Plasmid transcriptional repressor CopG oligomerises to render helical superstructures unbound and in...

doi:10.1006/jmbi.2001.4760 available online at http://www.idealibrary.com on J. Mol. Biol. (2001) 310, 403±417

Plasmid Transcriptional Repressor CopGOligomerises to Render Helical SuperstructuresUnbound and in Complexes with Oligonucleotides

M. Costa1, M. SolaÁ 1, G. del Solar2, R. Eritja1, A. M. HernaÂndez-Arriaga2

M. Espinosa2, F. X. Gomis-RuÈ th1* and M. Coll1*

1Institut de Biologia Molecularde Barcelona, CID-CSICJordi Girona, 18-2608034, Barcelona, Spain2Centro de InvestigacionesBioloÂgicas, CSIC, VelaÂzquez144, 28006 Madrid, Spain

E-mail addresses of the [email protected]; [email protected]

Abbreviations used: CopG, homotranscriptional repressor; Pcr, copG-rimperfect repeat; ds, double-strandehelix; RHH, ribbon-helix-helix; a.u.,

0022-2836/01/020403±15 $35.00/0

CopG is a 45 amino acid residue transcriptional repressor involved in thecopy number control of the streptococcal plasmid pMV158. To do so, itbinds to a DNA operator that contains a 13 bp pseudosymmetric DNAelement. Binding of CopG to its operator results in repression, at thetranscriptional level, of its own synthesis and that of the initiator of repli-cation protein, RepB. Biochemical experiments have shown that CopGco-operatively associates to its target DNA at low protein:DNA ratios,completely protecting four helical turns on the same face of the doublehelix in both directions from the inverted repeat that constitutes theCopG primary target. This has been correlated with a CopG-mediatedDNA bend of about 100 �. Here, we show that binding of CopG to DNAfragments containing the inverted repeat just at one end led to nucleationof the protein initiating from the inverted repeat. Nucleation extended tothe entire fragment, with CopG-DNA contacts occurring on the sameface of the DNA helix. The protein, the prototype for a family of homolo-gous plasmid repressors, displays a homodimeric ribbon-helix-helixarrangement. It polymerises within the unbound crystal to render a con-tinuous right-handed protein superhelix of homodimers, around which abound double-stranded (ds) DNA could wrap. We have solved the crys-tal structure of CopG in complex with a 22 bp dsDNA oligonucleotideencompassing the cognate pseudosymmetric element. In the crystal, oneprotein tetramer binds at one face of the DNA with two parallelb-ribbons inserted into the major groove. The DNA is bent about 50 �under compression of both major and minor grooves. A continuousright-handed complex helix made up mainly by protein-protein andsome protein-DNA interactions is observed. The protein-protein inter-actions involve regions similar to those observed in the oligomerisationof the native crystals and those employed to set up the functional tetra-mer. A previously solved complex structure of the protein with a 19 bpdsDNA had unveiled a left-handed helical superstructure just made upby DNA interactions.

# 2001 Academic Press

Keywords: CopG; plasmid; protein-DNA complex; transcriptionalrepressor; X-ray crystal structure
*Corresponding authors
ding authors:ic.es

dimeric 45 residueepB promotor; IR,d; HTH, helix-turn-asymmetric unit.

Introduction

Plasmids are extrachromosomal DNA elementsthat exhibit controlled autonomous replicationinside a prokaryotic or eukaryotic cell. In manycases, they are transmissible to other cells by intraand inter-speci®c horizontal transfer, as observedduring conjugation. They may provide the hostwith advantageous functions, such as drug-resist-ance or metabolic pathways useful under adverse

# 2001 Academic Press

404 Superhelical Oligomerisation of CopG Repressor

environmental conditions,1 but are also associatedwith acceptor menaces, such as tumour inductionin plant cells.2 To co-exist stably with their hosts,plasmids must be kept at a constant copy num-ber.3 ± 5 Therefore, plasmid replication must beregulated following ®ne-tuning mechanisms. Thisevent is controlled in multicopy plasmids thatreplicate by the rolling circle mechanism throughthe availability of a plasmid-encoded nickingenzyme (Rep), responsible for introducing a siteand strand-speci®c nick at the origin of replicationof the leading strand, termed dso, in this way initi-ating DNA replication.

One plasmid that has been studied extensively isthe streptococcal 5536 bp pMV158 broad-hostrange replicon. This plasmid regulates the avail-ability of the 24.5 kDa RepB initiator protein bymeans of the products of two genes, rnaII andcopG. The former encodes a 50 nt antisense RNA,capable of pairing with the initiation of translationregion of the rep mRNA and therefore trans-actingat the post-transcriptional level.6,7 The copg genecodes the transcriptional repressor CopG, a homo-dimeric 45 residue protein constituting the smallestnatural transcriptional repressor biochemicallycharacterised to date and the prototype for theCop-family of homologous bacterial plasmidrepressors.6,8 ± 11 By binding to the single copG-repBpromoter (Pcr) region around a 13 bp imperfectrepeat (IR) element, CopG represses its own syn-thesis and that of RepB.9 The DNA around the tar-get of CopG includes several A-tracts spaced 10 bpupstream of the IR, resulting in an intrinsic curva-ture that is enhanced by the addition of the CopGprotein.12 Measurement of the CopG-generatedbend gives an angle of about 100 � (G.d.S., unpub-lished results). At low protein:DNA ratios,hydroxyl radical and DNaseI footprint experimentsunveiled a co-operative protection of the DNA,with the protein covering the major groove of thedouble helix along a region that comprises foursuccessive DNA turns, two on each side of the IRcentre.9 This phased CopG-mediated protectionincludes the ÿ35 box of the promoter, the overlap-ping IR and the ÿ10 region, thus hindering thebinding of RNA polymerase to the Pcr promoter.8,12

