Nucleic Acids Research

Nucleic Acids Research 2006 34(5):1450-1458; doi:10.1093/nar/gkl015

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Published online 9 March 2006

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Article

Structures of repressors bound to direct and inverted DNA repeats explain modulation of transcription

Wilhelm Andreas Weihofen, Aslan Cicek, Florencia Pratto¹, Juan Carlos Alonso¹ and Wolfram Saenger^*

Institut für Chemie und Biochemie/Kristallographie, Freie Universität Berlin Takustr. 6, 14195 Berlin, Germany ¹Departamento de Biotecnología Microbiana, Centro Nacional de Biotecnología CSIC, 28049 Madrid, Spain

^*To whom correspondence should be addressed. Tel: +49 30 838 53412; Fax: +49 30 838 56702; Email: saenger{at}chemie.fu-berlin.de

Received December 20, 2005. Revised February 14, 2006. Accepted February 14, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION SUPPLEMENTARY DATA REFERENCES

 
Repressor regulates transcription of genes required for copy number control, accurate segregation and stable maintenance of inc18 plasmids hosted by Gram-positive bacteria. belongs to homodimeric ribbon-helix-helix (RHH₂) repressors typified by a central, antiparallel ß-sheet for DNA major groove binding. Homodimeric ₂ binds cooperatively to promotors with 7 to 10 consecutive non-palindromic DNA heptad repeats (5'-^A/_TATCAC^A/_T-3', symbolized by ) in palindromic inverted, converging () or diverging () orientation and also, unique to ₂ and contrasting other RHH₂ repressors, to non-palindromic direct () repeats. Here we investigate with crystal structures how ₂ binds specifically to heptads in minimal operators with () and () repeats. Since the pseudo-2-fold axis relating the monomers in ₂ passes the central C–G base pair of each heptad with 0.3 Å downstream offset, the separation between the pseudo-2-fold axes is exactly 7 bp in (), 0.6 Å shorter in () but would be 0.6 Å longer in (). These variations grade interactions between adjacent ₂ and explain modulations in cooperative binding affinity of ₂ to operators with different heptad orientations.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION SUPPLEMENTARY DATA REFERENCES

 
Gene expression in prokaryotes is primarily regulated by helix–turn–helix proteins that bind specifically to palindromic operators whereas recognition of arrays of direct or inverted repeats by transcriptional, homodimeric ribbon-helix-helix (RHH₂) repressors like protein is less frequent (1). Structures are known for RHH₂ repressors Arc (2), CopG (3) and MetJ (4) bound to their cognate operators that are bent by 50° to 60°. MetJ₂ binds symmetrically to two to five consecutive 8 bp long palindromic repeats. By contrast, CopG₂ and Arc₂ bind asymmetrically to half sites of palindromic operators that are spaced by 10 and 11 bp, respectively. When bound to these operators, interactions between adjacent RHH₂ contribute to high affinity and cooperative association.

Repressor is a global regulator of and encoded by broad-host-range and low-copy number plasmids belonging to the inc18 family that are stably maintained in Gram-positive bacteria (5–7). was originally isolated from Streptococcus pyogenes plasmid pSM19035 where ₂ controls promoter regions located upstream of genes involved in plasmid copy number control (PcopS), plasmid partitioning (P) and post-segregational killing (P) if the plasmid is lost. These promoters comprise arrays of ten, nine or seven consecutive 7 bp repeats (heptads, symbolized by ), organized as: PcopS, (); P, () and P, () (1), see Supplementary Figure 9.

Binding of ₂ to a single heptad or to heptads separated by one or more additional base pair is poor (k_D >500 nM), but tight if operators include at least two consecutive heptads and tightens further with increasing number of heptads. In addition, the affinity depends on heptad arrangement as shown by 6-fold reduced affinity of ₂ for diverging repeats () (k_D 120 nM) compared to heptads in direct () or converging () arrangement (k_D 20 nM) (8). Multiple repeat binding sites are also found for eukaryotic operators that interact cooperatively with monomeric and therefore asymmetric transcription factors (1). However, these repeats show different base pair spacings and protein–protein interactions in direct and inverted orientation (9).

In wild type (wt) ₂, the N-terminal 20 residues of the 71 residues long monomers are probably unstructured as suggested by secondary structure prediction (10) and were cleaved during crystallization (20₂). The structure features a typical RHH-fold comprising a 2-fold symmetrical ß-sheet with antiparallel pairing of residues 28–32 of each monomer followed by -helices 1 (34–46) and 2 (51–66) (11).

