enough to disrupt the two-stranded -helical coiled-coil(34) .

The molar ellipticity at 220 nm of the 1013-1056 peptide, [], steadily increased of 45% during the solvent change from plain HO to 50% TFE, reflecting the induction of helical conformation by TFE (data not shown). Above 50% TFE, the ellipticity, [], no longer increased. The ratio []/[] decreased from a value above 1 at 0-20% TFE to about 0.95 at 30%. Accordingly, the -helical coiled-coil structure appears to be stable up to 20% TFE and, above this percentage, disrupted into isolated -helices that may recover their standard parameters.

Guanidine Hydrochloride and Urea Unfoldings

The GdnHCl and urea unfoldings allowed us to estimate the stability of the coiled-coil formed by the 1013-1056 peptide by considering the transition midpoints. [GdnHCl] and [urea] values at which 50% of the peptide is unfolded were found equal to 2.2 M and 1.9 M, respectively (Fig. 5). The free energy, GHO, derived from the unfolding curve, was 17.2 kcal/mol for GdnHCl and 7.4 kcal/mol for urea (Fig. 5, inset). We note the good agreement for unfolding between the free energy determined from the thermal (6.8 kcal/mol) and that from urea (7.4 kcal/mol). On the other hand, GdnHCl, which is a charged molecule, could potentially mask electrostatic interactions and increase hydrophobic interactions but would become a denaturant at higher concentrations(39) . Thus, the results of GdnHCl unfolding might traduce the contribution of hydrophobic forces to the coiled-coil stability. On the other hand, the urea is uncharged and the stability values of urea unfolding would in principle reflect the contribution of both hydrophobic and electrostatic interactions to the stability of the coiled-coil.


Figure 5: GdnHCl () and urea () denaturation profiles for the 1013-1056 peptide coiled-coil in water at pH 10, 20 °C. Inset, the linear dependence of Gversus GdnHCl () and urea () concentrations allows a simple determination of GHO



Dimer Stability

While a comparative study on the effects of GdnHCl and urea is always rather difficult, the stability values obtained from urea and temperature unfolding should be consistent, which is the case in the present study. Here, both the transition midpoints and the free energies provided by unfolding experiments showed that the coiled-coil formed by the 1013-1056 peptide is more stable in GdnHCl than in urea. The difference of free energy between the two denaturants could be explained by intra- or interhelical destabilizing electrostatic interactions arising in the peptide.

Values of -6.8 and -7.4 kcal/mol for the dimerization free energy, G, were found by thermal and urea unfolding, respectively, for the 1013-1056 peptide of human topoisomerase II . Remarkably, the above values are similar to those recently determined (-7.6 kcal/mol) for the yeast DNA topoisomerase II entire molecule from sedimentation equilibrium in the analytical centrifuge(32) . However, conditions adopted in experiments with the peptide and the protein are different.


CONCLUSIONS

The present study decisively indicates that the topoisomerase II 1013-1056 fragment has the potential to adopt a native-like structure favorable to dimer formation, without the participation of the entire protein(40) . Actually, a similar conclusion has been reached for thermolysin fragment 255-316 in its dimeric state, which is essentially identical to that of the corresponding chain region in the x-ray structure of thermolysin(41, 42) . Moreover, the unfolding thermodynamic parameters of this fragment dimer are similar to those normally observed for small and globular proteins(43) . The same proved true for chromogranin A and one of its constitutive peptides from a comparative thermodynamic study of their oligomerization properties (44) .

Our results show that the human topoisomerase II 1013-1056 fragment associates into a stable two-stranded -helical coiled-coil structure. The dimeric state of the fragment appears to be stabilized mostly through hydrophobic interactions. The sensitivity reflected by the -helix against peptide concentration shows that coiled-coil formation plays a non-negligible role toward the folding and stability of the fragment.

A striking feature of the present work consists of the free energy G, which is found to be approximately the same for the 1013-1056 fragment and the entire yeast topoisomerase II (32) . Notwithstanding the particular experimental conditions used for the fragment, we are tempting to speculate on the involvement of 1013-1056 segment as a unique region for the dissociation/reassociation process of topoisomerase II, outward interactions with DNA. This proposal is consistent with the results of Caron et al.(29) , which have pointed out that topoisomerase II dimerization arises in the C-terminal half of the protein and is further supported by the fact that this dimerization would occur in a strongly conserved region of topoisomerase II.

Furthermore, our results do not allow retention of the 994-1021 segment, which partly overlaps the preferred 1013-1056 segment, as a possible candidate to participate in the coiled-coil (leucine-zipper) structure.

Our study thus provides an additional example tending to prove that model studies on fragments are relevant for the folding or interactions of intact proteins(45, 46) . Based on a number of experimental results, there is real hope that some deeper understanding of the structural and energetic properties of protein domains can be gained by looking closer the folding and self-association processes present in isolated polypeptide fragments.


FOOTNOTES

*
This work was supported by the Laboratoire L. Lafon and the Association de la Recherche contre le Cancer. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom reprint requests should be addressed. Tel.: 33-1-45-59-49-85; Fax: 33-1-46-78-41-20.

The abbreviations used are: RP, reverse-phase; TFE, trifluoroethanol; PAGE, polyacrylamide gel electrophoresis; GdnHCl, guanidine hydrochloride.


ACKNOWLEDGEMENTS

We thank J. P. Levillain and M. Le Maout for skilled technical assistance in peptide synthesis and Profs. Ph. Laurent and D. Bellet for valuable discussions.


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