This protein oligomerisation is apparently facili-tated by its binding to DNA, as the free proteinremains dimeric in solution up to a concentrationof 780 mM.13

We recently reported the structure of the nativeunbound protein and of a complex with a 19 bpdouble-stranded (ds) DNA fragment encompassingthe 13 bp IR. In these structures, a right and a left-handed helical superstructure, respectively, wereobserved within the crystal14 made up by protein-protein (unbound structure) and DNA-DNA inter-actions (complex). In the present work, we havecontinued the structural studies of this transcrip-tional repressor with a complex with a 22 bpdsDNA fragment containing the primary recog-nition sequence. An oligomerisation behaviour toproduce a right-handed superhelix is observed in

the crystal, in this case made up mainly by pro-tein-protein interactions. In addition, hydroxyl rad-ical footprinting experiments on a DNA fragmentending just at the IR showed a regular pattern ofprotected regions that extended through the entirefragment. The protection pattern exhibited a regu-lar spacing of 5-6 bp, indicative of the generationof a highly ordered multiprotein-DNA complex,nucleating from the 13 bp cognate region. A modelof the possible CopG-DNA interactions in vitro isdiscussed.

Results and Discussion

Protein structure

The polypeptide chain starts with a segment inextended conformation (strand bI, see Figure 1(a))reaching Leu9, followed by helix aA (Glu11-Met24), with its axis rotated � 70 � away from thestrand direction.14 At this point, a glycine-mediatedturn leads to helix aB, reaching from Lys28 to theC-terminal end. Both helices form an angle of�90 �, owing to the presence of Gly25. This biheli-cal topology corresponds to a helix-turn-helix(HTH) motif already described for proteinsinvolved in DNA major groove recognition.15 ± 17

Taken together with strand bI, however, this struc-tural motif is enlarged to generate a ribbon-helix-helix (RHH) motif, as found in repressors of theMetJ/Arc superfamily, namely both 53 residue Arcand 82 residue Mnt repressors of Salmonella typhi-murium bacteriophage �22, Escherichia coli 104 resi-due methionine repressor protein MetJ, and TraYand TrwA proteins of conjugative plasmids.14,18 ± 25

In agreement with hydroxyl radical footprintingexperiments, two CopG protomers (molecules Aand B) associate intimately in a symmetrical man-ner via a local 2-fold axis (Figure 1(b)) forming atwo-stranded antiparallel b-sheet or b-ribbon, pre-sented to the surface of the dimer and responsiblefor the main interaction with target DNA (seebelow).

Interactions of CopG with double-strandedoligonucleotides

The presently solved structure of CopG in com-plex with a 22 bp dsDNA (provided with A-Toverhangs) containing the 13 bp pseudosymmetricelement (see Figure 2(a)) unveils that the minimalfunctional oligomerisation state is a dimer ofhomodimers having the same shape and inter-protomer interactions as found in the nativeunbound structure (Figure 2(b)). Two and a halfcomplexes are present in the asymmetric unit(Figure 2(c); see Materials and Methods for num-bering). The chain tracing and the interactionsobserved for each complex are very similar (seeMaterials and Methods), so just the complex madeup by protein chains D, E, F, and G with DNAstrands W and X is discussed. The latter is com-posed of two dimers related by a local 2-fold axis

Figure 1. (a) CopG monomer as a ribbon cartoon superimposed with the Connolly dot surface of the proteinatoms. The three structural elements of the RHH motif, strand bI, helix aA, and helix aB are labelled. (b) Ribbon plotof the CopG dimer. The DNA-contacting b-ribbon is presented to the exterior. (c) A representation of the arrangementof CopG molecules in the unbound crystal structure. The structurally equivalent dimers are either of type AB or CC.Symmetry-related molecules are denoted by apostrophes. Crystallographic 2-fold axes are displayed with ellipses attheir ends, local dyads as continuous (molecule A to molecule B transformation) and broken (molecule B to moleculeC transition) lines. The functional tetramer as found in the protein-DNA complexes is A0B0A00B00. Axial (d) and lateral(e) stereo views of the continuous superhelix of CopG molecules in the unbound crystal, with the distances (30-35 AÊ )between vicinal surface-exposed b-ribbons indicated. The helical pitch is � 40 AÊ and the diameter 85 AÊ .

Superhelical Oligomerisation of CopG Repressor 405

interacting in the same manner as two AB dimersdo in the unliganded structure (based on crystallo-graphic contacts; see Figure 1(c)). No signi®cant

deviations are observed in the main chains.Although each CopG dimer has local 2-fold sym-metry, the two b-strands contact different bases,

Figure 2. (a) Sequence of the double-stranded deoxyribooligonucleotide encompassing the 13 bp cognate IRsequence employed for co-crystallisation in the present work. The black box signi®es the centre of the operator.(b) Stereo ribbon plot of the CopG dimer of dimers (in brown/blue and magenta/blue) in complex with the 22 bpdsDNA superimposed with its semitransparent Connolly surface. (c) Schematic organisation of the asymmetric unitcontents (bright colours) transformed into their symmetry-equivalents via a crystallographic dyad (light colours).


not matching the (pseudo)2-fold symmetry (50-TGCA-30) of the DNA sequence. The protein-DNAinteraction induces a bend of 50 � in the nucleicacid moiety due to the marked compression ofboth minor and major grooves facing the protein.The DNA backbone follows a smooth path in gen-eral, except for the zone close to the centre of theoperator, where the minor groove is compressedand the base-pairs are somewhat inclined.