We describe here the crystal structures of an N-terminal deletion mutant (see Results) with 19 residues removed, hereafter 19, in complex with two minimal operators comprising two heptads in () and () arrangement. It was of interest to elucidate the structural determinants for high specificity, affinity and cooperative binding of repressor to minimal binding sites and to extrapolate these to natural operators with different heptad arrangements.

Unintentionally, both complexes cocrystallized with free operator DNA. Free ()-DNA allowed us to compare structural changes in DNA induced by repressor binding, whereas free ()-DNA was ill-defined in the electron density and could not be fully modeled. The complex between 19₂ and ()-DNA dissociated during gel filtration and could not be crystallized.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION SUPPLEMENTARY DATA REFERENCES

 
Plasmid construction
For expression of 19 in Escherichia coli, wt gene missing the first 19 codons was cloned into NcoI–BamHI-cleaved pET28a (Novagen) to render pET28a-19. The described (1) pHP14-borne gene (pHP14) was modified to pHP1419 containing promoter P, the ribosomal binding site and the Met start codon fused to codon 20 of gene. pHP14 mutants pHP14Thr29Ala and pHP14His38Val were generated by site-directed mutagenesis. The plasmids were transferred into Bacillus subtilis strain BG511 (P:lacZ, recA4) as described (1).

ß-galactosidase assay
ß-galactosidase assays (Table 1) were performed as described (1) except that the centrifuged B.subtilis cells were resuspended and lysed by the addition of 0.1% SDS (final concentration 0.0025%) and chloroform.

Preparation of protein–DNA complexes
19₂ was expressed in E.coli according to (1), the cell paste was resuspended in buffer A [50 mM Tris–HCl (pH 7.5), 50 mM NaCl] and lysed (French Press). The crude extract was processed (1), except that after the phosphocellulose step the fractions were pooled, diluted 5-fold with buffer A and loaded on a POROS 20 HE column (Applied Biosystems). 19₂ was eluted with 50–1000 mM NaCl gradient in buffer A. Concentrated fractions were gel filtrated on Superdex75 (GE Healthcare) run with buffer B [20 mM Tris–HCl (pH 7.5), 300 mM NaCl].

Complementary oligonucleotides were purified by high-performance liquid chromatography (HPLC), mixed at 1:1 molar ratio, hybridized and purified using a MonoQ column (GE Healthcare). Eluted DNA was dialyzed against 20 mM Tris–HCl (pH 7.5), 100 mM KCl and 30 mM NaCl. 19₂ was added at 2.1:1 molar ratio and purified by gelfiltration (SuperdexS75). Fractions of 19₂/DNA were concentrated to 10.5 mg/ml for crystallization.

Crystallization
Using hanging-drop vapor diffusion, crystals with space group C2 grew from drops made of 2 µl of [19₂]₂-() solution or 2 µl of [19₂]₂-() (bp A9–T6' exchanged by bp G9–C6') solution and 2–3 µl precipitant solution [150 mM KH₂PO₄ (pH 7.0), 2.4 M Na₂-malonate, 2% 6-amino-caproic acid].

Crystals of [19₂]₂-() grew in space group P2₁ under similar conditions when precipitant solution contained 150 mM Na/KPO₄ (pH 7.0), 2.4 M Na₂-malonate and 2 to 3% 2-methyl-2,4-pentanediol. In all cases crystal quality was improved by micro-seeding.

Data collection, structure determination and refinement
X-ray data were collected at 100 K at the Protein Structure Factory beamline BL14.1 of Free University Berlin at BESSY and processed with HKL2K (12); Table 2.

The structure of [19₂]₂-() was determined by molecular replacement in PHASER (13) with 20₂ (PDB code 1IRQ) modeled to 8 bp idealized B-DNA. After manual building of [19₂]₂-() and restrained refinement in REFMAC5 (14), F_o-F_c maps showed additional electron density for another DNA molecule but not for additional 19₂. This second DNA molecule was build manually starting from ideal B-form DNA. In the final model the asymmetric unit consists of one [19₂]₂-() and one free ()-DNA. 19 molecules A' and B could be modeled with all residues 19–71, but A and B' only with residues 23–71 and 25–71, respectively, see Figures 1 and 2 for assignment of A, A', B, B'.

