letters

872 nature structural biology • volume 5 number 10 • october 1998

Structures of HhaImethyltransferasecomplexed with substratescontaining mismatches atthe target base

Three structures have been determined for complexes betweenHhaI methyltransferase (M.HhaI) and oligonucleotides con-taining a G:A, G:U or G:AP (AP = abasic or apurinic/apyrimi-dinic) mismatch at the target base pair. The mismatchedadenine, uracil and abasic site are all flipped out of the DNAhelix and located in the enzyme’s active-site pocket, adoptingthe same conformation as in the flipped-out normal substrate.These results, particularly the flipped-out abasic deoxyribosesugar, provide insight into the mechanism of base flipping. Ifthe process involves the protein pushing the base out of thehelix, then the push must take place not on the base, but ratheron the sugar-phosphate backbone. Thus rotation of the DNAbackbone is probably the key to base flipping.

Base flipping is a phenomenon in which a DNA nucleotide isrotated completely out of the double helix, breaking the base-pairing hydrogen bonds and interrupting the base stack. Baseflipping was first observed in a structure for HhaI DNA methyl-transferase (Mtase) bound to its substrate DNA1. Subsequently itwas demonstrated in several systems — another Mtase2 andthree DNA repair enzymes3–5 — and has been suggested forabout a dozen other Mtase and DNA repair-related systems (ref.6 and refs therein). Base flipping provides a mechanistic linkbetween DNA Mtases and DNA repair enzymes. It has beenobserved directly in the interactions with DNA of T4 endonucle-ase V3, human uracil glycosylase4 and E. coli mismatch-specificuracil glycosylase5.

Furthermore, human7 and bacterial Mtases8–11 bind to DNAsubstrates containing mismatches at the target base within therecognition sequences. Gel shift assays8 showing the equilibriumbinding of M.HhaI to the DNA mismatches at the target baseindicated that the G:U mismatch and abasic site had a Kd ~10-fold less than the G:C target, while the G:T mismatch had a Kd

five-fold less than the normal target. Thus, M.HhaI has a higherbinding affinity for DNA mismatches than the normal substrate.Under the equilibrium conditions and the limited concentrationrange during gel electrophoresis, these relative Kd values areprobably more significant than the absolute values (in the rangeof nM to less than 1µM). The ability to bind a mismatch at thetarget base may be related to the monomeric nature of DNAMtases which methylate only one DNA strand at a time.However, it is worth noting that the mismatch and DNA damagerecognition at the target base pair by Mtases is within a specificsequence context. That M.HhaI does not show much bindingspecificity for the target base may reflect a need to leave the baseunencumbered by recognition contacts. Flipping of the targetbase is simply needed to facilitate catalysis at the concave cata-lytic pocket12,13.

We report here three well-refined ternary structures of M.HhaIcomplexed with the methylation end-product S-adenosyl-L-homo-cysteine (AdoHcy) and oligodeoxynucleotides containing a G:A,G:U, or G:AP (abasic) mismatch at the target base pair at resolu-tions of 2.87 Å, 2.76 Å and 2.39 Å respectively (Table 1). The suc-

cess of crystallization of these M.HhaI–DNA mismatch complexes,with mM concentrations of protein and DNA, was likely to bedependent on the off-rate that M.HhaI dissociates from DNA mis-matches. To slow the dissociation rate significantly8,9, the crystal-lization was performed in the presence of the methylationend-product AdoHcy. The t1/2 and koff for the normal G:C, the mis-match G:U and the abasic site were ~260 min and 2.65 × 10–3 min–1

respectively, in the presence of AdoHcy8. Interestingly, the off-rateof the G:T mismatch was too rapid to be measured by gel shiftassays even in the presence of AdoHcy8. The faster off-rate of theG:T mismatch may account for the failure to obtain crystals of theM.HhaI–AdoHcy–G:T mismatch complex, although the G:T mis-match has five-fold higher binding affinity by M.HhaI when com-pared with the normal G:C base pair.