The recognition of the bases by CopG dimer DEon the left side of the operator (according toFigures 2(a) and 3(a)) is characterised by the fol-lowing contacts: Arg4D NZ-Thy216W O4, Arg4DNZ1-Gua206X O6, Arg4D NZ1-Gua217W O6, Thr6DOg1-Thy205X O4, Arg4E NZ1-Ade203X N7, Arg4ENZ2-Thy204X O4, Leu5E Cd1-Thy216W C30, Thr8ECg2-Thy216W C5A. Accordingly, only residuespointing outwards of the protein dimer b-ribbonare involved. On the right side in relation to thecentral G-C base-pair (Figures 2(a) and 3(b)), pro-tein (CopG dimer FG)-base interactions are Arg4FNZ2-Gua208W N7, Arg4F NZ1-Gua215X O6, Arg4FNZ1-Cyt209W N4, Arg4F NZ1-Thy214X O4, Thr6FOg1-Thy207W O4, Arg4G NZ1-Gua206W N7,Arg4G NZ2-Ade217X N6, Thr6G Og1-Cyt216X N4,and Thr6G Cg2-Thy214X C5A.

Further contacts occur between the protein partand the DNA backbone, Thr8D Og1-Ade203X O2P,Lys28D N-Thy216W O1P, Ser29D Og-Thy216WO2P, Lys2E Nz-Gua202X O1P, Thr6E N-Gua217WO2P, Thr8E Og1-Ade215W O2P, Ser27E Og-Ade203O1P, Lys28E Nz-Thy204X O1P, and Ser29E Og-Ade203X O1P (left side), and Thr8F Og1-Cyt205WO2P, Ser27F Og-Gua213 O1P, Lys28F N-Thy214O2P, Ser29F N-Thy214 O2P, Lys2G Nz-Cyt204WO1P, Thr6G N-Gua215X O2P, Thr8G Og1-Gua213X

O1P, Lys28G Nz-Gua206W O1P, Lys28G Nz-Cyt205 O30, and Ser29G Og1-Cyt205W O1P (rightside). These interplays are not directly dependenton the DNA sequence, but involve DNA phos-phate group oxygen atoms and protein residuescoming from the N-terminal strand, turn aAaB,and the beginning of aB of each protein monomer.This results in N-capping of helices aB of each ofthe four CopG monomers D, E, F, and G by phos-phate oxygen atoms belonging to nucleotidesThy216W, Ade203X, Thy214X, and Cyt205W,respectively. This kind of interaction has beenobserved in Arc and MetJ,19,20 and in other other-wise unrelated DNA-binding proteins, such as cat-abolite-repressor protein CAP.26

Accordingly, the recognition of both sides inrelation to the centre of the operator is asymmetric,with more protein-base contacts on the right side(nine) than on the left side (eight). This asymmetryhad been observed in the Arc-operator complex19

and in the structure of the complex of CopG with a19 bp dsDNA.14 All in all, despite the fact that theprogram used for calculating the DNA curvaturein the complex (see Materials and Methods) gave avalue of � 60 � for the latter and of �50 � for thepresently described complex, the structures ofthese complexes are very similar and practicallysuperimposable in the common parts (the rms�value for the common 169 Ca atoms is 0.58 AÊ ).Signi®cant deviations occur only in both ends ofthe oligonucleotide, in one case involved in inter-actions with vicinal (symmetry equivalent) mol-ecules to form a continuous dsDNA ®lament in thecrystal (CopG/19 bp dsDNA), in the other caseleading to electrostatic repulsion with neighbour-ing DNA ends (CopG/22 bp dsDNA; see below).

Figure 3. Stereo stick representation displaying the detailed interaction of CopG with the left-hand side (a) andthe right-hand side (b) of the 22 bp dsDNA (green backbone, red bases) with respect to the centre of the operator(bp Gua211X-Cyt213W). Only the interacting zones of the protein part (brown main chain and colour-codedside-chains, with blue positively charged side-chains and negative ones in red) are displayed for clarity.


Oligomerisation in the unboundcrystal structure

In the asymmetric unit of the unbound crystals,a third CopG molecule (termed C) is observedbesides the A and B molecules establishing adimer.14 This molecule forms a further dimer oftype molecule C-molecule C via a crystallographic

dyad with interactions as found in the AB dimer(see Figure 1(c)-(e)). Accordingly, there is for-mation of dimers either via a local or a crystallo-graphic axis in this structure. The dimers AB andCC interact with a buried surface of 528 AÊ 2 and 56contacts below 4 AÊ . These include 14 van derWaals interactions and six hydrogen bonds, mainlythrough turn aAaB and helix aB of molecule B


with equivalent parts of a molecule C, with bothhelices running almost parallel. In addition, the ABdimer interplays with a crystallographically relatedvicinal AB dimer with a common surface of 526 AÊ 2

and 49 contacts below 4 AÊ (see Figure 1(c)). Theinteraction observed at this interface is similar to,but not identical with that observed for the AB-CCdimer-dimer interface. Mainly hydrophobic con-tacts between the side-chains of Met24, Val13,Leu26, Val34 and Ala30, and two hydrogen bondsare established. These contacts generate in the crys-tal a right-handed helical superstructure ofapproximately 85 AÊ diameter and 40 AÊ pitchencompassing 12 CopG molecules per turn, withthe DNA-binding b-ribbons pointing outwards andspaced 30-35 AÊ (see Figure 1(c)-(e)).