View larger version (24K): [in this window] [in a new window]   Figure 1 DNA used for cocrystallization and arrangement of (A) [192]2-() and (B) [192]2-() and free DNAs in the crystal asymmetric units (large boxes). Subunits sharing the same protein–DNA interactions 19A/B shown in blue and 19A'/B' in white ovals. Solid bars indicate subunits interacting through helices 1, 1'. The pseudo-2-fold axis relating 192 and bound heptads in [192]2-() indicated by black ellipses in (B). Heptads with sequence 5'-AATCACA/T-3' outlined by arrows, and nucleotides are numbered 0 to 17 and –1' to 16', respectively. Top strands of free DNA in green. Grey nucleotides C17 in both structures and 4 to 15 bp of free DNA in [192]2-() could not be modeled due to poor electron density.

 

View larger version (24K): [in this window] [in a new window]   Figure 2 (A) Structure of [192]2-(). DNA backbone trace in light grey for top strand and dark grey for bottom strand, see Figure 1. 19 monomers A/A' and B/B' in light and dark green and blue, respectively, helices 1 and 2 labeled with white letters. Helices 1' of A' and 1 of B related by a pseudo-2-fold axis perpendicular to the paper plane (red ellipse) form the 192···192 interface. (B) Superimposition of 19A/A' of [192]2-() (green) and 19A/A' of [192]2-() (grey) to show slight positional differences of 19 B/B' associated with palindromic symmetry in (). Pseudo-2-fold axes relating monomers in 192 indicated by dashed lines colored green for [192]2-() and red for [192]2-(). Helices 1, 1' involved in 192···192 interactions superimpose well allowing cooperative binding in both complexes. DNA in [192]2-() is locally kinked by 12° at the centre (bp G11–C4') of heptad A8–T14, and 192 are 0.6 Å closer (vertical distance between pseudo-2-fold axes) than in [192]2-(), see text. (C) Phosphate backbone of the 14 bp operator regions of operator DNA free () in green and 192-bound () in blue superimposed on ideal B-DNA (grey).

 
The structure determination of [19₂]₂-() mutant with bp A9–T6' replaced by bp G9–C6' used difference Fourier technique applied to the isomorphous crystal structure of [19₂]₂-(), see Table 2.

The structure of [19₂]₂-() was determined in MOLREP (15) using [19₂]₂-() as search model. One [19₂]₂-() was found, and after restrained refinement sparse electron density indicated only four additional bases for free ()-DNA that could not be modeled completely, Figure 1B. Molecule B' could be modeled with all residues, B with residues 25–71 and A, A' with residues 22–71.

For refinement of all three structures in Refmac5 TLS groups were assigned and refined for each polypeptide chain and oligonucleotide, see Table 2 for statistics. No non-crystallographic symmetry was used during refinements. Model quality was examined by Whatcheck and Procheck (16) showing that , torsion angles of most amino acids in all three structures are within the most favored, some are in additionally allowed and none are in forbidden areas of the Ramachandran plot. Figures were generated with MOLSCRIPT (17) and Raster3D (18). Analysis of DNA parameters used program Curves (19).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION SUPPLEMENTARY DATA REFERENCES

 
19₂ protein
Cocrystallization of wt ₂ with operator DNA yielded only crystals with poor X-ray diffraction, but was successful with 19. In vitro, 19₂ binds specifically to promoter PcopS with 2-fold lower affinity (k_D 12 nM) (see Supplementary Figure 10) compared to wt ₂ (k_D 6 nM) (8), and likewise plasmid-borne 19 gene product represses P utilization in vivo 2-fold weaker compared to wt gene (Table 1). This suggests that even without the N-terminal 19 residues, ₂ still binds strongly (only 2-fold weaker) and specifically to DNA heptads in both, in vitro and in vivo gene regulation.

View this table: [in this window] [in a new window]   Table 1 Utilization of P:lacZ in the presence of variants in B.subtilis cells

 
Crystal unit cells contain free and 19₂ bound operator DNA
The crystal structures of 19₂ bound to two minimal operators formed by 17 bp DNAs with C, G overhangs and comprising direct () and inverted () heptads (Figures 1 and 2) were determined by molecular replacement at 2.45 and 2.6 Å resolution, respectively (Table 2). The asymmetric units of both complexes contain two 19₂ bound to operator DNAs ([19₂]₂-() and [19₂]₂-()) which in turn interact with the ends to free operator DNAs () and (), respectively, to form pseudo-continuous DNA (Figures 1 and 3).