Well-defined electron densities (Fig. 1a) reveal that the mis-matched adenine, uracil and abasic sites are completely flippedout from the interior of the DNA duplex and are accommodatedby the enzyme’s active-site pocket (Fig. 1b), in a manner identicalto that previously seen for DNA containing the normal substratecytosine interacting with M.HhaI. The active-site pocket is locat-ed almost in the middle of the protein–DNA complex and is notinvolved directly in any crystal packing contacts (see Fig. 5 ofref. 14). Of course, the packing lattice prevents complex dissocia-tion. The orphan guanine of the complementary strand remainsstacked in the helix, the ribose ring has a C2'-endo pucker(pseudorotation angle of 157o to 163°) and a high-anti (-syncli-nal) glycosidic torsion angle of about -72° to -76°. Significantchanges in the protein side chains were only observed near theflipped abasic deoxyribose.

G:A mismatchThe flipped adenine of the G:A mismatch makes both hydrogenbonds and hydrophobic interactions with amino acids ofM.HhaI. In the plane of the ring (Fig. 2a), the amino group (N6)forms a hydrogen bond with the backbone carbonyl oxygen ofPhe 79. The N1 atom of adenine is within hydrogen bonding dis-tance (2.6 Å) to one of the carboxyl oxygens of the acidic aminoacid Glu 119. The N7 atom interacts with the side chains of Gln82 and Asn 304 through two water molecules. The aliphaticamino acid Val 121 sits perpendicular to the adenine ring, whiletwo sulfur atoms from Cys 81 and AdoHcy approach the C8 andN7 atoms at the edge of the ring respectively, from the oppositedirections (Fig. 2b). The ribose ring adopts a C3'-endo pucker(pseudorotation angle of 33°) and a high-anti (-synclinal) glyco-sidic torsion angle of -75°.

The comparative analysis of the structures of DNA Mtases(cytosine-C5 and cytosine-N4-specific versus adenine-N6-spe-cific) suggested that cytosine/adenine binding pockets, struc-turally and functionally, are remarkably similar to oneanother15–17. Thus, it is not a surprise that an adenine residue canbe accommodated in the catalytic pocket of M.HhaI, an enzymespecific for cytosine-C5 methylation. On the other hand, the for-mation of the pocket requires a massive conformational changeof the so-called ‘catalytic loop’1. The distance of 2.6 Å betweenOε2 of Glu 119 and N1 of adenine is a little more confined thanthe hydrogen bond between Glu 119 and N3 of cytosine toaccommodate the slightly bulkier adenine (Fig. 2a).

G:U mismatchThe flipped uracil of the G:U mismatch interacts with M.HhaI ina very similar way to the flipped cytosine. It stacks with Val 121,and forms hydrogen bonds through its O2 and N3-H atoms tothe side chains of Arg 165 and Glu 119 respectively (Fig. 3a). The

© 1998 Nature America Inc. • http://structbio.nature.com©

199

8 N

atu

re A

mer

ica

Inc.

• h

ttp

://s

tru

ctb

io.n

atu

re.c

om

letters

sugar ring adopts a C4'-exo pucker (pseudorotation angle of 63°)and a high-anti (-synclinal) glycosidic torsion angle of -81°.Interestingly, the O4 atom is located out of the plane of the uracilring, probably to release some electrostatic repulsion caused bythe lack of a proton with which a hydrogen bond can be formedwith the backbone carbonyl oxygen of Phe 79. The phenyl ring ofPhe 79 points away from the active-site pocket and is part of ahydrophobic core formed by many phenyl rings. Although theamino acids prior and after Phe 79 are a glycine and a proline(two conserved amino acids among DNA Mtases), the electrondensity in this region is well defined, indicating a relatively rigidregion that is essential for proper orientation of Cys 81. Thus, themain chain oxygen of Phe 79 cannot move the small distancenecessary to accommodate the uracil O4 atom. Except for thisrepulsive force, the uracil superimposes very well with the cyto-sine. The sulfur atom of Cys 81 and the C6 atom are 2.6 Å apart

nature structural biology • volume 5 number 10 • october 1998 873

(Fig. 3b), a distance that is close enough that the enzyme couldpossibly catalyze the methylation reaction18, in which the sulfuratom of Cys 81 covalently bonds to C6. We do not know if theout-of-plane bending of the uracil O4 atom is mechanistic,accounting for the low efficiencies of the methylation of uracilenzymatically8,9.