Oligomerisation in the complexcrystal structures

As described, two and a half complexes are pre-sent in the asymmetric unit in the structure ofCopG bound to the 22 bp dsDNA (see Materialsand Methods and Figure 2(c)). The complex madeup by protein chains D, E, F and G with DNAstrands W and X (DEFG_WX) interacts on one sidewith ABA0B0_YZ under burial of 707 AÊ 2 via 18 con-tacts of less than 4 AÊ including nine hydrogen

Figure 4. Stereo picture displaying the zones involved innative structure and the complexes with DNA to render theof one dimer. The interdimer regions are shown for dimer Aguishable from those of the active tetramer upon complex fowith CC0 of the native structure (cyan), dimer DE of DEFGAB of ABA0B0_YZ (green) and dimer FG of DEFG_WX with

bonds and one van der Waals interaction (Leu26B-Ley26E). The regions involved are mainly the endof helix aA, the glycine-mediated turn aAaB, andthe beginning of helix aB of both molecules B andE (Figure 4). Additionally, eight intercomplex inter-actions between protein side-chains of chargedresidues and nucleotide atoms are observed. Onthe other side (Figures 2(c) and 4), DEFG_WXapproaches HJKL_UV with a common surface of1111 AÊ under establishment of 16 contacts, in par-ticular ®ve hydrogen bonds, three van der Waalsinteractions (including Leu26G-Leu26 J) and onesalt-bridge. Further eight charged protein-DNAintercomplex interactions are present. Anunfavourable approach between the terminal back-bones of DNA strands X and V (Thy222X andThy221V) leads the ®nal bases at this complex jointto be disordered (see Materials and Methods). Thedifference in the interaction surfaces of DEFG_WXon either side is due to the DNA molecules that inone case interact with each other and in the otherdo not. The regions mainly involved in getting intouch with the neighbour are the same as thoseobserved in the DEFG_WX/ABA0B0_YZ interface(in this case belonging to molecules G and J; seeFigure 2(c)), but slightly out of phase (see Figure 4),probably due to the repulsion of the terminal DNAregions mentioned above.

the different dimer-dimer interactions observed in theactive tetramer or to oligomerise, after superimpositionB with A0B0 from the native structure (yellow; indistin-

rmation with both 19 bp and 22 bp dsDNAs), dimer AB_WX (complex with 22 bp dsDNA) interacts with dimerdimer HJ of HJKL_UV (magenta).


These complexes interact with each other andwith symmetry-equivalent complexes mainly viathe protein tetramers to render a continuous right-handed superhelix within the crystal of 105 AÊ

diameter and 235 AÊ pitch (see Figures 2(c) and5(b)). Although slightly different in orientation,nature and intimacy (much fewer contacts, seeabove), the regions involved in protein-proteininteraction with neighbour complexes are verysimilar to those observed in the dimer-dimer inter-actions that produce functional tetramers withineach complex and in the unbound structure (seeabove). The (arbitrarily) chosen length of the oligo-nucleotide means that, although an oligomerisationbased on protein contacts happens within the crys-tal, the repulsion between terminal phosphategroups and the slight dsDNA shift in the planeperpendicular to the superhelical axis does not per-mit the oligomerisation of the oligonucleotide tooccur.

The previously reported structure of CopG incomplex with a 19 bp dsDNA14 revealed that thecrystal symmetry interplays and those betweencomplexes are made exclusively by the DNA partprovided with G and C overhangs, forming aleft-handed continuous dsDNA. An extendedleft-handed superhelical twist due to the DNA-bending ability of the protein tetramer upon bind-ing was observed. In this case, the length of thedsDNA used for crystallisation places the centreof the IR at different faces of the DNA helix,thus preventing any additional protein-protein (i.e.intertetramer) contacts similar to those seen in the

Figure 5. Superhelical oligomerisation of CopG in its comperal stereo ribbon plots for each superhelix are displayed, soous left-handed helix is based on dsDNA (superimposed witno protein-protein contacts, whereas mainly protein-protein cwith the semitransparent Connolly surface of the protein par

unliganded or the presently described complexcrystal structure (Figure 5(a)). However, such inter-actions may occur in the case of other oligonucleo-tides or when dealing with (continuous) plasmidDNA containing the entire CopG target. In thiscase, we have preliminary evidence indicating thatthe DNA binds four dimers of the protein in ahighly co-operative way that would involveprotein-protein interactions (G.d.S., unpublishedresults).

CopG-oligomerisation on linear plasmid DNA

DNaseI hypersensitive sites are indicative of theDNA phosphodiester backbone being highlyexposed to solvent or of a minor groove that iswider than normal, as seen at the exterior face of abent DNA.27 Hydroxyl radical and DNaseI foot-prints generated by CopG on plasmid linear frag-ments containing the IR in a centred position (atthe molar protein:DNA ratio of 20:1), showed var-ious protected regions on the same face of thehelix, spanning ®ve successive helix turns.8 Thecontacted regions are distributed symmetrically onboth strands around the 2-fold axis of the IR(Figure 6(a)). At high CopG:DNA ratios (400:1),binding of the protein progresses in both direc-tions, but it shows some preference for binding toregions upstream of the IR (G.d.S., unpublishedresults). However, when the DNA fragmentemployed harboured the IR at one of the ends(Figure 6(c)), the CopG-generated hydroxyl radicalfootprints had a different pattern. In this case, even

lexes with (a) 19 bp dsDNA and (b) 22 bp dsDNA. Lat-as the helical pitches and diameters. In (a) the continu-

h its semitransparent Connolly surface) interactions withontacts result in a right-handed superhelix in (b), shownt.

Figure 6 (legend opposite)


at relatively low protein concentrations (molarratio of 20:1), the appearance of a regular patternof CopG-DNA contacts, distributed every helixturn, was evident (Figure 6(b) and (d)). Successive

4-5 nt protected regions were separated by 6-7 ntunprotected regions, so that the length of therepeated unit was 11 bp (Figure 6(d)). Protectionson the top (coding) and bottom (non-coding)


strands were offset 3 nt (Figure 6(b)), indicatingthat the contacts on both strands are established onthe same DNA face. A regular protection patternwas also observed when the footprinting exper-iments were performed with DNaseI (not shown).Since a DNA fragment lacking the IR does notshow any nucleation pattern at a CopG:DNAmolar ratio of 20:1 (not shown), the contacts in theIR-ended fragment should initiate at the IR andprogress through the entire DNA fragment.Initiation of nucleation from the IR can be deducedfrom comparison of the protection patternsobtained in the presence of increasing concen-trations of CopG; as the protein:DNA ratiosincreased, the footprints reached regions more andmore distant from the IR (Figure 6(d)). Protectionsin the vicinity of the IR correspond, approximately,to footprints a, b, d, a0, c0, and e0, observed whenusing a DNA fragment containing the IR centredin the wild-type entire operator (compareFigure 6(a) and (c)). It is possible that the locationof the IR just at the very end of the DNA fragmenthinders an appropriate positioning of the proteinwithin its target, thus leading to the nucleationprocess, which is not seen in the wild-type DNAunder the same conditions (not shown). It is worthnoting that a guanine residue (Gua206; seeMaterials and Methods) contacted by Arg4 in theco-crystal, is missing in the fragment used(Figure 6(a) and (c)). Arginine-guanine contacts arethought to be especially strong, since the arginineside-chain is positively charged, while the guanine-cytosine base-pair is electrostatically polar, beingthe guanine negatively charged.28 The CopG-mediated nucleation on the IR-ended DNA frag-ment, in conjunction with the lack of nucleationwhen the protein binds to the wild-type sequence,allowed us to suggest that the interaction of CopGwith the DNA can modulate the protein-proteininteractions. This is supported by the ®nding thatthe CopG dimer-dimer interactions found in theasymmetric unit of the unbound crystal structure(AB and CC) were similar to, but not identicalwith those observed between two CopG dimersbound to the IR in both the 19 bp and 22 bpdsDNA co-crystals.

Figure 6. (a) Nucleotide sequence of the wild-type plasmbases whose deoxyribose groups are protected by CopG frindicated by a rectangle. (b) Footprint pattern of the 224 bpcated amounts of CopG (pmols of protomer) performed bamount of DNA used was 0.1 pmol, and the products weregel. As marker size controls, chemical reactions were performG). (c) Nucleotide sequence of the IR-containing end of the 2employed for footprinting experiments. The asterisks indicatBases whose deoxyribose groups are protected by CopG frThe regions a, a0, b, c0, d and e0 (protected by CopG from thin a central position (rectangle)) are indicated by brackets. (da 8 % (w/v) polyacrylamide denaturing gel to reveal the proNote that the ®rst nucleotide that can be read in the sequenc(pLS1 Accession number M29725).

Proposed model for the interaction ofCopG-DNA in vitro

The dimer and tetramer interactions observed inour unliganded crystal structure are likely to be ofintrinsic nature and not due to a crystal artefact(since they are observed in both crystallographicand non-crystallographic contacts). The complexstructures discussed con®rm the establishment ofthese interactions when setting up the functionaltetramer and the DNA-bending capacity of CopG.Due to the chosen length of the dsDNA moleculesemployed in co-crystallisation (several others werealso tried, unsuccessfully), the contacts betweenfunctional tetramers are impaired either due torepulsion of terminal DNA phosphate backbonegroups or due to productive interactions of theDNA parts, rendering a continuous DNA superhe-lix. In addition, the oligomerisation pattern foundwith the IR-ended DNA fragment showed that suc-cessive CopG molecules bind to the same face ofthe DNA helix. The diameter of the CopG dimer,as deduced from the Stokes radius of the nativeprotein, is about 32 AÊ ,13 a value close to the helicalpitch of the B-DNA. Thus, it appears that a closecontact between adjacent dimers and the DNAcould take place during the nucleation process.The trend for helical oligomerisation to generatecontinuous superhelical ®bres within the crystalsof the unbound CopG protein (displaying theDNA-binding regions on the surface at appropriateaverage distances), together with the superhelixobserved in the protein-DNA complex, suggeststwo working model hypotheses for CopG in vitro:at relatively low protein:DNA ratios (around 20:1),protection extends to about ®ve turns of the DNAhelix major groove, rendering a model with twovicinal tetramers bending the dsDNA for about120 �, as determined by DNA band-shift exper-iments (see Figure 7(a)). At higher protein:DNAratios (about 400:1), where protection is extendedto include successive turns of major groove DNA(see Figure 6), additional dimers or tetramers puta-tively could associate on both sides of this initialcomplex, leading to a strongly bent underwoundDNA forming a loop of 82 AÊ diameter, witharound 74 bp of the DNA establishing one right-

id DNA region contacted by CopG. Brackets show theom hydroxyl radical cleavage. The position of the IR is

NcoI-ApaLI DNA fragmenting the presence of the indi-y protection against cleavage by hydroxyl radical. The

separated on a 20 % (w/v) polyacrylamide denaturinged with the same DNA fragment (C � T, C, A � G, and

24 bp NcoI-ApaLI DNA fragment from plasmid pLS1D24e the bases added by ®lling-in with the Klenow enzyme.om hydroxyl radical cleavage are indicated by wedges.e chemical cleavage in DNA fragments containing the IR) The same reaction products as in (b) were separated ontection patterns in the fragment regions distal to the IR.

e of both strands corresponds to the pLS1 coordinate 578

Figure 7. Hypothetical working models of CopG in itsinteraction with plasmid dsDNA in vitro. (a) At lower(e.g. 20:1) protein:DNA ratios, four CopG dimers(characterised by their solid coloured Connolly surfaces)cover four double-helix turns bending the DNA by120 �. (b) At higher (e.g. 400:1) protein:DNA ratios, bind-ing progresses in both directions. Each dimer is rep-resented by the Connolly surface coloured in magentaand yellow. (c) Side-view with the protein Connollysurface coloured according to electrostatic potential(positive in blue, negative in red). Note that the exteriorsurface is positively charged and provides an idealcomplement for the negative DNA backbone.