View this table: [in this window] [in a new window]   Table 2 Crystallographic data and refinement statistics

 

View larger version (87K): [in this window] [in a new window]   Figure 3 2F0-Fc electron density distribution contoured at 1.2 level; view perpendicular to crystallographic b, c planes with the c-axes vertically oriented. (A) Parallel free DNAs (yellow) in [192]2-()() are at 5 Å shortest distance to each other and stack at both ends with 192-bound DNAs (magenta). Free DNA is not involved in any further crystal contacts. (B) Discontinous electron density for free DNA in [192]2-()(). Modeled complexes of [192]2-() and modeled bases of free DNA (see Figure 1B) in yellow. The central parts of free DNA marked by dotted lines (4 to 15 bp, Figure 1B) could not be modeled due to patchy electron density. The shortest distance between free DNAs is 8 Å and they are at an angle of 40°.

 
On the ‘left’ sides of free DNA (Figure 1), nucleotides C17 of free and 19₂-bound DNA are not in helical arrangement and not seen in the electron density as they are disordered but were confirmed by MALDI-TOF-spectrometry of dissolved crystals (data not shown). Both, [19₂]₂-bound and free DNAs, stack with bp G16–C-1' that are related by pseudo-2-fold symmetry.

On the ‘right’ sides of free DNA (Figure 1), the two 3'-G16' overhangs lie in the minor groove of the adjacent duplex and interact with both 5'-G0 to form two consecutive G16'*(G0–C15') base-triplets (Supplementary Figure 11) with similar geometry as reported (20–22).

In the crystals of 19₂ bound to () in space group C2 and bound to () in space group P2₁ (Table 2), both [19₂]₂–DNA complexes interact by protein–protein contacts to form layers parallel to the crystallographic a, b planes. The crystallographic a, b-axes in space group P2₁ correspond to b, a-axes in C2 thus reflecting space group and lattice packing similarities. The pseudo-continuous DNA helices (Figures 1 and 3) are oriented in c-direction. The doubled c-axis in C2 relative to that in P2₁ is due to the C-centering, and the DNA helices are parallel to the c-axis at shortest inter-helix distance of 5 Å in [19₂]₂-()() (base-triplets indicated by ), possibly stabilized by bridging water. By contrast, in [19₂]₂-()() the pseudo-continuous DNA helices are parallel to the crystallographic a, c plane but inclined at an angle of 40° towards the c-axis and at least 8 Å apart, and poor electron density (Figure 3B) indicates that they are partially disordered as shown by B factors >100 Ų. Consequently, bp 4 to 15 of free ()-DNA in [19₂]₂-()() could not be modeled (Figures 1B and 3B).

In both crystal unit cells (space groups C2 and P2₁, Table 2), the DNA-bound 19₂ show minor structural changes compared to the X-ray structure of free 20₂ (11). This concerns the loop connecting -helices 1 and 2 [residues 46–48, 2.0 Å root mean square (r.m.s.) deviation for superimposed C-Atoms] and to a minor extent the ß-sheet (residues 27–32, 0.8 Å r.m.s. deviation).

Since gel permeation chromatography of [19₂]₂-() at 20 mM Tris–HCl (pH 7.5), 150 mM NaCl indicated an apparent molecular mass of 40 kDa (calculated M_w = 33.4 kDa), we assume that under crystallization conditions with 2.4 M Na₂-malonate, the complexes associate pairwise through base-triplets G16'^*(G0–C15') to form the complex [19₂]₂-()()-[19₂]₂. If one [19₂]₂-() of this complex forms layers mediated by protein–protein contacts as in the present structures, it appears that the other [19₂]₂-() has to release [19₂]₂ for packing reasons, thereby giving rise to the crystallized [19₂]₂-()(). The other crystal contact with only DNA–DNA stacking interactions is more flexible than the base-triplets and permits formation of a regular crystal lattice. The same crystallization scenario applies for [19₂]₂-().

Protein–DNA interactions
Formation of G16'^*(G0–C15') base-triplets induces distortions at the 5' ends of heptads A1–A7 as indicated by different, partly water-mediated interactions to 19₂ compared to heptads A8–A14 in () and A8–T14 in (), Figures 4 and 5. For this reason, we focus here on protein–DNA interactions for the less distorted dimer 19B/B' bound to heptad A8–A14 of [19₂]₂-() (Figures 2A and 4(left)). In both structures, for each 19₂-bound heptad the direct (not water-mediated) protein–DNA contacts are comparable.