G:AP mismatchThe interaction of the flipped deoxyribose of the G:AP mismatchwith M.HhaI involves two sets of water molecules (Fig. 4). Atwo-water network connects the 5' phosphate, the O4* of theabasic deoxyribose, and the side chain of Arg 165. A directhydrogen bond is observed between O4* and the Arg 165 Nη1(NH2 group). The pseudorotation angle of the deoxyribose ringis 33°, indicative of a C3'-endo pucker. Although O4* has beensuggested to be a weak hydrogen bond acceptor19, such a hydro-

Fig. 1 a, Close up of the 5'-GCGC-3'/5'-GXGC-3' sequence, where X = A(top), U (middle), and AP (bottom). The difference electron density (Fo - Fc,αc), calculated using Phases37 and displayed using O38, is shown in green andcontoured at 5.0σ above the mean, where the mismatched nucleotide (ade-nine, uracil, or abasic site) was omitted in the structure factor (Fc, αc) calcu-lation. b, Ribbon39 diagram of the complex of M.HhaI (in black) bound to aDNA duplex containing an abasic site. The DNA is in magenta, with six phos-phates and sugar rings in green: three on the 5' side and three on the 3' sideof the abasic site. Arg 165 is also shown in black. c, Phosphodiester back-bone contacts between M.HhaI and the oligonucleotide containing an aba-sic site. The distance cut off is 3.0 Å for hydrogen bonds (solid lines) and 3.35Å for non-bonded contacts (dashed lines). The phosphate groups and watermolecules are labeled as ‘P’ and ‘W’ respectively. Mainly, interactionsinvolve six phosphates (white letter P on black background) of the dam-aged strand: three on the 5' side and three on the 3' side of the flipped site.The figure was modified from the output of NUCPLOT40. For specific basecontacts between M.HhaI and DNA, see Fig. 8 of ref 1.

a b

c

© 1998 Nature America Inc. • http://structbio.nature.com©

199

8 N

atu

re A

mer

ica

Inc.

• h

ttp

://s

tru

ctb

io.n

atu

re.c

om

letters

gen bond appears in each of the M.HhaI ternary structures con-taining either a normal substrate or its analog20. A more extensivewater network connects the 3' phosphate, the main chain car-bonyl oxygen of Phe 79, and side chains of Glu 119 and Asn 304.Interestingly, Arg 165 and Glu 119 form an ion pair(Arg 165:Nε . . . Oε2:Glu 119) that was present in the M.HhaIstructure without DNA21, but absent in all other M.HhaI–DNAcomplexes examined thus far. Clearly, the presence of a flipped-out base breaks the salt bridge and allows the side chain of Arg165 to undergo a conformational change, enabling both Glu 119and Arg 165 to interact with the base. This ion pair, observed inboth the uncomplexed structure21 and the complexed structurewith the abasic site, does not indicate a pre-formed active-sitepocket; as reasoned above the formation of the pocket requires aconformational change of the catalytic loop. In addition, as aresult of the absence of a flipped-out base and the side chainconformational change of Arg 165, multiple conformations areobserved for the side chains of Cys 81, Ser 87, Val 121, and Thr250; these amino acids participate in catalysis of the

874 nature structural biology • volume 5 number 10 • october 1998

cytosine/uracil ring (Cys 81), stabilizing the flipped base (Val 121and Cys 81; Figs 2b, 3b), and DNA backbone interactions (Ser 87and Thr 250; Fig. 1c).

G:G or G:T mismatchWe were unable to obtain crystals of complexes of M.HhaI andoligonucleotides containing either a G:T or a G:G mismatch,probably due to the short lifetime of these complexes (the off-rate of the G:T mismatch from M.HhaI was too rapid to be mea-sured by a gel shift assay8). When compared to adenine anduracil, guanine and thymine have an amino group at the 2-posi-tion and a methyl group at the 5-position respectively. A model-ing study (not shown) suggests that the extra group would causesteric collision between the flipped nucleotide and the proteinside chains in the active-site pocket, resulting in the rapid releaseof the substrate.