handed superhelical turn of around 40 AÊ superhe-lical pitch (based on the native protein structure).This model would be further based on the stronglyDNA-complementary positively charged outer pro-tein oligomer surface (Figure 7(c)). This DNA struc-ture is reminiscent of the nucleosome structure, inthis case a left-handed DNA superhelix of approxi-mately 85 AÊ diameter, 26.5 AÊ superhelical pitchand about 88 bp per turn.29

A series of transcriptional regulatory proteinshave been reported to distort DNA sequencesupon binding, resulting in DNA wrapping or loop-ing.30 Among them is 9.5 kDa protein HU fromBacillus stearothermophilus that binds non-speci®-cally to DNA and forms nucleoprotein structures

similar to eukaryotic nucleosomes.31 ± 34 This pro-tein is proposed to recognise the DNA as a dimervia two distorted b-ribbons, each coming from amonomer, that bind to two successive turns of theminor groove under strong bending and negativeDNA supercoiling. Eight to ten HU dimers bind to80-100 bp. The protein is even capable of stimulat-ing the ring closure of small DNA fragments thatare 99-126 bp long, which do not close in itsabsence.35

Transcriptional regulator proteins of the Lrp-type family belonging to the helix-turn-helix motifDNA-binding proteins36 achieve a similar effect.E. coli Lrp is responsible for both positive andnegative regulation of many promoters throughbinding of extremely long regions of DNAupstream of these promoters. It has been proposedto condense DNA into a histone-like nucleoproteincomplex whose conformation changes in responseto inducing ligands.37 Two further members of thisfamily, for which similar hypotheses have beenestablished, are Rhodobacter capsulatus/Agrobactertumefaciens response regulator PutR, protecting 110nt upstream of the putA transcription site fromDNaseI, despite showing only modest binding co-operativity38 and Pseudomonas putida BkdR. Thisprotein is a positive transcriptional regulator of thebkd operon, which encodes a multimodular dehy-drogenase that catabolises branched chain keto-acids. Further members of this growing familyinclude, among many uncharacterised sequence-related proteins, AsnC of E. coli, Grp of Zymomonasmobilis, and E. coli OxyR transcriptional regulatorthat, under oxidative stress induces the expressionof a set of antioxidant defence genes.38

A further example is provided by 12 kDa proteinP6 from B. subtilis phage �29 that binds to DNAforming a large nucleoprotein complex, in whichDNA wraps a multimeric protein core with onesuperhelical turn of 63 bp.39,40

Preliminary results indicated that binding ofCopG to negatively supercoiled or relaxed DNA,followed by treatment with topoisomerase I,results in negative supercoils (G.d.S. and J. Roca,unpublished results). Such an ability of CopG isshared with the histones and HU. This wouldagree with the CopG-mediated generation of a left-handed DNA superhelix, in apparent con¯ict withthe superhelix observed in the crystals. However,the TopoI-mediated reduction in the linking num-ber of the DNA molecules bound to CopG, com-pared to the unbound molecules, can be the resultof variations in the twist of the DNA and/or in theDNA supercoiling. To harmonise these obser-vations with a positive right-handed superhelix(Figure 7(b)), CopG should mediate a decrease inthe twist of the DNA that should overcompensatethe supercoiling introduced by the protein. Furtherexperiments on the changes of the DNA topologymediated by CopG would be needed. It is worthnoting that these CopG-mediated topologicalchanges are observed only at high protein concen-trations; that is, in conditions where most of the


DNA would be covered by the protein. An alterna-tive possibility is that the dimer-dimer interactionsin the nucleated CopG-DNA are different fromthose observed in the unbound crystal, so that gen-eration of a left-handed superhelix cannot be dis-carded. Be that as it may, as a transcriptionalregulator protein, CopG should have a de®ned tar-get, so that nucleation along the plasmid DNAwould be unlikely at physiological conditions. Theprotein should recognise speci®cally its target toavoid transcription from promoter Pcr, rather thanexhibiting a structural role. It would be most inter-esting to know how CopG restricts its nucleationto the operator/promoter region. It is possible thatthe contacts between the central CopG tetramer(bound to the IR) and further CopG dimers boundto the adjacent regions of the operator differ fromthose observed in the presently solved structures.

Materials and Methods

Protein purification, crystallisation anddata collection

CopG was produced by heterologous overexpression,puri®ed and crystallised unbound and in a complex witha 19 bp dsDNA as described.14,41 The complex betweenCopG and a 22 bp dsDNA (concentration 10 mg mlÿ1)encompassing the 13 bp IR (sequence provided with A-Toverhangs; see Figure 2(a)) was crystallised after mer-ging protein with annealed dsDNA following a 1:4(dsDNA:protein) stoichiometry. Equivolumetric dropswere equilibrated at 4 �C by the hanging drop vapour

Table 1. Data collection and processing and ®nal model re®n

Structure

SpacegroupCell constants (AÊ ) a

Wavelength (l in AÊ )No. of measurementsNo. of unique reflectionsWhole resolution range (AÊ )

Completeness (%)Rmerge (%) b

Average intensity [I/s(I)]Last resolution shell (AÊ )

Completeness (%)Rmerge (%) b

Average intensity [I/s(I)]B-Factor (Wilson) (AÊ 2)Average multiplicityResolution range used for refinement (AÊ )No. of reflections used (percentage of total)Crystallographic Rfactor (free Rfactor)

c

No. of protein/DNA atoms per a.u. (atoms with 50 % occupancy)No. of solvent moleculesr.m.s. deviation from target values

Bonds (AÊ )Angles (deg.)Bonded B-factors (AÊ 2)

Average temperature factor non-solvent atoms (AÊ 2)

a Values for a, b, c.b Rmerge � �hkl�ijIi(hkl) ÿ hI(hkl)ij/�hkl�i Ii(hkl), where Ii is the ith mc Rfactor � �hkljjFobsj ÿ k jFcalcjj/�hkljFobsj; free Rfactor, same for a te

cycle last-but-one).

diffusion method against 30 % (w/v) 2-methyl-2,4-penta-nediol, 0.2 M NaCl, buffered with 0.1 M sodium acetate,pH 4.6. These crystals, diffracting beyond 3 AÊ resolution,belong to orthorhombic space group P21212 with cellconstants a � 213.4 AÊ , b � 76.0 AÊ , c � 50.5 AÊ . Consider-ations about the Matthews coef®cient (VM) suggested thepresence of two to three complexes per asymmetric unit(a.u.). Diffraction data were collected at 100 K on a MARResearch image plate at the EMBL X31 X-ray diffractionbeamline (DESY, Hamburg), and processed withMOSFLM v. 6.042 and programs of the CCP4 suite43 (seeTable 1).

DNA footprinting assays

Hydroxyl radical footprintings were performed essen-tially as described.9 The DNA employed consisted of a224 bp NcoI-ApaLI fragment from plasmid pLS1�24, aderivative of plasmid pLS1. The DNA was ®rst digestedwith ApaLI, which recognises a single site in the DNA ofthe plasmid. The overhanging ends were ®lled-in withthe Klenow enzyme and the DNA was labelled in eitherstrand as described.8 Various protein:DNA ratios wereemployed, and the reaction products were separated on20 % or 8 % (w/v) polyacrylamide denaturing gels andrevealed by autoradiography. Chemical DNA sequencereactions, performed with the same DNA fragment, wereemployed as markers to position the CopG-protectedregions.

Structure solution and refinement

The structure was solved by molecular replacementcalculations using AMoRe44 with data in the 10 AÊ to

ement statistics

CopG/22 bp dsDNA

P21212213.40, 76.04, 50.52

1.0527103,43217,592

43.85-2.9293.60.1065.9

3.07-2.9258.60.4711.680.85.9

40-2.9517,507 (96.9)0.230 (0.307)5924 (844)

115

0.0091.3402.766.3

easurement of re¯ection I(hkl).st set of 7 % re¯ections (>500) not used during re®nement (till

Table 2. Molecular replacement calculations

Solution No. a b g x y z CC-Ia Rfactorb CC-Fa CC-I

Rfactor

backgroundRotationfunction

Rotation function calculation#3 13.1 64.9 87.7 20.1 54.9 33.9 17.5#4 152.3 78.7 194.1 20.1 54.9 32.6 17.4#5 156.8 56.0 167.7 19.4 55.0 29.9 17.1#14 22.1 78.8 39.5 19.2 55.2 33.5 15.5#24 9.0 60.4 108.8 19.7 55.0 33.3 14.5

One-body translation search#4 152.3 78.7 194.1 0.4419 0.4257 0.2027 29.1 52.8 32.5 26.7 53.6

Two-body translation search>4 152.3 78.7 194.1 0.4419 0.4257 0.2027 29.1 52.8 32.5#3 13.8 63.2 85.2 0.2476 0.5990 0.3054 37.9 49.6 41.4 30.1 52.2

Three-body translation search>4 152.3 78.7 194.1 0.4419 0.4257 0.2027 29.1 52.8 32.5>3 13.8 63.2 85.2 0.2476 0.5990 0.3054 37.9 49.6 41.4#24 9.1 58.5 108.3 0.3650 0.1755 0.5110 43.6 47.5 47.4 38.5 50.8

Four-body translation search>4 152.3 78.7 194.1 0.4419 0.4257 0.2027 29.1 52.8 32.5>3 13.8 63.2 85.2 0.2476 0.5990 0.3054 37.9 49.6 41.4>24 9.1 58.5 108.3 0.3650 0.1755 0.5110 43.6 47.5 47.4#14 22.5 78.7 39.3 0.1563 0.2096 0.0949 48.3 45.6 53.5 43.0 48.5

Rigid-body refinement of four-body translation search (``fiting'')#4 150.9 79.4 195.9 0.4424 0.4268 0.2025 29.1 52.8 32.5#3 11.5 61.1 86.3 0.2489 0.6043 0.3013 37.9 49.6 41.4#24 14.2 55.9 110.4 0.3670 0.1709 0.5162 43.6 47.5 47.4#14 30.0 78.2 40.6 0.1547 0.2123 0.1018 62.4 39.3 64.2

Five-body translation search>4 150.9 79.4 195.9 0.4424 0.4268 0.2025 29.1 52.8 32.5>3 11.5 61.1 86.3 0.2489 0.6043 0.3013 37.9 49.6 41.4>24 14.2 55.9 110.4 0.3670 0.1709 0.5162 43.6 47.5 47.4>14 30.0 78.2 40.6 0.1547 0.2123 0.1018 62.4 39.3 64.2#5 159.8 58.2 166.6 0.5372 0.1543 0.8425 69.0 35.8 70.9 60.5 40.6

Rigid-body refinement (``fiting'')#4 150.6 79.3 196.2 0.4424 0.4270 0.2022 29.1 52.8 32.5#3 11.4 61.4 86.5 0.2491 0.6037 0.3000 37.9 49.6 41.4#24 14.2 55.4 110.7 0.3670 0.1714 0.5166 43.6 47.5 47.4#14 29.8 78.2 40.8 0.1550 0.2134 0.1032 62.4 39.3 64.2#5 160.0 58.1 166.5 0.5380 0.1584 0.8408 69.8 35.3 72.0

a, b and g are in Eulerian angles, and x, y and z are in fractional cell coordinates. Only correct solutions in the rotation function list are displayed. # refers to the number of a correct rotationfunction solution and > to ®xed positions of previously found correct solutions when searching for a new one. Rigid body re®nement was performed with the ®ting routine of AMoRe.44 Thevalues of CC-I and Rfactor for the highest peak in the background are displayed for comparison.

a Correlation coef®cient based on intensities (CC-I) or structure factor amplitudes (CC-F) as de®ned previously.44

b Rfactor � (�hkljjFobsj ÿ kjFcalcjj/�hkljFobsj) � 100. For the rotation function, this value is calculated after data expansion to P1.


4.5 AÊ resolution range. For the translational searches, thecentred-correlation translation function was employedfor the ®rst one-body search and the phased-translationfunction for the remaining scours. The starting modelconsisted of a CopG dimer in complex with a 9 bpdsDNA molecule extracted from the recently solvedstructure of CopG with a 19 bp dsDNA 14 (PDB accesscode 1b01). The correct solutions of the computedrotation function were those at positions 3, 4, 5, 14 and24, respectively. Table 2 provides a detailed summary ofthese calculations that con®rmed P21212 as the correctspace group and the presence of ®ve solutions of thesearching model per a.u. This corresponds to two and ahalf actual CopG/22 bp dsDNA complexes. Accord-ingly, a crystallographic 2-fold axis runs through thecentre of the dsDNA moiety (molecules Y and Z, seebelow) in one of the complexes and, as the sequence isnot perfectly palindromic, the DNA is present in dualoccupancy (see Figure 2(c)). This effect has beenobserved in the structure of CopG with the 19 bp dsDNA14 and other reported protein/DNA complexes.45,46

This was accounted for in the construction of the dsDNAin the two orientations and half occupancy for all atomsfor this particular complex. The other two present in theasymmetric unit are fully occupied (see Figure 2(c)). Themodel was improved and completed in successive cyclesof manual model building using program Turbo-Frodo47

on a SGI Graphic Workstation and maximum-likelihoodcrystallographic re®nement, including bulk solvent cor-rection, positional and constrained temperature factorre®nement, employing CNS v. 0.9/1.0.48 NOE-likerestraints for the DNA base-pairs and NCS restraints forboth protein and DNA parts were applied. At the ®nalstages, solvent molecules were introduced where appro-priate if present in (2Fobs ÿ Fcalc) and (Fobs ÿ Fcalc) elec-tron density maps at 1 s and at 2.5 s, respectively.Table 1 provides a summary for the re®nement and ®nalmodel parameters.

The ®nal model comprises the following molecules.Protein chains A and B (de®ned from Lys2A to Gln43A,and Met1B to Lys41B, respectively) and DNA strands Y(Ade202Y to Cyt222Y) and Z (Ade201Z to Thy221Z) areconverted via a crystallographic 2-fold axis into the sym-metry-equivalent primed forms (see Figure 2(c)) and setup one complex (ABA0B0_YZ). CopG molecules E,D,G,F(Met1E to Glu44E, Met1D to Gln43D, Met1G to Arg45G,and Met1F to Arg45F, respectively) interact with DNAstrands W and X (Thy201W to Cyt222W and Ade201X toThy222X) to set up complex DEFG_WX. Finally, proteinchains J, H, K, and L (Met1 J to Glu44 J, Lys2H toArg45H, Met1 K to Gln43 K, and Met1L to Glu44L,respectively) determine the third complex present in thea.u. (HJKL_UV) together with DNA strands U (Ade202Uto Thy221U) and V (Ade201V to Thy221V). The rms�values for the common 163 and 169 Ca atoms betweencomplexes are 0.53 AÊ (DEFG_WX to ABA0B0_YZ) and0.46 AÊ (DEFG_WX to HJKL_UV), respectively. DNAnucleotides Thy221U, Thy221V and Thy222X are de®nedonly for their phosphate groups by electron density. Inall, 115 solvent molecules (labelled 501-615) havebeen further assigned. All de®ned residues belongingto the ten protein chains are in allowed regions of aRamachandran plot.

Miscellaneous

Figures and solid surfaces have been calculated withGRASP,49 VOLUMES/MOLSCRIPT/BOBSCRIPT,50,51 inpart rendered with Raster3D,52 and Turbo-Frodo. Acces-

sibility surface calculations have been computed withCNS v. 1.048 using a probe radius of 1.6 AÊ . Superimposi-tions have been calculated with Turbo-Frodo. DNA cur-vature and axis were determined with CURVES.53 Modelquality control checks were performed with WHAT IF.54

The bent DNA model including the CopG operatorsequence (Figure 7(b) and (c)) was kindly constructed byC.S. Tung.55,56

Protein Data Bank accession numbers

The ®nal co-ordinates of the complex of CopG withthe 22 bp dsDNA have been deposited with the ProteinData Bank at the European Bioinformatics Institute,EMBL Outstation Hinxton, GB (accession no. 1ea4).

Acknowledgements

We thank Rosa PeÂrez-Luque for help in crystallisationwork, Maite Alda in the overproduction and puri®cationof CopG, and Paloma Acebo for participating at theearly stages of this work. Research was supported bygrants PB98-1631 and 2FD97-0518 from the Ministerio deEducacioÂn y Cultura, Spain, by grant 1999SGR188 andthe Centre de RefereÁncia en Biotecnologia, both from theGeneralitat de Catalunya, by grants BMC2000-0550 andB10200-1659 from the Ministerio de Ciencia y Tecnolo-gõÂa, and by grant CAM07/B/49/99 from the ComunidadAutoÂnoma de Madrid. Data collection at the proteincrystallography EMBL beamline X31 (DESY, Hamburg)was supported by a European Union Large InstallationProject (Ref. HPRI-CT-1999-00017). Special thanks toChang-Shung Tung, Los Alamos National Laboratory,for his interest in the project and help in DNA model-ling.

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Edited by R. Huber

(Received 19 February 2001; received in revised form 7 May 2001; accepted 7 May 2001)

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