Proc. Nadl. Acad. Sci. USAVol. 91, pp. 4840-4844, May 1994Biochemistry

Double-stranded DNA templates can induce a-helical conformationin peptides containing lysine and alanine: Functional implicationsfor leucine zipper and helix-loop-helix transcription factors

(proenstrucn re/a-helix/transcriptional regulation/circular dichroism)

NEIL P. JOHNSON*, JOEL LINDSTROM, WALTER A. BAASE, AND PETER H. VON HIPPELInstitute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, OR 97403

Contributed by Peter H. von Hippel, February 15, 1994

ABSTRACT Transcription factors of the basic-leucinezipper and basic-helixloop-helix families specifically recog-nize DNA by means of intrinsicaly flexible peptide domainsthat assume an a-helical conformation upon binding to targetDNA sequences. We have investigated the nonspecific interac-tions that underlie specific DNA recognition. Circular dichro-Ism measurements showed that 20-bp double-stranded DNAoligonucleotides can act as templates to promote random coil-a-helix transitions in short peptides contag alanine andlysine. This conformational change takes place without alteringthe structure of the DNA, and neither specific peptide-DNAcontacts nor cooperative interactions between peptides arenecessay. The conformational change does require (i) double-stranded (but not single-stranded) oligodeoxynucleotides ineither the B or the B' conformation and (ii) peptides that canform positively charged amphipathic a-hellces. In 10 nimNa2EIPO4 (pH 7.5; 100C), the excess free-energy contribution ofthe DNA template to the stability of the a-helical form of theoligopeptides tested was AG, = -0.15 (± 0.07) kcal/mol perlysine residue. The implications of these results for the ther-modynamics and kinetics of DNA target site selection bybasic-4eucine zipper and basic-helix4oop-helix regulatoryproteins are dud.

Eukaryotic regulatory proteins of the basic-leucine zipper(bZIP) and basic-helix-loop-helix (bHLH) families bind asdimers to symmetric DNA sites. Each of the monomerscontains a 60- to 100-aa sequence composed oftwo domains,both of which are required for efficient specific complexformation. One, the HLH or the ZIP domain, provides theprotein-protein interactions involved in dimer formation.The second selectively binds the DNA target sequence. ThisDNA-binding motif generally contains several basic aminoacid residues and has a flexible conformation in the absenceofDNA. On binding to the major groove ofthe specific DNAtarget sequence it forms an amphipathic a-helix (i.e., ana-helix with all of the positively charged residues located onone side), thereby introducing sufficient topological con-straints to selectively recognize complementary hydrogenbonding and van der Waals sites within the DNA double helix(1-10).

Specific DNA-binding proteins distinguish their target se-quences from a vast number of competing nonspecific sitesby means of large free energy differences between specificand nonspecific protein-DNA complexes. Rigid DNA-binding domains, such as the helix-turn-helix (HTH) motif,drive selective recognition by imposing hydrogen-bondinginteractions that destabilize nonspecific protein-DNA com-plexes relative to those with the correct target sequence(11-13). Proteins with flexible DNA-binding domains (such

as bZIP and bHLH) might be expected to avoid unfavorableinteractions and consequently to bind more tightly at non-specific sites than proteins with rigid DNA-binding motifs.Furthermore, binding free energy must be used to driveunstructured binding domains into the bound conformation;if this structure-forming reaction occurs only during bindingat the regulatory site, then specific complexes would beselectively destabilized relative to nonspecific complexes.Hence, specific recognition of DNA target sites by flexiblebinding domains would be significantly more thermodynam-ically "expensive" than recognition by initially structuredmotifs, in that a larger amount of the protein would have tobe synthesized and bound at nonspecific DNA sites tomaintain binding equilibrium at the specific sites (12-14).Finally, the rate of specific target location by a regulatoryprotein might also be significantly decreased if the bindingdomain of the protein can assume its docking and recognitionconformation only on arrival at the specific DNA targetsequence (15).