Relationship to DNA repair enzymesIn light of the fact that the non-substrate adenine and the poor

Fig. 2 M.HhaI–AdoHcy–G:A mis-match. a, Detailed structure in thevicinity of the mismatched adenine,displayed using MOLSCRIPT41.Atomic bonds are depicted as greensticks for DNA and white sticks forprotein. Nitrogen, oxygen, carbon,sulfur and phosphorus atoms areshown as cyan, red, white, yellowand magenta balls respectively.Specific interactions are displayed asdouble dashed lines; some arelabeled with the interatomic dis-tances in Å. The oxygen atoms ofwater molecules are labeled as H2O.The two hydrogen atoms at N6position of adenine were builtusing X-PLOR36. The flipped cyto-sine (in black sticks) of theM.HhaI–AdoHcy–G:C complex18 issuperimposed on the flipped adenine with the Cyt-N4 coincidingwith the Ade-N6. b, A view perpen-dicular to (a), looking edge-on tothe flipped adenine ring.

Fig. 3 M.HhaI–AdoHcy–G:U mis-match. a, Detailed structure inthe vicinity of the mismatcheduracil. Color scheme and label-ing are the same as in Fig. 2a.The hydrogen atom at the N3position of uracil was built usingX-PLOR36. The flipped cytosine(in black sticks) of theM.HhaI–AdoHcy–G:C complex18

is superimposed on the flippeduracil. b, A view perpendicularto (a), looking edge-on to theflipped uracil ring.

a b

a b

© 1998 Nature America Inc. • http://structbio.nature.com©

199

8 N

atu

re A

mer

ica

Inc.

• h

ttp

://s

tru

ctb

io.n

atu

re.c

om

letters

substrate uracil residue can be accommodated in the catalyticpocket, the DNA Mtases may be more related to repair enzymessuch as 3-methyladenine DNA glycosylase II22,23 and endonucle-ase III24,25, which have broad substrate specificities. On the otherhand, the methylation reaction is specific in that catalysis occursonly when the flipped base is cytosine or uracil (at low efficien-cy8,9), which more closely resembles uracil DNA glycosylase(UDG) in which the binding pocket for uracil is designed toexclude non-uracil bases in DNA and uracil in RNA. A flippeduracil has also been observed in the cocrystal structure of a dou-ble-mutant of human UDG with uracil-containing DNA4. Eventhough the catalytic efficiency of this double-mutant was severe-ly reduced, the N–C1* glycosidic bond of the flipped nucleotidehad been cleaved and free uracil was bound in the catalytic pock-et. More recently, a G:U mismatch was used in the cocrystalstructure of E. coli mismatch-specific uracil DNA glycosylase(MUG), where the flipped-out nucleotide shows only electrondensity for the deoxyribose, suggesting that base-excision hadtaken place and free uracil had diffused out of the enzyme5.

The interactions observed between M.HhaI and the flippedadenine are completely different from those observed in T4endonuclease V3, a cyclobutane pyrimidine dimer-specific DNAglycosylase/AP lyase. This enzyme moves the adenine oppositethe 5' pyrimidine of the dimer into a binding pocket on the sur-face of the enzyme. The flipped adenine is sandwiched betweentwo layers of atoms (most of which are polar side chains andwater molecules) arranged in parallel with the base plane.Vassylyev et al.3 suggested that the nature of these interactions(weak van der Waals forces) makes the pocket non-specific and,thus, any base can be tolerated. Recently this non-specificity hasbeen demonstrated by fluorescence spectroscopy analysis26.

Base flipping mechanism: active or passive?Two theories can explain the mechanism of base flipping — activeor passive. The former suggests that base flipping is an activeprocess in which the base is first pushed out of the helix by appro-priate amino acid(s) on the protein, and then pulled into theactive-site pocket of the enzyme where it remains trapped duringthe reaction. This ‘push and pull’ mechanism has been suggestedfor human UDG4. In the context of M.HhaI, the ‘push’ requiredcould be supplied by the side chain of Gln 237 which penetrates

nature structural biology • volume 5 number 10 • october 1998 875

into the DNA duplex and occupies the space left by the flippednucleotide. However, mutating Gln 237 to the any of the other 19amino acids does not affect sequence specificity but does greatlyreduce stability of the protein–DNA complex27. A three-step path-way13 has been proposed for the DNA Mtases, in which they firstrecognize the target base pair within the recognition sequence andincrease the interstrand phosphate–phosphate distance nearby.Afterward, they initiate base flipping by protein invasion of theDNA, and finally trap the flipped DNA structure. The local open-ing at the target base pair, which provides a path for the base torotate out of the helix, may be facilitated by already weakenedhydrogen bonds, such as those resulting from mismatched or dam-aged bases, or by the transient hydrogen bond breakage of ‘breath-ing’28 that occurs spontaneously in DNA molecules. However,mismatched bases should be partially paired and remain in thedouble helix29. As observed for DNA Mtases8–11, the tighter bindingto DNA containing mismatches at the target pairs may simply indi-cate a reduced need of binding energy required to flip out a base —that is, an easier flipping. In human UDG4, the local compressionof the intrastrand phosphate-phosphate distance flanking theflipped-out nucleotide may favor the extrahelical position in thecatalytic pocket.