View larger version (32K): [in this window] [in a new window]   Figure 4 Schematic representation of interactions between 192 and DNA in [192]2-() (left) and [192]2-() (right). The orientation of heptads is indicated by blue arrows and palindromic symmetry by the black ellipse. Yellow bases specifically interact with 192. Residues labeled green for 19 A/B and black for 19 A'/B'. Hydrogen bonds to phosphate oxygens (red) in thin black lines, specific hydrogen bonds to bases in the major groove in black arrows, blue lines indicate water (blue dots) mediated hydrogen bonds. K28 (K28') and Arg31 (Arg31') interact differently in subunits A/B and A'/B'.

 

View larger version (41K): [in this window] [in a new window]   Figure 5 Stereo view of specific interactions between 19 B/B' and bound heptad A8–A14 in [192]2-(). Water molecules; red spheres, hydrogen bonds; dashed lines, phosphorus atoms; magenta, directly contacted bp C11–G4' and base G2' in yellow.

 
In the major grooves, base pair specific interactions are formed with Thr29 and Arg31 located on the ß-sheet. Thr29O and Thr29'O of 19B and B' bind specifically to the central bp G4'–C11, and Arg31N, hydrogen bond with base G2' (Figure 4(left)). In contrast, the corresponding Arg31' of 19B' hydrogen bonds with N' to Thr29O of 19B and with N1',2' through three water molecules to bases G4', A5' and A9, Figures 4 and 5.

To see whether 19₂ would bind symmetrically to a palindromic heptad featuring two G to provide both, Arg31 and Arg31', with potential binding partners, we determined the 2.9 Å resolution crystal structure of [19₂]₂ in complex with a mutated () where bp A9–T6' was replaced by G9–C6' (heptad sequence: ⁸AGTCACA¹⁴, Table 2). However, the same interaction pattern was found as in the original heptad. This agrees with similar (2-fold weaker) binding of wt ₂ to mutated () with the same replacement in the first heptad (¹AGTCACA⁷) compared to the original operator (8). A referee suggested to test the binding of 19₂ to heptads with pseudo-palindromic symmetry, 5'-TGTCACA-3'. In view of the binding geometry of 19₂ to ⁸AGTCACA¹⁴, we question whether this would provide novel knowledge. This is because the 5'-AAT- or 5'-AGT-ends of the heptads contact 19₂ in all cases exclusively through unspecific interactions with phosphate groups or are mediated by water molecules (Figure 4A and B). Hence, base pair exchanges in this part of the heptads should not significantly affect binding of 19₂.

Backbone phosphates of all four heptads contact helices 1 and 2 of 19 subunits with pseudo-2-fold symmetry (Figure 4). In 19B, the 5'-phosphate of A9 caps the N-terminus of 2 by hydrogen bonding to peptide amides of V51 and K52 in a pattern known for RHH₂ proteins and other repressors (4,23), and His37N, K41N of 1 bind to the 5'-phosphate of T10. Corresponding residues of 19B' interact with 5'-phosphates of G2' and T3'. However, K28N located on the ß-sheet of 19B forms a salt-bridge with the 5'-phosphate of G4' whereas K28'N on 19B' is >5 Å away from the corresponding 5'-phosphate of C11.

The asymmetry in the binding of each 19₂ to its particular heptad is reflected by superimposition of C atoms of monomers (A on A' and B on B', Figure 2A) in each 19₂ showing 0.6 Å r.m.s. deviation partly associated with structural differences in the loops connecting helices 1 and 2 (residues 46–48) and in the ß-strands. When 19₂ dimers are superimposed on each other in the same orientation (A on B and A' on B'), i.e. the bound heptads point in the same direction, r.m.s. deviation of only 0.3 Å confirms that all 19₂-heptad interactions are similar.

Thr29 is essential for specific operator binding
To test the importance of Thr29 for heptad sequence recognition in vivo studies were conducted showing that ₂Thr29Ala failed to completely repress promoter P utilization (Table 1). Binding of ₂Thr29Ala to PcopS operator DNA embedded in 300 bp DNA was tested by electrophoretic mobility shift assays (EMSA) (Supplementary Figure 10). Protein–DNA complexes formed with 19₂ but Thr29Ala required 100-fold higher concentration (compared to 19₂) that yielded prominent but unspecific binding to PcopS as confirmed by DNase I footprinting (data not shown).