In order to understand the thermodynamic and kineticbases of specific binding by bZIP and bHLH transcriptionfactors, it is important to know whether their flexible DNA-binding motifs are also driven into an a-helix when theprotein is bound at nonspecific DNA sites. To this end weasked whether DNA-induced a-helix formation must bestabilized with the assistance of a specific complementaryhydrogen-bonding docking matrix or whether it could occurat nonspecific peptide-DNA interactions. We show here thatsmall peptides with appropriately placed alanine and lysineresidues do indeed have the potential to form a-helices byinteracting nonspecifically with double-stranded DNA, sug-gesting that helical DNA-binding motifs may be generallypresent in nonspecific as well as specific complexes betweenbZIP or bHLH transcription factors and DNA. This resulthas important thermodynamic, kinetic, and structural con-sequences for transcriptional regulation which are consid-ered in the Discussion.

MATERIALS AND METHODSPreparation and Characterization of Oligopeptides and Oli-

gonudeotides. The peptides designated A2K2 and A2KA3K(Fig. 1) were synthesized on an Applied Biosystems model430A apparatus in the University of Oregon BiotechnologyLaboratory by means of standard tert-butoxycarbonyl chem-istry and were purified by reverse-phase HPLC on a C18 resinwith elution by a 0-70% acetonitrile gradient in 0.1% tri-fluoroacetic acid. Purity was evaluated by amino acid se-

Abbreviations: bZIP, basic-leucine zipper; bHLH, basic-helix-loop-helix; HTH, helixturn-helix.*Permanent address: Laboratoire de Pharmacologie et de Toxicol-ogie Fondamentales du Centre National de la Recherche Scienti-fique, UPR 8221, 205 route de Narbonne, 31077 Toulouse, France.

4840

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Proc. Nati. Acad. Sci. USA 91 (1994) 4841

5'-TATATATATAATTAATTAAT-3' dsAT3'-ATATATATATTAATTAATTA-5'5'-TATATATATAATTAATTAAT-3' ssAT

(dA)20(dT)20

acetyl-YAAKKAAKKAAKKAA-amide A2K2

acetyl-YAAKAAAKAAKAAAKA-amide A2KA3K

acetyl-YKAAAAKAAAAKAAAAK-amide A4K

acetyl-KAKAK-amide AK

(Y = tyrosine, A = alanine, K = lysine)

FIG. 1. Oligonucleotides and oligopeptides used in this study andabbreviations used to designate these in the text.

quencing and molecular weights were confirmed by fast-atombombardment mass spectroscopy. The peptide designatedA4K was the gift of S. Padmanabhan and Robert Baldwin(Stanford University) and the peptide designated AK waskindly provided by Warner Peticolas (University of Oregon).

Peptides (except AK) were synthesized with an N-terminaltyrosine residue to facilitate concentration determinationfrom the specific tyrosine absorbance at 275 nm in 6 Mguanidinium chloride (16). Concentrations ofAK were esti-mated from the absorbance at 220 nm by comparison with theabsorbance of the tyrosine-containing peptides at this wave-length after correction for tyrosine residue absorbance (17).Molar ellipticities of the peptides were independent of con-centration over the range 1-40 pM.

Oligodeoxynucleotides were purchased from Macromolec-ular Resources (Colorado State University) or were synthe-sized in the University of Oregon Biotechnology Laboratoryand purified by reverse-phase HPLC. DNA concentrationswere determined from UV absorbance by using the nearest-neighbor approximation (18) to calculate extinction coeffi-cients. The double- or single-stranded nature of the oligonu-cleotides was confirmed by thermal melting. Solutions wereprepared with filtered deionized water (Barnstead NanopureII). Concentrations of oligonucleotides and oligopeptides areexpressed in units of molecules.CD Spectra and Titratbons. CD spectra were measured from

194 to 340 nm by a JASCO J600 spectropolarimeter coupledwith a Hewlett-Packard HP89100A temperature controller(19). Spectra were scanned at 1 nm/min with an 8-sec timeconstant. The CD spectrum of buffer alone was subtractedfrom the experimental spectra after alignment at 340 nm. CDdifference spectra were calculated by subtracting spectra ofthe oligonucleotides alone from those of the oligonucleotide/oligopeptide mixtures. Final spectra were averages of six toeight runs (uncertainty, ±0.2 millidegree at wavelengthsgreater than 210 nm).Automatic titrations of stirred 2 ,uM DNA solutions in a