The ‘passive’ theory suggests that during the normal breathingof DNA, the bases naturally spend some time in an extra-helicalor spontaneously flipped-out position, or series of such posi-tions, and it is this transient conformation in DNA that is recog-nized and caught by the protein (for example see ref. 30).Experimental support for the passive theory comes from NMRspectroscopy and molecular dynamics of a synthetic 11 base pairduplex DNA fragment containing an abasic site31: the deoxyribosering is in two configurations, within or out of the helix, while theadenine residue in the position opposite the abasic site is predomi-nantly stacked in the helix. Furthermore, the DNA structural dis-tortion induced by an intrahelical abasic site is minimal whereasthere is a considerable distortion in the DNA induced by the pres-ence of an extrahelical abasic site31.

Based on the crystal structure of the major human AP endonu-clease (HAP1), it was suggested that the extrahelical abasic ribosering serves as the specific recognition structure to interact withamino acid Phe 266 of HAP130. However, this hypothesis wasrecently refuted: site-specific DNA structural modifications and an

Fig. 4. M.HhaI–AdoHcy–G:APmismatch. a, Detailed struc-ture in the vicinity of themismatched abasic site.Color scheme and labelingare the same as in Fig. 2a.b, A view perpendicular to(a), looking edge-on to theflipped sugar ring.

a b

© 1998 Nature America Inc. • http://structbio.nature.com©

199

8 N

atu

re A

mer

ica

Inc.

• h

ttp

://s

tru

ctb

io.n

atu

re.c

om

letters

Phe 266 to Ala mutation of HAP1 indicate that the ring structure ofan abasic site is not a critical element in target recognition32. Thefact that human and E. coli AP endonucleases recognize a variety ofDNA structural conformations induced by different sequence con-texts and abasic site chemistries, but not a budged abasic site (thatextrudes from a 10 base pair helix and is highly flexible33), providescompelling evidence that these enzymes are not in search of a spe-cific extra-helical structural form32.

While our data do not prove the active theory, they do provideclear support for it and offer a new perspective on the mecha-nism. In the M.HhaI–AdoHcy–G:AP complex, the conformationin the region near the abasic site is much more B-like, compara-ble to the distortion induced by a spontaneously flipped-outabasic site in free DNA31. However, the backbone sugar andphosphodiester of the flipped-out abasic site in theM.HhaI–AdoHcy–G:AP complex is in the same conformation asin the flipped-out normal cytosine and mismatched adenine anduracil by M.HhaI. A recent NMR dynamics study of M.HhaIshows that the initial product of binding is a complex containingnormal B-DNA34. Only later does a conformational change takeplace that results in the flipped base. However, we do not knowwhether the DNA repair enzymes such as human UDG and DNAMtases use the same mechanistic pathways for base flipping.

876 nature structural biology • volume 5 number 10 • october 1998

M.HhaI–DNA backbone interactionsFinally, it appears that the base per se is not the target for thestructural change in the DNA. Since both uracil and adeninemismatches are also flipped, there is clearly nothing specialabout the cytosine, or any of the bases8–11, that is required forflipping. Thus, we conclude that it is the backbone that is tar-geted for rotation by the enzyme and the base is merely carriedalong with it. The recent structure for E. coli MUG5 also has thebackbone of the AP site in a flipped conformation, although itcould be argued that this merely reflects the end product of thereaction. Like M.HhaI, T4 endonuclease V can flip bases otherthan its normal substrate adenine26 (also cited as publisheddata of M. Maeda in ref. 3), again suggesting that the base is notimportant for the damage recognition, but rather facilitatescatalysis. Similar results have been shown for the human and E.coli AP endonucleases, E. coli exonuclease III and endonucleaseIV where the base opposite an abasic site is not essential for theinitial recognition32.