19₂···19₂ interactions
Protein–DNA interactions in both complexes bury 1610 Ų of solvent accessible surface area. Another 550 Ų are buried by interaction of pseudo-2-fold axis related 1 helices of adjacent monomers A' and B in [19₂]₂-() and A' and B' in [19₂]₂-(), respectively (Figure 1 and Figure 2A and B). In detail, bifurcated hydrogen bonds between His38N and Ala45'O/Lys46'O are augmented by hydrophobic contacts between Ile42, Ala45 of both subunits (Figure 6). Positions of His38 and His38' are identical in both complexes whereas the hydrophobic side-chain of Ile42 adopts different rotamers without affecting the size of the buried interface. These interactions ensure cooperative binding when several ₂ associate with multiple heptad repeats as found in natural operators.

View larger version (45K): [in this window] [in a new window]   Figure 6 Interactions at the 192···192 interface (A) between helices 1' and 1 of subunits A' and B of [192]2-(), (see Figure 2A). The 192 are related by a pseudo-2-fold axis perpendicular to the paper plane (red ellipse). Residues with contacts <4 Å to neighbor helix are depicted with side-chains, colors as in Figure 2. Bifurcated hydrogen bonds between His38 (His38') and Ala45O/Lys46O to Ala45'O/Lys46'O of the other subunit in dashed lines. (B) Superimposition as in Figure 2B with view on the dimer–dimer interface to show that dimer–dimer interactions are comparable in both structures.

 
To test the importance of His38, plasmid-borne His38Val was constructed to remove the bifurcated hydrogen bonds but to maintain the hydrophobic character of the interface. ₂His38Val repressed P utilization in vivo with 7-fold lower efficiency than wt ₂ (Table 1), indicating the important role of His38 for cooperative binding between ₂ and multiple consecutive heptads.

Conformation of free and 19₂-bound DNA
The DNAs in [19₂]₂-() and [19₂]₂-() are nearly straight B-form with average helical twist of 36° (range 23° to 43°; Supplementary Figure 12), but show distinct features. The major groove width in [19₂]₂-() shows strong modulation depending on nucleotide sequence, being 13 to 14 Å except for the ⁷AAAT¹⁰ tract (11 to 12 Å) compared to 11.7 Å for ideal B-DNA (24). In contrast, the major groove width in [19₂]₂-() is more continuous (13 to 14 Å) due to the palindromic symmetry (Figure 7A). In the ⁷AAAT¹⁰ tract of [19₂]₂-() and in the ⁷AAGT¹⁰ tract of [19₂]₂-() the minor groove is narrowed to 2.7 and 4 Å, respectively, compared to 5.7 Å in ideal B-DNA (24) due to an average negative base pair inclination of –8° (Figure 7C), as frequently found for A-tracts (25). This is associated in ⁷AAAT¹⁰ of [19₂]₂-() with binding of a single spine of four water molecules, one in each base step as reported in (26). Strong opposite buckles of the C–G bp in the ⁴CAC⁶ (–15°, 15°) and ¹¹CAC¹³ (–10°, 15°) segments of [19₂]₂-() (Figure 7B) widen the major groove to 13 Å to accommodate the ß-sheet, and adjacent (up- and downstream) minor grooves are narrowed (Figure 7A). Similarly, in [19₂]₂-() the C–G bp in segment ⁴CAC⁶ show comparable buckles of –10°, 19° but in ⁹GTG¹¹ the buckles are reduced to –8°, 5°. Of the four heptads, three feature two subsequent CA steps that are known for their ability to bend B-DNA through positive roll (20,27,28). However, the CA steps do not exhibit unusual structure except for a moderate slide movement of 1 Å at the central C–G bp where 19₂ binds to both bases (Supplementary Figure 12).

View larger version (35K): [in this window] [in a new window]   Figure 7 Comparison of operator DNA free and in complex with 192. (A) Minor (m) and major (M) groove widths along the central 11 bp of each operator, as measured by shortest P-P distances (less two phosphate group radii, 5.8 Å) indicated by horizontal dotted black lines for ideal B-DNA (24), 5.7 Å and 11.7 Å, respectively, Note minor groove narrowing in the 7AAAT10 tract of in 192-bound and free () DNA and reduced narrowing in 7AAGT10 tract of (192)2-(). (B) Base pair buckle (°) and (C) inclination angles (°) of each operator.