1-cm cell were carried out with 2- or 4-p1 incrementalvolumes of 100 or 250 puM peptide solutions by use of asyringe coupled to a micrometer driven by a stepper motor(200 steps per turn), which was installed in the enlargedsample chamberofaJASCO J500 spectropolarimeter. Duringtitration, the ellipticity at 223 nm was collected as a functionof total peptide concentration on a Hewlett-Packard HP-87computer. The dilution-corrected CD signal due to oligonu-cleotide alone was subtracted and the initial slope of theresulting titration curve, S, in degrees/(pM oligopeptide-cm)was used to calculate the a-helical content of each peptide:

% a-helix = (S x 100)/(n x 10-6) x (1/33,000), [ll

where n is the number of amino acid residues in the peptideand [01223 = -33,000 deg-cm2/dmol is the molar ellipticity for

a peptide residue in a fully a-helical conformation (20). Thisvalue was determined experimentally from the CD spectra ofseven peptides, all 15-17 amino acid residues long andcontaining mainly alanine and lysine as well as an N-terminaltyrosine residue. The peptides were made fully a-helical bydissolving in aqueous 2,2,2-trifluoroethanol. Consequently,the above value of [Oln3 should include, to a first approxi-mation, the contribution of tyrosine to the CD spectrum,although a tyrosine residue at this position may lead to anunderestimation of the helix content of such peptides (17).The values of helix content that we present are based on theassumption that the fraction of a-helical peptide is propor-tional to [O]n3. We have also assumed that the relationshipbetween [O]l3 and percent helicity is the same for the freepeptide and for the peptide complexed with a double-stranded DNA template.

RESULTSIn this paperwe ask whetherDNA oligonucleotides can serveas templates to induce a-helix formation in basic peptides(see Fig. 1 for oligomer sequences and abbreviations). Wehave used low salt and low temperature conditions to stabi-lize potential ordered forms. The induction of a-helicity incomplexed peptides has been examined for two double-stranded oligonucleotide templates with different secondarystructures. The CD spectrum of (dA2o)-(dT20) at 10°C in 10mM Na2HPO4 (pH 7.5) shows three maxima (at 282,258, and216 nm) and three minima (at 268, 247, and 207 nm). Thisspectrum resembles that obtained with poly(dA)-poly(dT) inthe B' form (21). This conformation is characterized by alarge propeller twist of the base pairs and unusual bifurcatedhydrogen bonds (22). The CD spectrum ofdsAT showed twomaxima (at 271 and 218 nm) and a single minimum (at 248nm); it is expected to assume a B-form conformation asjudged from the CD of poly(dA-dT)poly(dA-dT) (21) andx-ray structures of polynucleotides related to dsAT (22). Thetemplate activity of these double-stranded oligonucleotideswas compared with that ofthe single-stranded ssAT oligomer(see Fig. 1).The amino acid sequences ofpeptides A2K2 and A2KA3K

were designed to mimic certain potentially important featuresofthe DNA-binding domains ofthe bZIP andbHLH proteins.Alanine residues favor the a-helical conformation in shortpeptides in aqueous solution (23), while lysine residues helpto make these peptides reasonably soluble and can interactelectrostatically with the negatively charged phosphates ofthe DNA backbone. Deletion analysis'experiments and ho-mology arguments have shown that the DNA-binding motifsof bZIP (3) and bHLH (5) proteins contain 15-20 aa. Con-served residues occur every 3-4 aa along the peptide back-bone and include the positively charged side chains. In ana-helical conformation (3.6 aa per turn), residues that occurat such separations fall on the same side of the a-helix.Electrostatic repulsion opposes a-helix formation in thesepeptides in the absence ofa bound oligonucleotide. Howeverin an a-helical peptide, this separation of charged residuescorresponds closely to the distance between adjacent phos-phate groups in B-form DNA, and complexation with DNAmay stabilize the a-helical conformation.The four lysine residues of A2KA3K are separated by

three, two, and three alanine residues, whereas the A2K2molecule contains three pairs of lysine residues separated bypairs of alanine residues. Thus, when these peptides assumean a-helical conformation in the major groove of DNA, thebasic lysine residues are spaced to form favorable charge-charge interactions with the negatively charged phosphategroups of the double-stranded DNA. In contrast, the basicresidues of a-helical A4K and AK are not appropriatelyspaced to make such stabilizing contacts. In keeping with

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4842 Biochemistry: Johnson et al.

these expectations we find that the amplitude of the CDspectra near 220 nm, for an equimolar mixture of A2KA3Kand dsAT from which the spectrum of dsAT has beensubtracted (lower curves in Fig. 2), is approximately doubledrelative to the spectrum of A2KA3K alone (upper curves).Similar increases in CD amplitudes were also observed forcomplexes ofA2KA3K with (dA20)-(dT20), and for complexesof A2K2 with both of these oligonucleotides (Fig. 2b). Incontrast, equimolar mixtures of (dA20)-(dT20) or dsAT withA4K and AK displayed CD spectra that were identical to thesum of the component spectra (data not shown).The peptides that we have used do not contribute to the

molar ellipticity at 260-300 nm, where optical activity reflectsoligonucleotide base stacking. No intensity was observed inthe CD difference spectrum at these wavelengths for any ofour peptide/oligonucleotide mixtures (Fig. 2), indicating thatDNA base stacking was not perturbed by peptide binding.Instead, these CD difference spectra are characteristic ofsmall alanine/lysine-containing peptides in an a-helical con-formation (20). Although we cannot rule out a contributionfrom the oligonucleotide to the 200- to 240-nm region of theCD spectrum, it seems reasonable, judging from the shapesand intensities ofthe difference spectra, to assign the spectralchanges observed in Fig. 2 to an increase in the a-helicalcontent of the peptides.To further investigate this helix-induction phenomenon,

we titrated oligonucleotides with peptides and followed themolar ellipticity at 223 nm ([01223) as a function of totalpeptide concentration (Fig. 3). The CD signal of the oligo-nucleotide alone, corrected for dilution, has been subtractedfrom these data. Linear titration curves were obtained whenpeptides were added to solutions containing buffer only (Fig.3a), indicating that helix formation was not stabilized byassociations between peptide molecules. Molar ellipticitiescalculated from the slope of these titration curves were thesame as those obtained from the CD spectra of the peptidesalone. However, when the peptides were titrated into someof the oligonucleotide solutions, the values of [O]2l obtainedfell below the straight line obtained for titration into bufferalone (Fig. 3 b-d). Control CD spectra showed that theadditional negative ellipticity observed at 223 nm corre-sponded to a CD difference spectrum similar to that of Fig.2. Thus we interpret the changes in the slopes of the titrationcurves of Fig. 3 in terms of DNA-induced increases in thea-helix content of certain peptides. Replicate determinations

.-l

E

E0

0--.

x

a)-

200 250 200 250 300

0

-0.5

Wavelength (nm)

FiG. 2. CD spectra and difference spectra of the alanine/lysinepeptides in the presence and absence of various DNA oligonucleo-tides. (a) Spectrum of 1 MuM A2KA3K alone (e) and as a differencespectrum of 1 uM dsAT and equimolar A2KA3K from which thespectrum of 1 pM dsAT alone has been subtracted (U). (b) Spectrumof 0.8 pM A2K2 alone (e) and as a difference spectrum of 0.8 pM

(dA)2o(dT)2o and equimolar A2K2 from which the spectrum of 0.8pM (dA)20j(dT)20 alone has been subtracted (m). Experiments wereperformed at pH 7.5 and 10"C in the presence of either 50 mMNa2HPO4 (a) or 5 mM Na2HPO4/50 mM NaCl (b).

0)

~0

E-l

0 (a) buffer b

-5

-10

;.-1 5

-20

-25

-3C0

0 (c) (dA)(dT)20 (d) ssAT-5

-10

-15

-20

-25

-30 .... ,_0 2 4 6 8 0 2 4 6 8 1

Peptide (EM)0

FIG. 3. Typical data for the titration of the indicated oligonucle-otides or buffer only with A2K2 (e, top curve, except fordwhere thispeptide is not shown; see text), A4K (*, bottom curve), orA2KA3K(i, middle curve) in 10 mM Na2HPO4 (pH 7.5, 100C). Om, ellipticityat 223 nm in a 1-cm-path-length cell. Nucleotide concentrations were2 pM single-stranded (ssAT) or double-stranded [dsAT and (dA)20(dT)20] oligonucleotide templates.

of the initial slope from such titrations were used to computethe a-helix content of each peptide in the presence andabsence of the various oligonucleotide templates (Table 1).The binding ofA2KA3K to ssAT did not change the helix

content of the peptide compared with buffer (401% and 34%,respectively; Table 1). In contrast, titration of dsAT or(dA2o) (dT2o) with this peptide resulted in initial slopes lessthan those obtained in the equivalent buffer titration (60%16a-helical content; Table 1). Thus, A2KA3K became morea-helical in the presence of double-stranded DNA (but not inthe presence of single-stranded DNA). Similar analysesshowed that the a-helix content ofA2K2 increased from 10%to 33% in the presence of dsAT and (dA2o)(dT20) (Table 1).Precipitation occurred during titration of ssAT with A2K2(data not shown). Prior to precipitation the amplitude of theCD spectrum of ssAT decreased at its 270-nm peak andincreased at its 250-nm trough, indicating that this peptidefurther unstacked the single-stranded DNA. This behaviorwas not observed on the addition ofA2K2 to double-strandedDNA.

Identical titration curves were obtained for the addition ofA4K to buffer or to oligonucleotide solutions (bottom trace inall panels of Fig. 3). The CD signal decreased linearly as afunction ofthe concentration ofadded peptide, and the slopeindicated that this peptide remained about 50% helical in thepresence or absence of oligonucleotides (Table 1).

Table 1. a-Helix content of peptides with and without DNAoligonucleotides in 10 mM Na2HPO4 (pH 7.5, 100C)

% helix (mean ± range)In With With With

Peptide buffer dsAT ssAT (dA2h)(dT20)A2K2 10.2 ± 0.6 33.3 ± 4.0 Pptn 31.0 ± 2.4A2KA3K 33.7 ± 3.0 60.9 ± 9.4 39.8 ± 1.5 57.5 ± 3.3A4K 46.9 ± 2.7 52.2 ± 3.7 50.8 ± 0.8 51.6 ± 1.6

Pptn, precipitation.

1 l l l l l l.5- a b

0 -.5-_

.5-

2

____A ____A ____A Iii0 11 t111

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Proc. Natl. Acad. Sci. USA 91 (1994) 4843

DISCUSSIONWe have shown that, at low salt concentrations and temper-atures, double-stranded (but not single-stranded) DNA tem-plates can promote a-helix formation in the oligopeptidesA2K2 and A2KA3K (Table 1). In contrast, none of the DNAtemplates we tested was able to induce an increase in thea-helical content of the peptide A4K. Clearly this does notreflect conformational constraints within A4K, since thispeptide becomes 80%o a-helical when charge repulsion be-tween lysine residues is screened by addition of 1 M NaCl,and becomes fully a-helical in trifluoroethanol (20). Thelysine residues ofA4K are separated by four alanine residuesand thus A4K cannot form an amphipathic a-helix, whereasA2K2 and A2KA3K do form such helices. The bZIP DNA-binding domain of the GCN4 dimer, a positively chargedpeptide that has evolved for specific binding and carriesresidues other than alanine and lysine, can also assume anamphipathic, partially a-helical conformation on nonspecifictemplates (7). These results suggest that the potential forDNA-directed a-helix induction is general for positivelycharged peptides that can form amphipathic a-helices. Theability to drive helix formation in these peptides does notappear to be very sensitive to the structure or the flexibilityof the double-helical DNA template, at least with the oligo-mers tested (Table 1). Likewise, the unchanged CD spectrain the vicinity of 260 nm (Fig. 2) indicate that peptide helixinduction does not alter the conformations of the oligonucle-otide partners.We have used a simple two-state thermodynamic model to

estimate the contribution of the double-stranded oligonucle-otide templates to the free energy of the coil = helixtransition of the peptides that we have studied:t

cD c+D 4h+D hD. [2]

In this model K1 = [h]/[c], K2 = [hD]/[h][D], and K3 =

[cD]/[c]JD], with c representing a peptide molecule in therandom coil (unfolded) form, h a peptide molecule in thea-helical form, and D a double-stranded DNA templatemolecule. cD and hD represent complexes ofDNA templateswith peptides in the coil and helical forms, respectively.These equilibria describe the binding of peptide to DNA inthe limit at which a single peptide interacts with a singleDNAtemplate molecule to form a 1:1 complex. Our results showthat double-stranded DNA templates preferentially bind thepeptide in the a-helical conformation and shift the equilibriaof Eq. 2 to the right.Based on Eq. 2, the overall equilibrium between peptide in

the coil and helix forms can be written:

K = ([h] + [hD])/([c] + [cD]). [3]

In the absence of DNA, K becomes K1, which may becalculated from the measured helix content of the peptidefree in solution (Table 2). K is determined from the helixcontent of the peptide in the presence of excess DNA. Fromthe equilibrium binding constants we may write

tClearly, as Baldwin and coworkers (24) have shown, such a modelis not adequate, in general, to describe helix = coil transitions insolution in short peptides of this sort. Distributions of partiallyhelical molecules are actually obtained at the midpoint of thetransition, and the use of a two-state model can significantlyunderestimate helix content under some conditions. Thus the ap-proximations involved in using the two-state model must be bornein mind in evaluating our quantitative results. Nevertheless, thetwo-state model may be reasonably appropriate in the situationdescribed here, since a peptide bound to a double-stranded DNAtemplate is more likely to be primarily helical than the same peptidefree in solution.

K = Kl(l + K2[D])/(l + K)[D]), [4]

and the free energy of the coil = helix transition (AG =-RTlnK) is

AG = AG(0) + AGC,,(D), [15

where AG(0) = -RTlnK1 is the free energy ofhelix formationin the absence ofDNA, and lGe,(D) = -RTln{(1 + K2[D])/(l+ K3[D])} is the excess free energy of the coil -- helix

transition due to DNA binding. The contribution ofthe DNAtemplate to the free energy of helix formation is then

[6]

Hence the double-stranded oligonucleotide template contrib-utes a standard free energy (AGC) of approximately -0.8 (±0.1) kcal/mol of oligopeptide to the helix formation ofA2K2and approximately -0.6 (± 0.3) kcal/mol of oligopeptide tothe helix formation of A2KA3K (Table 2). This correspondsto an average AG..of -0.15 (± 0.07) kcal/mol for each lysineresidue in the peptide. We have observed (data not shown)that helix induction in the A2K2 peptide is suppressed by 0.1M NaCl, as expected for a stabilization mechanism that isprimarily based on electrostatic interactions between prop-erly placed lysine residues and DNA phosphate groups.From Eq. 4, K/K1 = K2/K3 for K3[D] >> 1. Since at high

DNA/peptide ratios [D] is the total nucleotide templateconcentration (2 IpM double-stranded oligonucleotide), K3must be >> 5 x 105 M-1. The peptides that we have examinedin this study contained four or six lysine residues. Conse-quently, K3, the equilibrium constant for binding a peptide toDNA in the random coil form at 100C in 10mM Na2HP04(pH7.5), can be estimated, to a first approximation, as 6 x 109permol of oligonucleotide from the binding constant of (Lys)s toDNA under equivalent solvent conditions (25). If this esti-mate is correct, the helix - coil equilibrium in the presenceand in the absence of DNA is simply related to the relativebinding of h and c to DNA, K/K1 = K2/K3, and we canestimate that the binding constant of dsAT to the peptidesA2K2 and A2KA3K in the a-helical conformation is about 4times greater than that for the binding of dsAT to theequivalent random-coil peptides.The free energy of binding of our peptides in the random-

coil form to DNA can be estimated as -13 kcal/mol ofoligonucleotide from the binding constant of (Lys)s to DNAunder our experimental conditions (25). In contrast, thecontribution of the double-stranded DNA templates to thefree energy of a-helix formation for these peptides is quitesmall: approximately -0.75 kcal/mol for a peptide carryingfive lysine residues that can form an amphipathic a-helix.These relative free energies are consistent with genetic data(27) suggesting that the flexibility ofthe DNA-binding domainmay be important in specific recognition of transcriptionfactors of this type. The need to accommodate stericallydemanding peptide-DNA interactions, such as hydrogen-bond formation, could easily provoke minor shifts in theposition and orientation of the peptide within the majorgroove of DNA, allowing similar positively charged DNA-reading heads to recognize different members of a family ofrelated regulatory DNA sites.

Table 2. Thermodynamic parameters for the coil = helixequilibrium in the presence and absence of dsAT

Parameter A2K2 A3KA2K A4KIn buffer (K1) 0.114 ± 0.007 0.51 ± 0.07 0.88 ± 0.10With dsAT (K) 0.50 ± 0.09 1.56 ± 0.81 1.09 ± 0.18AG., kcal/mol -0.8 ± 0.1 -0.6 ± 0.3 0

Biochemistry: Johnson et al.

AGex = -RTln(KIK,).

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4844 Biochemistry: Johnson et al.

The induction of a-helical conformations by nonspecificDNA sequences might be thermodynamically important forspecific binding of regulatory proteins in at least two ways.(i) If helix formation occurs only upon contact with the DNAtarget sequence, the binding free energy diverted to thispurpose will decrease specific binding affinity relative tononspecific binding affinity. On the other hand, if the bindingdomain assumes a helical conformation at both specific andnonspecific sites, this extra cost of specific binding isavoided. (ii) In the HTH proteins, unfavorable interactionsbetween the rigid DNA-binding domain and DNA at nonspe-cific sites cannot be avoided and the resulting unfavorablefree energy drives specific binding of the protein (11, 14). Incontrast, a flexible DNA-binding motif might decrease spec-ificity by avoiding these unfavorable interactions at nonspe-cific sites and minimizing their destabilizing impact. DNA-induced a-helix formation at the protein reading head maypermit bZIP and bHLH DNA proteins to resolve this appar-ent contradiction between flexibility and specificity by intro-ducing topological constraints that prevent the DNA-bindingdomain from avoiding unfavorable contacts at nonspecificDNA sequences.

Finally, the presence of the same conformation at nonspe-cific and specific sites would permit the retention ofthe mainstructural elements of the recognition domains in a scanningor sliding approach to the specific target site along the DNA.The ability to slide, dock, and recognize on moving fromnonspecific to specific sites without major conformationalrearrangements should speed binding to the regulatory targetsequence (15).What evolutionary advantages might lead to the selection

of transcription factors with flexible DNA-binding domains?The thermodynamic bases for specific binding would remainunchanged if binding to both specific and nonspecific sitesrequired the induction of a-helix. However, interactions withboth types of sites would be destabilized by the presence ofan unstructured binding domain in the unbound protein,thereby increasing the free protein concentration required tomaintain binding equilibrium. For example, ifthe free-energycost of putting the recognition domain into an a-helix were 1kcal/mol (a reasonable value judging from our results withmodel peptides), then 5 times more "free" protein wouldhave to be available in solution for a protein with a flexiblebinding domain than for a peptide with a rigid DNA-bindingsite. Increased concentrations of free protein could signifi-candy drive intermolecular associations, such as the forma-tion of homo- and heterodimers by bZIP and bHLH proteinsthat are important for their regulation (26). These interactionswould be less accessible to an equivalent transcription factorwith a rigid DNA-binding domain.

It may be significant for this argument that regulatoryproteins with flexible DNA-binding domains are more com-mon in eukaryotic than in prokaryotic cells. Transcriptionalactivation (regulated by the formation of multiprotein com-plexes) is also more common in eukaryotes. Transcriptionalactivation mechanisms generally allow more complex regu-lation of gene expression, and the resulting increase inadaptive cellular response may account, in part, for theevolution of regulatory proteins with flexible DNA-bindingdomains in higher organisms.

We thank Deborah McMillan of the University of Oregon Bio-

technology Laboratory for synthesizing some of the oligopeptidesused in this study and Robert Baldwin, S. Padmanabhan, and WarnerPeticolas for supplying additional peptides (see Materials and Meth-ods). We are very grateful to Robert Baldwin and John Schellman forhelpful discussions. These studies were supported in part by U.S.Public Health Service Research Grants GM15792 and GM29158 (toP.H.v.H.) and by a grant to the Institute of Molecular Biology fromthe Lucille P. Markey Charitable Trust. P.H.v.H. is an AmericanCancer Society Research Professor of Chemistry. N.P.J. gratefullyacknowledges additional support from the Centre Nationale de laRecherche Scientifique.

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