If backbone rotation is the key to base flipping, then it sug-gests that amino acids in the enzyme that interact with theDNA backbone will be good targets for site-specific mutagene-sis to probe their functions (Fig. 1c). For instance, mutations ofArg 165, which interacts with O4* of the deoxyribose, may lead

Table 1 Structure determination

Statistics of X-ray data collectionMismatch G:A G:U G:X (X = AP)DNA sequence TGTCAGCGCATGG TGTCAGCGCATGG TGTCAGXGCATGG

CAGTCGAGTACCT CAGTCGUGTACCT CAGTCGCGTACCTDiffraction limit (Å) 23.0–2.87 23.1–2.76 23.0–2.39Observations 55,718 42,797 61,880<I/σ(I)> 13.3 13.2 16.8Unique Reflections 13,031 13,839 22,634Completeness (%) 90.0 85.2 91.6I/σ(I) cutoff 2 1 0Rlinear

1 0.066 0.095 0.043Non-hydrogen atomsProtein 2,606 2,606 2,606DNA 494 492 484AdoHcy 26 26 26Water 139 114 243

Refinement statisticsG:A G:U G:AP

Resolution Reflections Rfactor2 Resolution Reflections Rfactor Resolution Reflection Rfactor

(Å) (complete) (Å) (complete) (Å) (complete)23.0–5.71 1,896 (97) 0.200 23.1–5.50 1,947 (89) 0.186 6.00–4.23 2,978 (99) 0.1505.71–4.55 1,844 (98) 0.132 5.50–4.37 1,905 (91) 0.133 4.23–3.58 2,942 (99) 0.1514.55–3.98 1,794 (97) 0.124 4.37–3.82 1,884 (91) 0.140 3.58–3.21 2,906 (99) 0.1883.98–3.61 1,761 (97) 0.135 3.82–3.48 1,871 (90) 0.147 3.21–2.95 2,904 (99) 0.2083.61–3.36 1,745 (96) 0.150 3.48–3.23 1,761 (88) 0.186 2.95–2.76 2,859 (98) 0.2203.36–3.16 1,635 (91) 0.174 3.23–3.04 1,693 (83) 0.218 2.76–2.61 2,738 (94) 0.2353.16–3.00 1,397 (77) 0.182 3.04–2.89 1,540 (76) 0.239 2.61–2.49 2,108 (72) 0.2413.00–2.87 985 (55) 0.199 2.89–2.76 1,238 (61) 0.266 2.49–2.39 1,544 (53) 0.240

23.0–2.87 13,027 (90) 0.156 23.1–2.76 13,839 (85) 0.174 6.00–2.39 20,979 (90) 0.186

1Rlinear = Σ I - <I>/Σ <I>, where I is the measured intensity of each reflection, <I> is the intensity averaged from multiple observations of symmetryrelated reflections, and σ is the r.m.s. deviation from the mean.2Rfactor = Σ Fo - Fc /Σ Fo, where Fo and Fc are the observed and calculated structure factor amplitudes respectively.

© 1998 Nature America Inc. • http://structbio.nature.com©

199

8 N

atu

re A

mer

ica

Inc.

• h

ttp

://s

tru

ctb

io.n

atu

re.c

om

letters

to mutants that retain the ability to form an initial bindingcomplex but may be impaired in flipping. The construction ofsuch mutants is currently underway.

MethodsThe oligonucleotides were from New England Biolabs. The abasicsite was introduced using a dSpacer CE phosphoramidite fromGlen Research. It introduces a furan ring joined through the nor-mal 5'-3' phosphodiesters. It should be noted that the abasic sitedoes not contain the O1* atom, which would be present if theabasic site had arisen through a glycolytic cleavage from a siteoriginally carrying a base. The ternary complexes were crystallizedusing either ammonium sulfate1,18,20 (G:A and G:U) or polyethyleneglycol-800014 (G:AP) as the precipitating agents. The X-ray diffrac-tion intensities, integrated and scaled by HKL and Scalepack35,were measured on a MarResearch imaging-plate diffractometer atbeamline X12-C in the National Synchrotron Light Source ofBrookhaven National Laboratory. Bulk solvent correction was car-ried out using X-PLOR36, in the resolution range of 23–6.5 Å (1297and 1177 reflections for the G:A and G:U complexes respectively).During refinement, the flipped uracil and adenine bases were notsubjected to any planar or angular constraints. However, as a con-trol, an additional round of refinement was applied using the pla-nar constraints which resulted in the same out-of-plane O4 atomin the flipped-out uracil.