 
In the crystal lattice, free ()-DNA contacts 19₂-bound ()-DNA with both ends to form pseudo-continuous helices (Figure 1) and is not distorted by any further crystal contacts (Figure 3A). Interestingly, free ()-DNA shows comparable structural features as found for 19₂-bound ()-DNA (Figure 2C). In the ⁷AAAT¹⁰ tract of free ()-DNA, base pair inclination angles are negative, and the minor groove is narrowed to 3.5 Å whereas the major groove is overall widened to an average of 12.5 Å. Additionally, strong opposite buckles are observed for C–G base pair in both CAC segments that are comparable to 19₂-bound ()-DNA (Figure 7B). These observations suggest that the described significant deviations from ideal B-DNA are a consequence of nucleotide sequence and not induced by binding to 19₂ (Figures 2C and 7).

However, conformational changes upon repressor binding concern base pair parameters helical twist, roll and slide (Supplementary Figure 12). In free ()-DNA these parameters cluster around values assigned to ideal B-DNA, but show a negative roll of –6° and positive slide of 1 Å for the central G11'–C4 and G4'–C11 bp of both heptads, and helical twist angles for bp A5–T10' and A12–T3' decrease to 25° in 19₂-bound ()-DNA. Since these deviations are similar for both heptads of [19₂]₂-() they can be attributed to 19₂ binding. Such comparison is not possible with free ()-DNA as it could not be modeled completely.

Comparison of [19₂]₂-() and [19₂]₂-()
The pseudo-2-fold axis relating the monomers in 19₂ passes with 0.3 Å downstream (5'3') offset through the central G–C bp of each heptad. Consequently, these symmetry axes are separated by 7 bp in [19₂]₂-() but they are 0.6 Å closer in [19₂]₂-(), see Figure 2B. Despite the different dimer–dimer separations helices 1 and 1' forming the 19₂...19₂ interfaces superimpose well in both complexes, consistent with similar dissociation constants (k_D 20 nM). In contrast, the separation between the two 19₂ will be 0.6 Å wider in diverging heptads [19₂]₂-(). Assuming that the interaction pattern between 19₂ and heptads with () orientation is similar as with () and () heptad orientations, the expected 0.6 Å longer 1···1 contacts are probably less favorable and diminish cooperativity. This agrees with the 6-fold weaker affinity of wt ₂ to heptads in () arrangement, the finding that [19₂]₂-() dissociated during gel filtration, and the drastically reduced binding affinity to heptad repeats spaced by one or more additional base pair (8).

Whereas DNA in [19₂]₂-() is nearly straight, the superimposition in Figure 2B reveals a kink of 12° at the G11–C4' bp at the centre of the heptad bound to dimer B/B' of [19₂]₂-(). This kink is associated with 12° rotation of dimer B/B' of [19₂]₂-(), the rotational pivot point being located in the 19₂···19₂ interface. By virtue of these two motions, the position and orientation of helix 1' of subunit B' remain almost as in [19₂]₂-() and similar 19₂···19₂ interactions explain comparable dissociation constants of both complexes.

Structural model of ₂-bound to natural operators
Extrapolation of the structures of [19₂]₂-() and [19₂]₂-() allowed modeling of 19₂ in complex with natural P promoter (Figure 8). The model implies that wt ₂ binds as left-handed matrix to right-handed, straight B-type operator DNA, each 19₂ being displaced relative to its neighbor by 7 bp and rotated by 252°. Figure 8B shows that the negatively charged sugar-phosphate backbone of DNA faces positively charged surface of 19₂.

View larger version (57K): [in this window] [in a new window]   Figure 8 Model of seven 192-bound heptads () of the natural promoter P. (A) DNA in space filling and 192 as orange/red ribbons, (B) DNA in stick and 192 in surface representation colored according to electrostatic potential (negative and positive charges red and blue, respectively). The model is based on the structures of [192]2-() and [192]2-(). Repressors form a left-handed protein-matrix winding around the nearly straight operator.