Coordinates. PDB accession numbers 7mht, 8mht, and 9mht havebeen assigned to the G:A, G:U, and G:AP complexes respectively.

AcknowledgmentsWe thank K. McCloy for protein purification and crystallization, and S. Kumarand X. Zhang for discussion and comments on the manuscript. M.O’G. wassupported in part by a National Institutes of Health fellowship (GM17052). Workwas supported in part by National Institutes of Health Grants to R.J.R. and X.C.

Margaret O’Gara1, John R. Horton2, Richard J. Roberts3

and Xiaodong Cheng2

1Present address: Pfizer Central Research, Discovery Biology, Ramsgate Road,Sandwich, Kent CT13 3NJ England. 2Department of Biochemistry, EmoryUniversity School of Medicine, 1510 Clifton Road, Atlanta, Georgia 30322, USA.3New England Biolabs, 32 Tozer Road, Beverly, Massachusetts 01915, USA.

nature structural biology • volume 5 number 10 • october 1998 877

Correspondence should be addressed to X.C. email: xcheng@emory.edu

Received 14 April, 1998; accepted 30 July, 1998.

1. Klimaπauskas, S., Kumar, S., Roberts, R.J. & Cheng. X. Cell 76, 357–369 (1994).2. Reinisch, K.M., Chen. L., Verdine, G.L. & Lipscomb, W.N. Cell 82, 143–153 (1995).3. Vassylyev, D.G. et al. Cell 83, 773–82 (1995).4. Slupphaug, G., Mol, C.D., Kavli, B., Arvai, A.S., Krokan, H.E. & Tainer J.A. Nature

384, 87–92 (1996).5. Barrett, T.E., Savva, R., Panayotou, G., Barlow, T., Brown, T., Jiricny, J. & Pearl, L.H.

Cell 92, 117–129 (1998).6. Roberts, R.J. & Cheng, X. Annu. Rev. Biochem. 67, 181–198 (1998).7. Smith, S.S. Prog. Nucleic Acid Res. Mol. Biol. 49, 65–111 (1994).8. Yang, A.S., Shen, J.-C., Zingg, J.-M., Mi, S. & Jones, P.A. Nucleic Acids Res.

23,1380–1387 (1995).9. Klimasauskas, S. & Roberts, R.J. Nucleic Acids Res. 23,1388–1395 (1995).

10. Cal, S. & Connolly, B.A. J. Biol. Chem. 272, 490–496 (1997).11. Allan, B.W., Beechem, J.M., Lindstrom, W.M. & Reich, N.O. J. Biol. Chem. 273,

2368–2373 (1998).12. Verdine, G.L. Cell 76, 197–200 (1994).13. Cheng, X. & Blumenthal, R.M. Structure 4, 639–645 (1996).14. O’Gara, M., Roberts, R.J. & Cheng, X. J. Mol. Biol. 263, 597–606 (1996).15. Schluckebier, G., O’Gara, M., Saenger, W. & Cheng, X. J. Mol. Biol. 247, 16–20 (1995).16. Malone, T., Blumenthal, R.M. & Cheng, X. J. Mol. Biol. 253, 618–632 (1995).17. Gong, W., O’Gara, M., Blumenthal, R.M. & Cheng, X. Nucleic Acids Res. 25,

2702–2715 (1997).18. O’Gara, M., Klimasauskas, S., Roberts, R.J. & Cheng, X. J. Mol. Biol. 261, 634–645 (1996).19. Saenger, W. Principles of nucleic acid structure. (Springer-Verlag, New York; 1984).20. Kumar, S. et al. Nucleic Acids Res. 25, 2773–2783 (1997).21. Cheng, X., Kumar, S., Klimaπauskas, S. & Roberts, R.J. Cell 74, 299–307 (1993).22. Yamagata, Y. et al. Cell 86, 311–319 (1996).23. Labahn, J. et al. Cell 86, 321–329 (1996).24. Kuo, C.F. et al. Science 258, 434–440 (1992).25. Thayer, M.M., Ahern, H., Xing, D., Cunningham, R.P. & Tainer, J.A. EMBO J. 14,

4108–4120 (1995).26. McCullough, A.K., Dodson, M.L., Scharer, O.D. & Lloyd, R.S. J. Biol. Chem. 272,

27210–27217 (1997).27. Mi, S., Alonso, D. & Roberts, R.J. Nucleic Acids Res. 23, 620–627 (1995).28. Chen, Y.Z. & Prohofsky, E.W. Biopolymers 35, 573–582 (1995).29. Bhattacharyya, A. & Lilley, D.M.J. J. Mol. Biol. 209, 583–597 (1989).30. Gorman, M.A. et al. EMBO J. 16, 6548–6558 (1997).31. Goljer, I., Kumar, S. & Bolton, P.H. J. Biol. Chem. 270, 22980–22987 (1995).32. Erzberger, J.P., Barsky, D., Scharer, O.D., Colvin, M.E. & Wilson, III, D.M. Nucleic

Acids Res. 26, 2771–2778 (1998).33. Lin, Z., Hung, K.-N., Grollman, A.P. & de los Santos, C. Nucleic Acids Res. 26,

2385–2391 (1998).34. Klimasauskas, S., Szyperski, T., Serva, S. & Wuthrich, K. EMBO J. 17, 317–324 (1998).35. Otwinowski, Z. & Minor, W. Meth. Enz. 276, 307–326 (1997).36. Brünger, A. T. X-PLOR, version 3.1: A system for X-ray crystallography and NMR.

(Yale University Press, New Haven, Connecticut; 1992).37. Furey, W. & Swaminathan, S. Meth. Enz. 277, 590–620 (1997).38. Jones, T.A., Zou, J.Y., Cowan, S.W. & Kjeldgaard, M. Acta Crystallogr. A47,

110–119 (1991).39. Carson, M. J. Appl. Crystallogr. 24, 958–961 (1991).40. Luscombe, N.M., Laskowski, R.A. & Thornton, J.M. Nucleic Acids Res. 25,

4940–4945 (1997).41. Kraulis, P.J. J. Appl. Crystallogr. 24, 946–950 (1991).

DNA bending determinesFos–Jun heterodimerorientation

Heterodimeric transcription factors can bind to palindromicrecognition elements in two opposite orientations with poten-tially distinct effects on transcriptional activity. We havedetermined the orientation of Fos–Jun binding at differentAP-1 sites using a novel gel-based fluorescence resonanceenergy transfer assay. The orientation preference of het-erodimer binding varied over a greater than 10-fold range.Single base pair substitutions that alter bending of flankingsequences reversed the orientation of heterodimer binding.Single amino acid substitutions that reduce the difference inDNA bending between Fos and Jun also reduced the orienta-tion preference. Consequently, indirect read-out mediated bydifferences in DNA structure can contribute to the structuralorganization of nucleoprotein complexes.

Many eukaryotic transcription factors form heterodimersbetween subunits with related DNA binding domains. The oppo-site sides of such heterodimeric complexes are likely to differ intheir abilities to interact with other proteins bound to the promot-er region. Thus, opposite orientations of heterodimer binding mayresult in different transcriptional activities.

Heterodimers formed between the Fos and Jun families of basicregion-leucine zipper (bZIP) proteins bind to AP-1 sites with aconsensus TGACTCA core recognition sequence1,2. X-ray crystal-lographic analysis of the bZIP domains of Fos and Jun bound to anAP-1 site indicated that Fos–Jun heterodimers could bind to theAP-1 site in both orientations3. Studies of DNA bending by Fos andJun indicated that heterodimers exhibited orientation preference inbinding to AP-1 sites, and that they could bind to different AP-1sites in opposite orientations4,5. Here we have examined the orien-tation of binding by heterodimers formed by the bZIP domains ofFos and Jun at several AP-1 sites using a novel fluorescence reso-nance energy transfer (FRET) assay.

FRET analysis of heterodimer orientation.To determine the orientation of Fos–Jun heterodimer binding,

© 1998 Nature America Inc. • http://structbio.nature.com©

199

8 N

atu

re A

mer

ica

Inc.

• h

ttp

://s

tru

ctb

io.n

atu

re.c

om