 

	DISCUSSION

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Implications of 19₂-DNA structures for regulation of transcription
The inc18 family plasmids harbor genes to control their copy number, accurate segregation and stable maintenance during cell division. Since expression of these genes is regulated by the common ₂ repressor, a unique mechanism has evolved to fine-tune repressor affinity for the different operators. How this is achieved is shown by the present study. It clearly indicates that the pseudo-symmetric ₂ binds with 0.3 Å downstream (5'3') offset relative to the center G–C base pair of the cognate heptad. Since the operators are nearly straight B-DNA, different heptad numbers and orientations lead to different distances between 1 helices of adjacent ₂, thereby modulating cooperative interactions between ₂ and different operators. The ability to bind to palindromic as well as to non-palindromic operators is a unique feature of ₂ and is not shared by other member of the RHH₂ family. We associate this with the interactions between 19₂ that are related by a pseudo-2-fold rotation axis (Figure 2A and Figure 6A and B) so that they interact comparably, no matter what the orientations of adjacent heptads are, (), () or (). It is unlikely that the deleted N-termini would contribute to 19₂ ···19₂ interactions as the N-termini of 19₂ point away from the 19₂ ···19₂ interface (Figures 2A and 6A).

When RHH₂ bind to DNA, we have to consider two different binding characteristics, the ‘variable’ binding of flexible side-chains of ß-sheets and 1 helices to major groove and phosphate groups, respectively, and ‘stiff’ capping of N-termini of 2 helices by rigid main-chain NH hydrogen bonding to phosphates. Although Arc₂, CopG₂ and MetJ₂ bend minimal cognate operators with two repressor binding sites by 50 to 60°, their cores do not rearrange significantly compared to free repressors and are structurally similar [r.m.s. deviations for superimposed C atoms 1.0 to 1.5 Å (3)]. In contrast, Arc₂, CopG₂ and MetJ₂ superimpose on DNA-bound 19₂ with higher r.m.s. deviations of 2.3 to 2.8 Å because the ß-sheet of 19₂ protrudes 3 Å less from the repressor surface and is closer to the N-termini of helices 2 (Supplementary Figure 13). Due to this geometry of 19₂, the N-termini of helices 2 are in the correct position to clamp the phosphate backbones of straight operator DNA when the ß-sheet is inserted into the major groove (Figures 2 and 4). In contrast, the other three repressors have to bend DNA around the more protruding ß-sheet to place the phosphate backbone in hydrogen bonding distance to N-H groups of the N-termini of their helices 2. It is notable that the distances between phosphates bound by 2 helices is 5 bp in ₂ (Figure 4) but 6 bp in the other three RHH₂ since DNA has to follow a longer path when bending around the protruding ß-sheet.

The N-termini of RHH₂ proteins are associated with different functions
The N-terminus in CopG₂ has no obvious function, in MetJ₂ it is involved in binding the corepressor S-adenosylmethionine whereas in free Arc₂ it is disordered but forms a 3₁₀ helix upon and contributes to DNA-binding. The N-terminal residues in ₂ do not contribute to DNA-binding in vitro and in vivo as shown in the present work. However, we have recently shown that protein , a homolog to ParA proteins and involved in active plasmid partitioning during cell division, is activated by wt ₂ but not by 19₂ (A. Cicek, F. Pratto, W. Weihofen, J.C. Alonso and W. Saenger, unpublished data). This suggests that ₂ is the yet missing ParB protein of inc18 family plasmids in the known ParA/ParB family of plasmid partitioning systems (29).

Direct and indirect readout of DNA sequence
Cocrystallized 19₂-bound and free ()-DNA show similar phosphate backbone conformation (Figure 2C) with significant deviations from ideal B-DNA (Figure 7), indicating that conformation is predominantly dependent on DNA sequence and not induced by 19₂ binding. This provides a good example for the ‘indirect’ readout of local DNA conformation by 19₂ that depends on the particular nucleotide sequence (30). The combination with ‘direct’ readout resulting from interactions of ₂ repressor amino acids with heptad bases increases protein–DNA-binding specificity and affinity. This view is consistent with studies in which the heptad nucleotide sequence was mutated base pair by base pair (8). Mutations at heptad positions 2, 3 and 5 (5'-AATCACA-3') resulted in at least 4-fold weaker binding to wt ₂, although these base pair are not directly contacted by repressor side-chains contrasting direct read out positions 4 and 6 that were much more sensitive against mutations.

Protein Data Bank accession codes
Atomic coordinates and structure factors have been deposited with the Protein Data Bank under accession codes 2bnw, 2bnz and 2cax.

	SUPPLEMENTARY DATA

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION