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Structure of a G-quadruplex–Ligand Complex
doi:10.1016/S0022-2836(02)01354-2
J. Mol. Biol. (2003) 326, 117–125
Structure of a G-quadruplex –Ligand Complex Shozeb M. Haider, Gary N. Parkinson and Stephen Neidle* Cancer Research UK Biomolecular Structure Group The School of Pharmacy University of London, 29-39 Brunswick Square, London WC1N 1AX, UK
Stabilisation of G-quadruplex structures formed from telomeric DNA, by means of quadruplex-selective ligands, is a means of inhibiting the telomerase enzyme from catalysing the synthesis of telomeric DNA repeats. In order to understand the molecular basis of ligand –quadruplex recognition, the crystal structure has been determined of such a complex, at ˚ resolution. This complex is between a dimeric antiparallel G-quad1.75 A ruplex formed from the Oxytricha nova telomeric DNA sequence d(GGGGTTTTGGGG), and a di-substituted aminoalkylamido acridine compound. The structure shows that the acridine moiety is bound at one end of the stack of G-quartets, within one of the thymine loops. It is held in place by a combination of stacking interactions and specific hydrogen bonds with thymine bases. The stability of the ligand in this binding site has been confirmed by a 2 ns molecular dynamics simulation. q 2003 Elsevier Science Ltd. All rights reserved
*Corresponding author
Keywords: G-quadruplex; acridine complex; crystal structure; molecular dynamics
Introduction Guanine-rich tracts of nucleic acids can form four-stranded structures under appropriate conditions of high Naþ or Kþ ionic strength.1,2 These structures, G-quadruplexes, adopt a variety of folds, dependent in part on whether they are intra- or intermolecular.3 All quadruplexes contain the basic repeating motif, the G-quartet, which comprises four guanine bases held in plane by Hoogsteen hydrogen bonding. Typically, three or four G-quartets are stacked one on top of each other within a quadruplex, held together by p – p non-bonded attractive interactions. The intermolecular quadruplex formed by two strands of the sequence d(T4G4T4) from Oxytricha nova, has been extensively characterised by NMR studies,4 – 6 and more recently, by X-ray crystallography,7,8 which has shown it to have an arrangement of two antiparallel and two parallel strands, with a diagonal loop containing four thymine residues at each end of the quadruplex (Figure 1). The formation of guanine – quadruplex structures has been implicated in the molecular pathology of fragile X syndrome,9 and quadruplexes have been described as the motif in a number of oligonucleotides that show potential therapeutic Abbreviations used: NOE, nuclear Overhauser enhancement. E-mail address of the corresponding author: [email protected]
effects (see, for example, Marchand et al.10). Guanine-rich sequences that can potentially form quadruplexes, also occur at the ends of chromosomes, which consist of tandem repeats of simple G-rich sequences, such as d(TTAGGG) in vertebrates and TnGn in ciliates.11,12 The extreme 30 terminus of this telomeric DNA is usually singlestranded, often of only a few tens of bases, although 100 – 200 nucleotides at the ends of human chromosomes are single-stranded.13 Telomeric DNA progressively shortens during successive rounds of replication as a direct consequence of the inability of DNA polymerase to fully replicate these single-stranded ends. Telomeric DNA in ca 80– 85% of tumour cells is maintained in length by the action of the reverse transcriptase enzyme complex telomerase,14 which catalyses the synthesis of further telomeric DNA repeats onto the single-stranded 30 end.15 Telomerase is not activated in human somatic cells and is therefore a potential target for selective anti-cancer therapy.16 – 18 One particular approach that has been developed19 for telomerase inhibition uses small-molecule ligands to stabilise the primer telomeric DNA strand into folded quadruplex structures, which are effective inhibitors of telomerase action, since the RNA template component of telomerase has an absolute requirement for a singlestranded rather than a folded 30 telomeric DNA primer.20 A number of quadruplex-stabilising ligands have now been described, which show a high correlation between telomerase inhibition
0022-2836/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved
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Figure 1. Schematic showing the fold in the intermolecular quadruplex formed by two molecules of d(GGGGTTTTGGGG) (4– 7).
and quadruplex affinity.21 – 24 These ligands have the common feature of extended planar aromatic electron-deficient chromophores, such as acridine, porphyrin or phenanthrene ring systems. Structure activity studies have also highlighted the need for substituents possessing cationic charge with acridines, anthraquinones and other tricyclic systems, and molecular modelling studies have been used to guide the design of a number of potent new acridine analogues.25 – 27 There has been no atomic resolution structure reported to date of a quadruplex – ligand complex, which would aid structure-based design studies. NMR analyses have been reported of parallel quadruplexes, complexes typically comprising four strands of sequence d(TTAGGGT), with the perylene ligand PIPER,28 and with a pentacyclic acridinium compound.29 Even though line-broadening effects have tended to limit the number of observable nuclear Overhauser enhancements (NOEs), structural interpretations, have concurred in suggesting that these ligands stack on the ends of the quadruplexes, and are stabilised by p – p interactions with the terminal G-tetrad. The presence of alkaline metal ions, needed to stabilise native quadruplexes, is also required for their ligand complexes. Systematic molecular modelling studies on quadruplex interactions with an amido-
Figure 2. The structure of 3,6-bis-[3-pyrrolidino-propionamide] acridine (BSU6039).
A G-quadruplex-Ligand Structure
anthraquinone ligand30 and the cationic tetrapyridyl porphyrin TMPy431 have explored alternative binding modes, in which there is intercalation between individual G-tetrads embedded within a quadruplex. Such alternative modes have been rejected30,31 on energetic grounds, and instead have enabled plausible molecular models to be developed,32 for example for the interaction of the natural product telomestatin (a potent telomerase inhibitor), with the intramolecular quadruplex formed from the sequence d[AGGG(TTAGGG)3]. We report here on the crystal structure of a complex between the Oxytricha dimeric intermolecular quadruplex formed from two strands of d(GGGGTTTTGGGG), and an acridine derivative di-substituted at the 3 and 6 positions with 3-pyrrolidinopropionamide side-chains (Figure 2). This compound was previously reported by us25,27,33 to be an effective telomerase inhibitor and quadruplexbinding agent at the mM level. The present crystallographic analysis reveals the position of ligand binding within this quadruplex, and also shows that the thymine loop in this quadruplex plays an active role in stabilising the complex. This structure thus represents a paradigm for ligand binding within quadruplex DNA diagonal loops.
Results The asymmetric unit of the structure contains one molecule of a hairpin dimer G-quadruplex. Each quadruplex comprises two strands of the sequence d(GGGGTTTTGGGG) with a diagonal fold topology, in which the thymine loops are lying diagonally across the top and bottom of the stack of guanine quartets and the strands of each sequence are antiparallel to each other, as shown in Figure 1. This fold is identical to the one found in the crystal structures of this native quadruplex7 and that found by NMR studies in both Naþ and Kþ environments.4 – 6 The quadruplex shows an alternating syn – anti arrangement of guanine glycosidic torsion angles along the strand and syn –syn – anti –anti arrangement within the quartet. All Gquartets exhibit N1 –O6 and N2 –N7 Hoogsteen hydrogen bond base-pairing with average dis˚ and 2.87 A ˚ , respectively. The tances of 2.84 A sugar conformations can be best described as C30 endo. One base in each of the terminal quartet is slightly more tilted than the others. This stacks effectively with the 30 thymine residue from the loop. The diagonal topology of the structure results in one wide, two medium and one narrow groove. The central core of the quadruplex binds four potassium ions, counteracting the highly electronegative central channel created by the O6 carbonyl oxygen atoms of the guanine residues. The potassium ions are arranged linearly along the helical axis of the quadruplex, at an average separation of ˚ , and each having a square anti-prismatic co3.4 A ordination geometry that was also observed in the native Kþ quadruplex structure.7 Three of the ions
A G-quadruplex-Ligand Structure
119
Figure 3. (a) Stereo plot of the structure of the complex between BSU6039 and the d(GGGGTTTTGGGG) quadruplex. (b) Sigma-weighted 2Fo 2 Fc electron density map in the plane of the acridine chromophore. (c) A view onto the plane of the acridine chromophore (coloured cyan), showing its stacking onto the adjacent G-quartet (blue). The two thymine residues involved in direct drug interaction are also shown (coloured red). (d) Water hydration pattern in one of the four grooves of the quadruplex, showing the spine of water molecules that starts from the pyrrolidino group at the top end of the groove. (e) A view of the bound drug molecule, sandwiched between a G-quartet and a loop thymine residue. The weak hydrogen bonding interaction between the protonated nitrogen atom in the pyrrolidino group and the O40 atom of a terminal guanine is shown by dotted lines. Three water molecules are also shown. One interacts with the protonated nitrogen atom in the pyrrolidino group at the other end of the drug molecules. The other two hydrogen bond to the amide nitrogen atoms of the drug side-chains.
are sandwiched between the planes of the four quartets and the fourth one is present in the thymine loop. Each of the potassium ions is co-ordinated to eight oxygen atoms, either O6 of guanine, O2 of thymine residues, or oxygen atoms from a water molecule. This is distinct from the native quadruplex structure, in which a linear array of five potassium ions were observed. In the present structure, the bound drug molecule has displaced the fifth potassium ion one of the two T4 loops. The equivalent potassium ion within the loop at the other end of the molecule co-ordinates two water molecules to complete its octahedral co-ordination shell, analogous to the arrangement observed in the native structure.7 The drug binding site The structure has a single drug molecule bound per quadruplex, i.e. as a 1:1 complex (Figure 3(a)), even though drug was in excess in the crystallising experiment. We suggest that the present complex is thermodynamically favoured for crystal packing reasons: it may be that other crystals observed, but not examined because of morphological twin-
ning, contained a different drug– nucleotide ratio. The electron density of the BSU6039 molecule is well-resolved (Figure 3(b)). It shows that the drug binds to the quadruplex within one of the two T4 loops, with the second thymine residue of the loop positioned in the plane of the acridine chromophore (see below). The acridine chromophore is bound in an intercalation-type arrangement reminiscent of intercalation into duplex DNA, due to its extended electron-deficient aromatic ring system, enabling it to form strong p – p stacking interactions with nucleobases (Figure 3(c)). One face of the acridine is stacked on two of the guanine bases in the terminal G-quartet, with its long axis aligned along these two guanine bases. In turn the thymine-3 base is stacked asymmetrically on the other face, ensuring that the acridine chromophore is effectively sandwiched between the guanine bases and the thymine. Thymine-2 is directly involved in ligand binding. The central ring nitrogen atom in the acridine chromophore forms a hydrogen bond interaction ˚ ). This obserwith the O2 atom of thymine-2 (2.95 A vation requires the acridine ring nitrogen atom to be protonated in this environment, in accord with
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A G-quadruplex-Ligand Structure
Table 1. The conformation of the thymine nucleotides in the two loops Loop 1 Backbone torsion angle 0
a (P –O5 ) b (O50 –C50 ) g (C50 –C40 ) d (C40 –C30 ) 1 (C30 –O30 ) z (O30 –P0 ) x (C10 –N1)
Loop 2
T1
T2
T3
T4
T1
T2
T3
T4
201 172 52 151 208 279 221
288 213 52 86 194 293 201
171 171 188 141 257 194 198
53 190 68 143 262 283 228
293 166 62 85 181 274 225
303 176 55 144 300 107 257
156 197 49 152 220 274 184
292 185 58 144 205 62 240
Loop 1 is the loop in which the drug is bound. Torsion angles in this loop that are significantly changed from corresponding values in loop 2 are highlighted in bold.
its pKa value of . 7 in solution. The amide oxygen atom of one of the two side-chain substituents also forms a strong hydrogen bond with the N3 ˚ ). Both atom of the same thymine base (2.71 A amide groups of the drug are in a cis orientation with respect to the ring nitrogen atom. The amide nitrogen atom of the second substituent is hydro˚ ), which is gen-bonded to a water molecule (3.04 A ˚ itself hydrogen-bonded (2.59 A) to a phosphate oxygen atom of thymine-4 in the loop. This water molecule is the first in an extended chain of six water molecules that reaches into the adjacent groove, although it only penetrates halfway down the groove, terminating in a hydrogen bond to a phosphate group (Figure 3(d)). The two aminoalkylamido side-chains of the drug both adopt extended conformations, which position them close to the grooves of the quadruplex, although neither side-chain actually penetrates its groove. Each protonated nitrogen atom in the terminal pyrrolidino groups is involved in
Figure 4. Superposition of the two strands that form the structure, highlighting the different conformation of the thymine loops. The strand coloured red is drugbound, and that coloured green is drug-free.
hydrogen bonding interactions. One is positioned directly above the 50 end of one strand (Figure 3(e)), and forms a weak hydrogen bond with the O40 atom of the terminal deoxyribose of guanine1. The other pyrrolidino ring nitrogen atom is hydrogen-bonded to a water molecule at the top end of a groove. This water molecule in turn hydrogen bonds to an exocyclic N2 atom of a guanine base in the first G-quartet. The water molecule also participates in a network of water molecules that lies along much of this groove, making hydrogen-bonded contact with base edges and phosphate groups. Loop and groove structure The four thymine nucleotides in each 12-mer sequence constitute the loop regions. These loops form the characteristic diagonal fold-over topology exhibited by this Oxytricha telomeric DNA sequence. There are two loops, one at each end of the molecule. These loops are conformationally equivalent in the native quadruplex structure,7 but here they adopt very different conformations. The loop with no drug bound is conformationally and structurally similar to that of the native structure. Thus, the first thymine stacks on the tilted guanine of the terminal quartet. The next thymine in the sequence stacks on the first. The third thymine loops back to stack on the adjacent guanine from the other strand. It also forms an O2 –N3 hydrogen bond with the first thymine. The fourth thymine swings back to stack on the second thymine. The conformation of the other thymine loop is completely different from this (Table 1 and Figure 4). One molecule of the drug molecule is bound in the loop. The terminal thymine in the loop, thymine-1, is swung out from the loop and hangs over the mouth of the wide groove. This thymine forms Thy – Thy base stacking interactions with thymine-4 from a symmetry-related quadruplex in the crystal lattice. Thymine-2 is in the position of thymine-3, as observed in the drug-free loop, and it stacks on the tilted guanine in the terminal Gquartet. This thymine also forms the crucial hydrogen bonds with the central nitrogen atom of the aromatic chromophore of the drug and also with the amide side-chain of the drug molecule. Thymine-3 performs the intercalative function, as outlined above, and thymine-4 is oriented away from the quadruplex, with no interactions being involved other than those in the crystal. The noninvolvement of two thymine bases in drug binding suggests that only the backbones of the residues are needed to maintain the topology of the loop, whereas the bases themselves are not involved. This suggests that loops are highly flexible and can adopt several conformations depending upon the particular interactions required to take place with a ligand molecule. All four grooves in the quadruplex are highly hydrated. The water molecules tend to cluster around the phosphate groups. Several water –
A G-quadruplex-Ligand Structure
121
Figure 5. (a) Trajectory of the distance O2(thymine-2)…N(acridine) during the time-course of the simulation. (b) Trajectory of the distance N3(thymine-2)…Oamide(acridine) during the time-course of the simulation. (c) Least-squares superposition of the dynamics snapshots of the BSU6039 molecules, sampled every 200 ps during the course of the simulation.
water bridges link the phosphate groups together. Water molecules bind preferentially to N2 exocyclic amino group of guanine residues as opposed to the N3 group. Almost complete spines of hydration are observed in several grooves. Their incompleteness is possibly due to the lack of highresolution diffraction data, errors in phases, or most likely, the disorder and mobility of some solvent molecules. The patterns observed are linear zig-zag connections of water molecules that also make contact with base edges. The most prominent pattern of hydration observed is water-mediated interaction between the N2 amino group of guanine and phosphate groups on the adjacent strand. The loops also have a considerable amount of hydration, thus contributing to the essential interactions that maintain the integrity of the loop conformations, especially for the non-drug bound one. Dynamic behaviour The structures gave very stable trajectories over the entire 2 ns time-scale of the dynamics simulation. The overall geometry of the quadruplex structure was well conserved and loop structures maintained. The drug molecule overall showed low geometric fluctuations in its thymine-loop binding site. The two strong hydrogen-bonding interactions between thymine-2 and the drug remained very stable throughout the 2 ns period of the simulations (Figure 5(a) and (b)). The length of the hydrogen bond between O2 of thymine-2 and the central ring nitrogen atom of the acridine ˚ , averaged over the chromophore, was 2.90(0.13) A
entire trajectory. The second hydrogen bond, between the amide oxygen atom in one of the side-chains and the ring N3 atom in thymine-2, ˚ . These strong has an average length of 3.02(16) A interactions serve to restrict the movement of the drug and thus locking it in the binding site, so ˚ over the 2 ns that its overall rmsd value is 1.06 A of the simulation. The most mobile parts of the drug are the side-chains (Figure 5(c)). At times during the simulation run, the nitrogen atom in the pyrrolidine ring of one side-chain approaches the O40 atom of guanine-1, to form the hydrogenbonding interaction that is observed in the crystal. However, this average distance over the entire tra˚ , with this distance extending jectory is 4.76(08) A ˚ at times during the course of the run. to about 7 A Snapshots of the drug were taken after every 200 ps and the structures overlaid to display the flexibility of the side-chains. The central chromophore is the most rigid part of the drug molecule with the flexibility increasing towards the sidechains. Two water molecules have also been observed to form hydrogen bridges with the amide nitrogen atom in both the side-chains. However, these water molecules are not stable throughout the trajectory. They are mobile and exchange positions, being replaced by others during the course of the simulation, so that solvent molecules were always present at any given time in this region during all the snapshots taken for the trajectory. This behaviour is analogous to the structured water molecules in the grooves observed in the crystal structure, where side-chain amide nitrogen atoms from the drug molecule form hydrogen bridges with solvent water molecules.
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A G-quadruplex-Ligand Structure
Table 2. Crystallographic data Space group ˚) Cell dimensions (A Wavelength ˚ 3/Da) VM (A ˚) Max. resolution (A Completeness (%) Net I/s(I) Overall Rmerge(I) Rmerge of high-resolution shell Completeness of high-resolution shell (%) Data redundancy Total no. of reflections collected No. of unique reflections used in refinement ˚) Resolution range of refinement (A R-factor (%) Rfree (%) ˚) rmsd bond distances (A rmsd bond angles (8) No. of DNA atoms No. of solvent molecules No. of potassium ions Drug molecules/AU ˚ 2) Average B factor (A G-quartet Thymine loop 1 Thymine loop 2 Solvent Potassium ions Drug
P21212 55.451, 42.736, 26.926 1.542 1.215 1.75 94 13.8 0.058 0.109 96.2 3.8 24,883 6540 10– 1.75 15.11 22.45 0.02 0.04 506 148 4 1 13.3 15.8 13.7 31.0 11.2 18.5
Discussion This crystal structure shows that the G-quadruplex-binding ligand based on a 3,6-di-substituted aminoalkylamido acridine has a preferred binding site within a loop in the Oxytricha intermolecular G-quadruplex structure. The dynamics simulations show that the ligand is firmly bound within the site, and that key stacking and hydrogen-bonding interactions are maintained. The observation that this site is not intercalated between G-quartets in the quadruplex, but is at the end of the four Gquartets in this structure, confirms and extends earlier both NMR and molecular modelling studies with a range of ligands and quadruplexes.28,29,31 It suggests that terminal stacking in general occurs with ligands possessing a planar aromatic moiety, and that the energetics are unfavourable for opening up an intercalation site that is embedded within a stack of G-quartets. The present structure also provides information on other factors involved in ligand binding to quadruplexes with diagonal-type loops, which can be formed with both Oxytricha and human telomeric DNA sequences.25,27,30,34 We see here a major reorganisation of loop conformation, with participation of bases not only in stacking either side of the chromophore, but also actively interacting with the ligand in the plane of the chromophore. It is thus advantageous for the chromophore to have appropriate atoms or groups that can participate in such interactions, and so enhance affinity and selectivity. It was initially conceived, when developing
quadruplex-binding ligands with amide groups, that these groups serve only to enhance the p-electron delocalisation of the chromophore. We see in this structure that they can play a more active role in directly hydrogen-bonding to a loop residue. The same geometry of binding involving in-plane base-chromophore hydrogen-bonding is not available to other quadruplex-binding ligands, such as di-substituted amidoanthraquinones,19,26 tetra-Npyridyl porphyrins,21,30 the substituted polycyclic compound PIPER,28 or the natural product telomestatin,32 which all lack a hydrogen-bond donor in an equivalent position on the chromophore. However, the flexibility of the tetranucleotide loop is such that alternative conformations are highly likely to be accessible, which might accommodate other ligands such as these, and thus establish other analogous modes of intermolecular interactions. The crystal structure shows that the side-chains of this ligand are too short to be interacting fully in the quadruplex grooves. The dynamics simulations demonstrate their mobility, and that the weak hydrogen bonding arrangements seen in the crystal can be considered to be time-averaged views of their flexibility. Longer side-chains would penetrate further into the grooves, and these may be held more firmly, especially if they are able to interact with phosphate groups. The structural information to date on quadruplexes formed from telomeric DNA sequences shows that they can be considered in two categories, (i) Having a pattern of anti/parallel strands with diagonal loops that are positioned above and below the plane of G-quartets in a quadruplex of the present Oxytricha sequence and the Naþ form of the human intramolecular quadruplex.34 (ii) Having all-parallel strands, with a very different pattern of external loops that are arranged in the groves rather than stacked on the G-quartets, as observed in the Kþ form of the d(TTAGGG) repeat intramolecular quadruplex.2 The present structure emphasises the general feature of chromophore stacking interactions with the terminal G-quartet and the unlikelihood of G-quartet intercalation, which is relevant to both categories. It does not show how the loops in allparallel structures such as in the Kþ form of the human telomere repeat quadruplex structures,2 may be involved in ligand binding, although analogous arrangements may be formed by the diagonal loop in the Naþ form of the quadruplex formed from human telomeric DNA repeats.34 The position of the bound ligand found here is in accord with that deduced from low-angle scattering data on an intermolecular parallel-stranded ligand complex31 involving a di-substituted
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A G-quadruplex-Ligand Structure
amidoanthraquinone, supporting the suggestion that chromophores generally prefer to bind on the exterior of guanine-tetrad stacks in a quadruplex, whether or not the strands are all-parallel. The type of arrangement found here is also likely to have relevance to ligand binding to loops in nontelomeric quadruplexes. This is exemplified by the guanine-rich sequence upstream from the promoter of the c-myc oncogene,35 which is stabilised as an intrastrand fold-back quadruplex by ligands such as the perylenediimide ligand PIPER.36
Materials and Methods Crystallisation and data collection The DNA sequence d(GGGGTTTTGGGG) was purchased from Oswel DNA Service (Southampton). The ligand, as the hydrochloride salt, was synthesised by Dr R. J. Harrison of this laboratory, and was analytically pure. Crystals were prepared by the hanging dropvapour diffusion method at 12 8C. The drop solution contained 5% (w/v) 2-methylpentane-2,4-diol, 10 mM MgCl2, 1.6 mM spermine, 0.5 mM DNA and 1 mM ligand, with 20 mM potassium cacodylate buffered at pH 7.0. This was equilibrated against a gradient of 30% (w/v) 2-methylpentane-2,4-diol in 20 mM KCl. Crystals grew in four to six weeks as cubic blocks of approximate dimensions 0.3 mm £ 0.3 mm £ 0.2 mm. Crystal parameters are given in Table 2. Crystals were harvested and directly flash-frozen in liquid nitrogen. Data were collected at 110 K using an in-house Rikagu RaxisIV image plate system on a rotating anode X-ray source, using Osmic optics to produce monochromatic radiation. Data were integrated with the DENZO/SCALEPACK software package.37 Structure determination and refinement The structure of the BSU6039-Quadruplex DNA complex was solved by molecular replacement by means of the program EPMR.38 The dimeric quadruplex molecule from the trigonal form of the native quadruplex DNA crystal structure that has been recently solved by us (RCSB Protein Data Bank code 1JPQ), was used as the search model.7 The molecular replacement rotation and translation searches each produced one clear solution for the orientation and position in the unit cell of the search molecule, indicating that there is one quadruplex per crystallographic asymmetric unit. Refinement on this starting structure was initially performed with the CNS program.39 Refinement converged after a few rounds of simulated annealing and individual temperature factor refinement. The overall correctness of the structure was judged by the decrease in the value of Rfree during this refinement. One loop was omitted at this stage. Subsequent refinement was carried out with the SHELX-97 program.† This resulted in significant decreases in the values of both the R-factor and Rfree and the resulting model was inspected manually with s-weighted omit and 2Fo 2 Fc electron density maps. These revealed a considerable † Sheldrick, G. M. (1997). SHELX-97, a crystallographic refinement program. The University of Go¨ttingen, Go¨ttingen, Germany.
amount of new electron density in one loop region, which corresponded in shape and dimensions to a molecule of the ligand. A number of other significant regions of electron density in the loop region indicated that the structure of the ligand-bound loop had changed substantially compared to its structure in the native quadruplex, and the conformation of all its residues had to be rebuilt manually. No extraneous density was observed in the other thymine loop, which behaved normally during the refinements. Water molecules were located from difference Fourier maps and judicious use of the automatic water selection procedure in SHELX-97, using standard hydrogen bond geometric criteria and Rfree to judge whether selected water molecules were real or not. Geometric parameters for the ligand were obtained from a molecular mechanics minimised structure. The complete ˚ resolution, complex was subsequently refined to 1.75 A to yield final values for the R-factor and Rfree of 15.11% and 22.45%, respectively. Refinement statistics are given in Table 2. Protein Data Bank accession numbers Co-ordinate and structure factor data have been deposited in the RCSB Protein Data Bank (accession code 1L1H). Molecular dynamics simulations The initial starting co-ordinates were taken from the crystal structure of the quadruplex complex. The structure was not subjected to any initial molecular mechanics energy minimisation prior to starting the simulations. A molecular model of the drug was constructed and partial charges calculated semi-empirically using MOPAC in the InsightII software. The AMBER 6.0 suite‡ was used to carry out all molecular dynamics simulations. The force-field parameters for the ligand were extrapolated from the existing values for analogous groups in the AMBER and CFF force fields. The DNA – drug complex was then solvated in a periodic TIP3 water box of dimen˚ £ 50 A ˚ £ 50 A ˚ that extended at least 10 A ˚ sions ,50 A from any solute atom. The X-ray structure had revealed a vertical alignment of consecutive potassium ions along the helical axis within the central core of the complex, between the guanine residues of each G-quartet. The ions were retained in the positions observed in the crystal structure. Additional positively charged potassium counter-ions were included in the system to neutralise the charge on the DNA backbone. These maintained consistency with the crystallisation conditions and hence simulated a uniform Kþ ionic environment. The counter-ions were automatically placed by the LEAP program throughout the water box, at grid points of negative Coulombic potential. The final system had a net zero charge. The resulting complex was then equilibrated with the explicit solvent molecules by 1000 steps of minimisation and 25 ps of molecular dynamics at 300 K. The entire complex was kept constrained, while allowing the ions and the solvent molecules to equilibrate. The complex was then subjected to a series of minimisations and dynamics calculations in which the constraints were gradually relaxed over six successive steps, until no ‡ P. A. Kollman et al. (1999). AMBER 6.0, University of California, San Francisco.
124
constraints at all were being applied. The final production run was performed without any restraints on the complex, for 2000 ps and co-ordinates were saved after every 10 ps for analysis of their trajectories. All calculations were carried out with the SANDER module of AMBER 6.0, and with the SHAKE algorithm enabled for ˚ non-bonded Lennard– Jones hydrogen atoms. A 10 A cut-off was used and the non-bonded pairs list updated every 20 steps. The particle-mesh-Ewald summation term was used for all simulations, in order to include all long-range electrostatic interactions in the calculations. ˚ , and the The PME charge grid spacing was , 1.0 A charge grid chosen to be products of the powers two, three and five to ensure efficiency for the fast-Fourier transform calculation. Trajectories were analysed using the CARNAL and RDPARM modules available in the AMBER 6.0 suite.
A G-quadruplex-Ligand Structure
11. 12. 13.
14.
15. 16.
Acknowledgements This work took place at the Institute of Cancer Research, who provided a research studentship to S.M.H. It, together with Cancer Research UK (programme grant SP13894) is thanked for supporting these studies. We are grateful to John Harrison for his synthesis of the acridine ligand, and together with Martin Read and Tony Reszka, for many useful discussions.
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topoisomerase I with Interaction of human nuclear topoisomerase I with guanosine quartet-forming and guanosine-rich single-stranded DNA and RNA oligonucleotides. J. Biol. Chem. 277, 8906– 8911. Blackburn, E. H. (2001). Switching and signalling at the telomere. Cell, 106, 661– 673. Cech, T. R. (2000). Life at the end of chromosomes: telomeres and telomerase. Angew. Chem. Int. Ed. 39, 34 –43. Wright, W. E., Tesmer, V. M., Huffman, K. E., Levene, S. D. & Shay, J. W. (1997). Normal human chromosomes have long G-rich telomeric overhangs at one end. Genes Dev. 11, 2801 –2809. Kim, N. W., Piatyszek, M. A., Prowse, K. R., Harley, C. B., West, M. D., Ho, P. L. C. et al. (1994). Specific association of human telomerase activity with immortal cell and cancer. Science, 266, 2011 – 2015. Greider, C. W. & Blackburn, E. H. (1985). Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell, 43, 405–413. White, L. K., Wright, W. E. & Shay, J. W. (2001). Telomerase inhibitors. Trends Biotechnol. 19, 114 – 120. Mergny, J.-L., Riou, J.-F., Maillet, P., Teulade-Fichou, M.-P. & Gilson, E. (2002). Natural and pharmacological regulation of telomerase. Nucl. Acids Res. 30, 839– 865. Neidle, S. & Parkinson, G. N. (2002). Telomere maintenance as a target for anticancer drug discovery. Nature Rev. Drug Discovery, 1, 383– 393. Sun, D., Thompson, B., Cathers, B. E., Salazar, M., Kerwin, S. M., Trent, J. O. et al. (1997). Inhibition of human telomerase by a G-quadruplex-interactive compound. J. Med. Chem. 40, 2113 – 2116. Zahler, A. M., Williamson, J. R., Cech, T. R. & Prescott, D. M. (1991). Inhibition of telomerase by Gquartet DNA structures. Nature, 350, 718– 720. Shi, D. F., Wheelhouse, R. T., Sun, D. & Hurley, L. H. (2001). Quadruplex-interactive agents as telomerase inhibitors: synthesis of porphyrins and structureactivity relationship for the inhibition of telomerase. J. Med. Chem. 44, 4509– 4523. Mergny, J.-L., Lacroix, L., Teulade, Fichou, M.-P., Hounsou, C., Guittal, L. et al. (2001). Telomerase inhibitors based on quadruplex ligands selected by a fluorescence assay. Proc. Natl Acad. Sci. USA, 98, 3062– 3067. Riou, J.-F., Guittat, L., Mailliet, P., Laoui, A., Renou, E., Petitgenet, O. et al. (2002). Cell senescence and telomere shortening induced by a new series of specific G-quadruplex DNA ligands. Proc. Natl Acad. Sci. USA, 99, 2672– 2677. Heald, R. A., Modi, C., Cookson, J. C., Hutchinson, I., Laughton, C. A., Gowan, S. M. et al. (2002). Antitumor polycyclic acridines. 8. Synthesis and telomerase-inhibitory activity of methylated pentacyclic acridinium salts. J. Med. Chem. 45, 590– 597. Read, M. A., Wood, A. A., Harrison, J. R., Gowan, S. M., Kelland, L. R., Dosanjh, H. S. & Neidle, S. (1999). Molecular modelling studies on G-quadruplex complexes of telomerase inhibitors: structureactivity relationships. J. Med. Chem. 42, 4538– 4546. Neidle, S., Harrison, R. J., Reszka, A. P. & Read, M. A. (2000). Structure-activity relationships among guanine– quadruplex telomerase inhibitors. Pharmacol. Ther. 85, 133– 139. Read, M. A., Harrison, R. J., Romagnoli, B., Tanious, F. A., Gowan, S. H., Reszka, A. P. et al. (2001). Structure-based design of selective and potent G
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28.
29.
30.
31. 32.
quadruplex-mediated telomerase inhibitors. Proc. Natl Acad. Sci. USA, 98, 4844– 4849. Fedoroff, O. Y., Salazar, M., Han, H., Chemeris, V. V., Kerwin, S. M. & Hurley, L. H. (1998). NMR-based model of a telomerase-inhibiting compound bound to G-quadruplex DNA. Biochemistry, 37, 12367– 12374. Gavathiotis, E., Heald, R. A., Stevens, F. G. & Searle, M. S. (2001). Recognition and stabilisation of quadruplex DNA by a potent new telomerase inhibitor: NMR studies of the 2:1 complex of a pentacyclic methylacridinium cation with d(TTAGGGT)4. Angew. Chem. Int. Ed. 40, 4749– 4751. Han, H., Langley, D. R., Rangan, A. & Hurley, L. H. (2001). Selective interactions of cationic porphyrins with G-quadruplex structures. J. Am. Chem. Soc. 123, 8902–8913. Read, M. A. & Neidle, S. (2000). Structural characterization of a guanine – quadruplex ligand complex. Biochemistry, 39, 13422– 13432. Kim, M. Y., Vankayalapati, H., Shin-ya, K., Wierzba, K. & Hurley, L. H. (2002). Telomestatin, a potent telomerase inhibitor that interacts quite specifically with the human telomeric intramolecular G-quadruplex. J. Am. Chem. Soc. 124, 2098– 2099.
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33. Harrison, R. J., Gowan, S. M., Kelland, L. R. & Neidle, S. (1999). Human telomerase inhibition by substituted acridine derivatives. Bioorg. Med. Chem. Letters, 9, 2463– 2468. 34. Wang, Y. & Patel, D. J. (1993). Solution structure of the human telomeric repeat d[AG3(T2AG3)3]. Structure, 1, 263– 282. 35. Simonsson, T., Pecinka, P. & Kubista, M. (1998). DNA tetraplex formation in the control region of c-myc. Nucl. Acids Res. 26, 1167– 1172. 36. Rangan, A., Federoff, O. Y. & Hurley, L. H. (2001). Induction of duplex to G-quadruplex transition in the c-myc promoter region by a small molecule. J. Biol. Chem. 276, 4640– 4646. 37. Otwinowski, Z. & Minor, W. (1993). Data Collection and Processing (Sawyer, L., Isaacs, N. W. & Bailey, S., eds), SERC Daresbury Laboratory, Warrington, UK. 38. Kissinger, C. R., Gehlhaar, K. & Fogel, D. B. (1999). Rapid automated molecular replacement by evolutionary search. Acta Crystallog. sect. D, 55, 484– 491. 39. Bru¨nger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P. & Grosse-Kunstleve, R. W. (1998). Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallog. sect. D, 54, 905–921.
Edited by A. Klug (Received 13 September 2002; received in revised form 14 November 2002; accepted 21 November 2002)
J. Mol. Biol. (2003) 326, 117–125
Structure of a G-quadruplex –Ligand Complex Shozeb M. Haider, Gary N. Parkinson and Stephen Neidle* Cancer Research UK Biomolecular Structure Group The School of Pharmacy University of London, 29-39 Brunswick Square, London WC1N 1AX, UK
Stabilisation of G-quadruplex structures formed from telomeric DNA, by means of quadruplex-selective ligands, is a means of inhibiting the telomerase enzyme from catalysing the synthesis of telomeric DNA repeats. In order to understand the molecular basis of ligand –quadruplex recognition, the crystal structure has been determined of such a complex, at ˚ resolution. This complex is between a dimeric antiparallel G-quad1.75 A ruplex formed from the Oxytricha nova telomeric DNA sequence d(GGGGTTTTGGGG), and a di-substituted aminoalkylamido acridine compound. The structure shows that the acridine moiety is bound at one end of the stack of G-quartets, within one of the thymine loops. It is held in place by a combination of stacking interactions and specific hydrogen bonds with thymine bases. The stability of the ligand in this binding site has been confirmed by a 2 ns molecular dynamics simulation. q 2003 Elsevier Science Ltd. All rights reserved
*Corresponding author
Keywords: G-quadruplex; acridine complex; crystal structure; molecular dynamics
Introduction Guanine-rich tracts of nucleic acids can form four-stranded structures under appropriate conditions of high Naþ or Kþ ionic strength.1,2 These structures, G-quadruplexes, adopt a variety of folds, dependent in part on whether they are intra- or intermolecular.3 All quadruplexes contain the basic repeating motif, the G-quartet, which comprises four guanine bases held in plane by Hoogsteen hydrogen bonding. Typically, three or four G-quartets are stacked one on top of each other within a quadruplex, held together by p – p non-bonded attractive interactions. The intermolecular quadruplex formed by two strands of the sequence d(T4G4T4) from Oxytricha nova, has been extensively characterised by NMR studies,4 – 6 and more recently, by X-ray crystallography,7,8 which has shown it to have an arrangement of two antiparallel and two parallel strands, with a diagonal loop containing four thymine residues at each end of the quadruplex (Figure 1). The formation of guanine – quadruplex structures has been implicated in the molecular pathology of fragile X syndrome,9 and quadruplexes have been described as the motif in a number of oligonucleotides that show potential therapeutic Abbreviations used: NOE, nuclear Overhauser enhancement. E-mail address of the corresponding author: [email protected]
effects (see, for example, Marchand et al.10). Guanine-rich sequences that can potentially form quadruplexes, also occur at the ends of chromosomes, which consist of tandem repeats of simple G-rich sequences, such as d(TTAGGG) in vertebrates and TnGn in ciliates.11,12 The extreme 30 terminus of this telomeric DNA is usually singlestranded, often of only a few tens of bases, although 100 – 200 nucleotides at the ends of human chromosomes are single-stranded.13 Telomeric DNA progressively shortens during successive rounds of replication as a direct consequence of the inability of DNA polymerase to fully replicate these single-stranded ends. Telomeric DNA in ca 80– 85% of tumour cells is maintained in length by the action of the reverse transcriptase enzyme complex telomerase,14 which catalyses the synthesis of further telomeric DNA repeats onto the single-stranded 30 end.15 Telomerase is not activated in human somatic cells and is therefore a potential target for selective anti-cancer therapy.16 – 18 One particular approach that has been developed19 for telomerase inhibition uses small-molecule ligands to stabilise the primer telomeric DNA strand into folded quadruplex structures, which are effective inhibitors of telomerase action, since the RNA template component of telomerase has an absolute requirement for a singlestranded rather than a folded 30 telomeric DNA primer.20 A number of quadruplex-stabilising ligands have now been described, which show a high correlation between telomerase inhibition
0022-2836/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved
118
Figure 1. Schematic showing the fold in the intermolecular quadruplex formed by two molecules of d(GGGGTTTTGGGG) (4– 7).
and quadruplex affinity.21 – 24 These ligands have the common feature of extended planar aromatic electron-deficient chromophores, such as acridine, porphyrin or phenanthrene ring systems. Structure activity studies have also highlighted the need for substituents possessing cationic charge with acridines, anthraquinones and other tricyclic systems, and molecular modelling studies have been used to guide the design of a number of potent new acridine analogues.25 – 27 There has been no atomic resolution structure reported to date of a quadruplex – ligand complex, which would aid structure-based design studies. NMR analyses have been reported of parallel quadruplexes, complexes typically comprising four strands of sequence d(TTAGGGT), with the perylene ligand PIPER,28 and with a pentacyclic acridinium compound.29 Even though line-broadening effects have tended to limit the number of observable nuclear Overhauser enhancements (NOEs), structural interpretations, have concurred in suggesting that these ligands stack on the ends of the quadruplexes, and are stabilised by p – p interactions with the terminal G-tetrad. The presence of alkaline metal ions, needed to stabilise native quadruplexes, is also required for their ligand complexes. Systematic molecular modelling studies on quadruplex interactions with an amido-
Figure 2. The structure of 3,6-bis-[3-pyrrolidino-propionamide] acridine (BSU6039).
A G-quadruplex-Ligand Structure
anthraquinone ligand30 and the cationic tetrapyridyl porphyrin TMPy431 have explored alternative binding modes, in which there is intercalation between individual G-tetrads embedded within a quadruplex. Such alternative modes have been rejected30,31 on energetic grounds, and instead have enabled plausible molecular models to be developed,32 for example for the interaction of the natural product telomestatin (a potent telomerase inhibitor), with the intramolecular quadruplex formed from the sequence d[AGGG(TTAGGG)3]. We report here on the crystal structure of a complex between the Oxytricha dimeric intermolecular quadruplex formed from two strands of d(GGGGTTTTGGGG), and an acridine derivative di-substituted at the 3 and 6 positions with 3-pyrrolidinopropionamide side-chains (Figure 2). This compound was previously reported by us25,27,33 to be an effective telomerase inhibitor and quadruplexbinding agent at the mM level. The present crystallographic analysis reveals the position of ligand binding within this quadruplex, and also shows that the thymine loop in this quadruplex plays an active role in stabilising the complex. This structure thus represents a paradigm for ligand binding within quadruplex DNA diagonal loops.
Results The asymmetric unit of the structure contains one molecule of a hairpin dimer G-quadruplex. Each quadruplex comprises two strands of the sequence d(GGGGTTTTGGGG) with a diagonal fold topology, in which the thymine loops are lying diagonally across the top and bottom of the stack of guanine quartets and the strands of each sequence are antiparallel to each other, as shown in Figure 1. This fold is identical to the one found in the crystal structures of this native quadruplex7 and that found by NMR studies in both Naþ and Kþ environments.4 – 6 The quadruplex shows an alternating syn – anti arrangement of guanine glycosidic torsion angles along the strand and syn –syn – anti –anti arrangement within the quartet. All Gquartets exhibit N1 –O6 and N2 –N7 Hoogsteen hydrogen bond base-pairing with average dis˚ and 2.87 A ˚ , respectively. The tances of 2.84 A sugar conformations can be best described as C30 endo. One base in each of the terminal quartet is slightly more tilted than the others. This stacks effectively with the 30 thymine residue from the loop. The diagonal topology of the structure results in one wide, two medium and one narrow groove. The central core of the quadruplex binds four potassium ions, counteracting the highly electronegative central channel created by the O6 carbonyl oxygen atoms of the guanine residues. The potassium ions are arranged linearly along the helical axis of the quadruplex, at an average separation of ˚ , and each having a square anti-prismatic co3.4 A ordination geometry that was also observed in the native Kþ quadruplex structure.7 Three of the ions
A G-quadruplex-Ligand Structure
119
Figure 3. (a) Stereo plot of the structure of the complex between BSU6039 and the d(GGGGTTTTGGGG) quadruplex. (b) Sigma-weighted 2Fo 2 Fc electron density map in the plane of the acridine chromophore. (c) A view onto the plane of the acridine chromophore (coloured cyan), showing its stacking onto the adjacent G-quartet (blue). The two thymine residues involved in direct drug interaction are also shown (coloured red). (d) Water hydration pattern in one of the four grooves of the quadruplex, showing the spine of water molecules that starts from the pyrrolidino group at the top end of the groove. (e) A view of the bound drug molecule, sandwiched between a G-quartet and a loop thymine residue. The weak hydrogen bonding interaction between the protonated nitrogen atom in the pyrrolidino group and the O40 atom of a terminal guanine is shown by dotted lines. Three water molecules are also shown. One interacts with the protonated nitrogen atom in the pyrrolidino group at the other end of the drug molecules. The other two hydrogen bond to the amide nitrogen atoms of the drug side-chains.
are sandwiched between the planes of the four quartets and the fourth one is present in the thymine loop. Each of the potassium ions is co-ordinated to eight oxygen atoms, either O6 of guanine, O2 of thymine residues, or oxygen atoms from a water molecule. This is distinct from the native quadruplex structure, in which a linear array of five potassium ions were observed. In the present structure, the bound drug molecule has displaced the fifth potassium ion one of the two T4 loops. The equivalent potassium ion within the loop at the other end of the molecule co-ordinates two water molecules to complete its octahedral co-ordination shell, analogous to the arrangement observed in the native structure.7 The drug binding site The structure has a single drug molecule bound per quadruplex, i.e. as a 1:1 complex (Figure 3(a)), even though drug was in excess in the crystallising experiment. We suggest that the present complex is thermodynamically favoured for crystal packing reasons: it may be that other crystals observed, but not examined because of morphological twin-
ning, contained a different drug– nucleotide ratio. The electron density of the BSU6039 molecule is well-resolved (Figure 3(b)). It shows that the drug binds to the quadruplex within one of the two T4 loops, with the second thymine residue of the loop positioned in the plane of the acridine chromophore (see below). The acridine chromophore is bound in an intercalation-type arrangement reminiscent of intercalation into duplex DNA, due to its extended electron-deficient aromatic ring system, enabling it to form strong p – p stacking interactions with nucleobases (Figure 3(c)). One face of the acridine is stacked on two of the guanine bases in the terminal G-quartet, with its long axis aligned along these two guanine bases. In turn the thymine-3 base is stacked asymmetrically on the other face, ensuring that the acridine chromophore is effectively sandwiched between the guanine bases and the thymine. Thymine-2 is directly involved in ligand binding. The central ring nitrogen atom in the acridine chromophore forms a hydrogen bond interaction ˚ ). This obserwith the O2 atom of thymine-2 (2.95 A vation requires the acridine ring nitrogen atom to be protonated in this environment, in accord with
120
A G-quadruplex-Ligand Structure
Table 1. The conformation of the thymine nucleotides in the two loops Loop 1 Backbone torsion angle 0
a (P –O5 ) b (O50 –C50 ) g (C50 –C40 ) d (C40 –C30 ) 1 (C30 –O30 ) z (O30 –P0 ) x (C10 –N1)
Loop 2
T1
T2
T3
T4
T1
T2
T3
T4
201 172 52 151 208 279 221
288 213 52 86 194 293 201
171 171 188 141 257 194 198
53 190 68 143 262 283 228
293 166 62 85 181 274 225
303 176 55 144 300 107 257
156 197 49 152 220 274 184
292 185 58 144 205 62 240
Loop 1 is the loop in which the drug is bound. Torsion angles in this loop that are significantly changed from corresponding values in loop 2 are highlighted in bold.
its pKa value of . 7 in solution. The amide oxygen atom of one of the two side-chain substituents also forms a strong hydrogen bond with the N3 ˚ ). Both atom of the same thymine base (2.71 A amide groups of the drug are in a cis orientation with respect to the ring nitrogen atom. The amide nitrogen atom of the second substituent is hydro˚ ), which is gen-bonded to a water molecule (3.04 A ˚ itself hydrogen-bonded (2.59 A) to a phosphate oxygen atom of thymine-4 in the loop. This water molecule is the first in an extended chain of six water molecules that reaches into the adjacent groove, although it only penetrates halfway down the groove, terminating in a hydrogen bond to a phosphate group (Figure 3(d)). The two aminoalkylamido side-chains of the drug both adopt extended conformations, which position them close to the grooves of the quadruplex, although neither side-chain actually penetrates its groove. Each protonated nitrogen atom in the terminal pyrrolidino groups is involved in
Figure 4. Superposition of the two strands that form the structure, highlighting the different conformation of the thymine loops. The strand coloured red is drugbound, and that coloured green is drug-free.
hydrogen bonding interactions. One is positioned directly above the 50 end of one strand (Figure 3(e)), and forms a weak hydrogen bond with the O40 atom of the terminal deoxyribose of guanine1. The other pyrrolidino ring nitrogen atom is hydrogen-bonded to a water molecule at the top end of a groove. This water molecule in turn hydrogen bonds to an exocyclic N2 atom of a guanine base in the first G-quartet. The water molecule also participates in a network of water molecules that lies along much of this groove, making hydrogen-bonded contact with base edges and phosphate groups. Loop and groove structure The four thymine nucleotides in each 12-mer sequence constitute the loop regions. These loops form the characteristic diagonal fold-over topology exhibited by this Oxytricha telomeric DNA sequence. There are two loops, one at each end of the molecule. These loops are conformationally equivalent in the native quadruplex structure,7 but here they adopt very different conformations. The loop with no drug bound is conformationally and structurally similar to that of the native structure. Thus, the first thymine stacks on the tilted guanine of the terminal quartet. The next thymine in the sequence stacks on the first. The third thymine loops back to stack on the adjacent guanine from the other strand. It also forms an O2 –N3 hydrogen bond with the first thymine. The fourth thymine swings back to stack on the second thymine. The conformation of the other thymine loop is completely different from this (Table 1 and Figure 4). One molecule of the drug molecule is bound in the loop. The terminal thymine in the loop, thymine-1, is swung out from the loop and hangs over the mouth of the wide groove. This thymine forms Thy – Thy base stacking interactions with thymine-4 from a symmetry-related quadruplex in the crystal lattice. Thymine-2 is in the position of thymine-3, as observed in the drug-free loop, and it stacks on the tilted guanine in the terminal Gquartet. This thymine also forms the crucial hydrogen bonds with the central nitrogen atom of the aromatic chromophore of the drug and also with the amide side-chain of the drug molecule. Thymine-3 performs the intercalative function, as outlined above, and thymine-4 is oriented away from the quadruplex, with no interactions being involved other than those in the crystal. The noninvolvement of two thymine bases in drug binding suggests that only the backbones of the residues are needed to maintain the topology of the loop, whereas the bases themselves are not involved. This suggests that loops are highly flexible and can adopt several conformations depending upon the particular interactions required to take place with a ligand molecule. All four grooves in the quadruplex are highly hydrated. The water molecules tend to cluster around the phosphate groups. Several water –
A G-quadruplex-Ligand Structure
121
Figure 5. (a) Trajectory of the distance O2(thymine-2)…N(acridine) during the time-course of the simulation. (b) Trajectory of the distance N3(thymine-2)…Oamide(acridine) during the time-course of the simulation. (c) Least-squares superposition of the dynamics snapshots of the BSU6039 molecules, sampled every 200 ps during the course of the simulation.
water bridges link the phosphate groups together. Water molecules bind preferentially to N2 exocyclic amino group of guanine residues as opposed to the N3 group. Almost complete spines of hydration are observed in several grooves. Their incompleteness is possibly due to the lack of highresolution diffraction data, errors in phases, or most likely, the disorder and mobility of some solvent molecules. The patterns observed are linear zig-zag connections of water molecules that also make contact with base edges. The most prominent pattern of hydration observed is water-mediated interaction between the N2 amino group of guanine and phosphate groups on the adjacent strand. The loops also have a considerable amount of hydration, thus contributing to the essential interactions that maintain the integrity of the loop conformations, especially for the non-drug bound one. Dynamic behaviour The structures gave very stable trajectories over the entire 2 ns time-scale of the dynamics simulation. The overall geometry of the quadruplex structure was well conserved and loop structures maintained. The drug molecule overall showed low geometric fluctuations in its thymine-loop binding site. The two strong hydrogen-bonding interactions between thymine-2 and the drug remained very stable throughout the 2 ns period of the simulations (Figure 5(a) and (b)). The length of the hydrogen bond between O2 of thymine-2 and the central ring nitrogen atom of the acridine ˚ , averaged over the chromophore, was 2.90(0.13) A
entire trajectory. The second hydrogen bond, between the amide oxygen atom in one of the side-chains and the ring N3 atom in thymine-2, ˚ . These strong has an average length of 3.02(16) A interactions serve to restrict the movement of the drug and thus locking it in the binding site, so ˚ over the 2 ns that its overall rmsd value is 1.06 A of the simulation. The most mobile parts of the drug are the side-chains (Figure 5(c)). At times during the simulation run, the nitrogen atom in the pyrrolidine ring of one side-chain approaches the O40 atom of guanine-1, to form the hydrogenbonding interaction that is observed in the crystal. However, this average distance over the entire tra˚ , with this distance extending jectory is 4.76(08) A ˚ at times during the course of the run. to about 7 A Snapshots of the drug were taken after every 200 ps and the structures overlaid to display the flexibility of the side-chains. The central chromophore is the most rigid part of the drug molecule with the flexibility increasing towards the sidechains. Two water molecules have also been observed to form hydrogen bridges with the amide nitrogen atom in both the side-chains. However, these water molecules are not stable throughout the trajectory. They are mobile and exchange positions, being replaced by others during the course of the simulation, so that solvent molecules were always present at any given time in this region during all the snapshots taken for the trajectory. This behaviour is analogous to the structured water molecules in the grooves observed in the crystal structure, where side-chain amide nitrogen atoms from the drug molecule form hydrogen bridges with solvent water molecules.
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A G-quadruplex-Ligand Structure
Table 2. Crystallographic data Space group ˚) Cell dimensions (A Wavelength ˚ 3/Da) VM (A ˚) Max. resolution (A Completeness (%) Net I/s(I) Overall Rmerge(I) Rmerge of high-resolution shell Completeness of high-resolution shell (%) Data redundancy Total no. of reflections collected No. of unique reflections used in refinement ˚) Resolution range of refinement (A R-factor (%) Rfree (%) ˚) rmsd bond distances (A rmsd bond angles (8) No. of DNA atoms No. of solvent molecules No. of potassium ions Drug molecules/AU ˚ 2) Average B factor (A G-quartet Thymine loop 1 Thymine loop 2 Solvent Potassium ions Drug
P21212 55.451, 42.736, 26.926 1.542 1.215 1.75 94 13.8 0.058 0.109 96.2 3.8 24,883 6540 10– 1.75 15.11 22.45 0.02 0.04 506 148 4 1 13.3 15.8 13.7 31.0 11.2 18.5
Discussion This crystal structure shows that the G-quadruplex-binding ligand based on a 3,6-di-substituted aminoalkylamido acridine has a preferred binding site within a loop in the Oxytricha intermolecular G-quadruplex structure. The dynamics simulations show that the ligand is firmly bound within the site, and that key stacking and hydrogen-bonding interactions are maintained. The observation that this site is not intercalated between G-quartets in the quadruplex, but is at the end of the four Gquartets in this structure, confirms and extends earlier both NMR and molecular modelling studies with a range of ligands and quadruplexes.28,29,31 It suggests that terminal stacking in general occurs with ligands possessing a planar aromatic moiety, and that the energetics are unfavourable for opening up an intercalation site that is embedded within a stack of G-quartets. The present structure also provides information on other factors involved in ligand binding to quadruplexes with diagonal-type loops, which can be formed with both Oxytricha and human telomeric DNA sequences.25,27,30,34 We see here a major reorganisation of loop conformation, with participation of bases not only in stacking either side of the chromophore, but also actively interacting with the ligand in the plane of the chromophore. It is thus advantageous for the chromophore to have appropriate atoms or groups that can participate in such interactions, and so enhance affinity and selectivity. It was initially conceived, when developing
quadruplex-binding ligands with amide groups, that these groups serve only to enhance the p-electron delocalisation of the chromophore. We see in this structure that they can play a more active role in directly hydrogen-bonding to a loop residue. The same geometry of binding involving in-plane base-chromophore hydrogen-bonding is not available to other quadruplex-binding ligands, such as di-substituted amidoanthraquinones,19,26 tetra-Npyridyl porphyrins,21,30 the substituted polycyclic compound PIPER,28 or the natural product telomestatin,32 which all lack a hydrogen-bond donor in an equivalent position on the chromophore. However, the flexibility of the tetranucleotide loop is such that alternative conformations are highly likely to be accessible, which might accommodate other ligands such as these, and thus establish other analogous modes of intermolecular interactions. The crystal structure shows that the side-chains of this ligand are too short to be interacting fully in the quadruplex grooves. The dynamics simulations demonstrate their mobility, and that the weak hydrogen bonding arrangements seen in the crystal can be considered to be time-averaged views of their flexibility. Longer side-chains would penetrate further into the grooves, and these may be held more firmly, especially if they are able to interact with phosphate groups. The structural information to date on quadruplexes formed from telomeric DNA sequences shows that they can be considered in two categories, (i) Having a pattern of anti/parallel strands with diagonal loops that are positioned above and below the plane of G-quartets in a quadruplex of the present Oxytricha sequence and the Naþ form of the human intramolecular quadruplex.34 (ii) Having all-parallel strands, with a very different pattern of external loops that are arranged in the groves rather than stacked on the G-quartets, as observed in the Kþ form of the d(TTAGGG) repeat intramolecular quadruplex.2 The present structure emphasises the general feature of chromophore stacking interactions with the terminal G-quartet and the unlikelihood of G-quartet intercalation, which is relevant to both categories. It does not show how the loops in allparallel structures such as in the Kþ form of the human telomere repeat quadruplex structures,2 may be involved in ligand binding, although analogous arrangements may be formed by the diagonal loop in the Naþ form of the quadruplex formed from human telomeric DNA repeats.34 The position of the bound ligand found here is in accord with that deduced from low-angle scattering data on an intermolecular parallel-stranded ligand complex31 involving a di-substituted
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A G-quadruplex-Ligand Structure
amidoanthraquinone, supporting the suggestion that chromophores generally prefer to bind on the exterior of guanine-tetrad stacks in a quadruplex, whether or not the strands are all-parallel. The type of arrangement found here is also likely to have relevance to ligand binding to loops in nontelomeric quadruplexes. This is exemplified by the guanine-rich sequence upstream from the promoter of the c-myc oncogene,35 which is stabilised as an intrastrand fold-back quadruplex by ligands such as the perylenediimide ligand PIPER.36
Materials and Methods Crystallisation and data collection The DNA sequence d(GGGGTTTTGGGG) was purchased from Oswel DNA Service (Southampton). The ligand, as the hydrochloride salt, was synthesised by Dr R. J. Harrison of this laboratory, and was analytically pure. Crystals were prepared by the hanging dropvapour diffusion method at 12 8C. The drop solution contained 5% (w/v) 2-methylpentane-2,4-diol, 10 mM MgCl2, 1.6 mM spermine, 0.5 mM DNA and 1 mM ligand, with 20 mM potassium cacodylate buffered at pH 7.0. This was equilibrated against a gradient of 30% (w/v) 2-methylpentane-2,4-diol in 20 mM KCl. Crystals grew in four to six weeks as cubic blocks of approximate dimensions 0.3 mm £ 0.3 mm £ 0.2 mm. Crystal parameters are given in Table 2. Crystals were harvested and directly flash-frozen in liquid nitrogen. Data were collected at 110 K using an in-house Rikagu RaxisIV image plate system on a rotating anode X-ray source, using Osmic optics to produce monochromatic radiation. Data were integrated with the DENZO/SCALEPACK software package.37 Structure determination and refinement The structure of the BSU6039-Quadruplex DNA complex was solved by molecular replacement by means of the program EPMR.38 The dimeric quadruplex molecule from the trigonal form of the native quadruplex DNA crystal structure that has been recently solved by us (RCSB Protein Data Bank code 1JPQ), was used as the search model.7 The molecular replacement rotation and translation searches each produced one clear solution for the orientation and position in the unit cell of the search molecule, indicating that there is one quadruplex per crystallographic asymmetric unit. Refinement on this starting structure was initially performed with the CNS program.39 Refinement converged after a few rounds of simulated annealing and individual temperature factor refinement. The overall correctness of the structure was judged by the decrease in the value of Rfree during this refinement. One loop was omitted at this stage. Subsequent refinement was carried out with the SHELX-97 program.† This resulted in significant decreases in the values of both the R-factor and Rfree and the resulting model was inspected manually with s-weighted omit and 2Fo 2 Fc electron density maps. These revealed a considerable † Sheldrick, G. M. (1997). SHELX-97, a crystallographic refinement program. The University of Go¨ttingen, Go¨ttingen, Germany.
amount of new electron density in one loop region, which corresponded in shape and dimensions to a molecule of the ligand. A number of other significant regions of electron density in the loop region indicated that the structure of the ligand-bound loop had changed substantially compared to its structure in the native quadruplex, and the conformation of all its residues had to be rebuilt manually. No extraneous density was observed in the other thymine loop, which behaved normally during the refinements. Water molecules were located from difference Fourier maps and judicious use of the automatic water selection procedure in SHELX-97, using standard hydrogen bond geometric criteria and Rfree to judge whether selected water molecules were real or not. Geometric parameters for the ligand were obtained from a molecular mechanics minimised structure. The complete ˚ resolution, complex was subsequently refined to 1.75 A to yield final values for the R-factor and Rfree of 15.11% and 22.45%, respectively. Refinement statistics are given in Table 2. Protein Data Bank accession numbers Co-ordinate and structure factor data have been deposited in the RCSB Protein Data Bank (accession code 1L1H). Molecular dynamics simulations The initial starting co-ordinates were taken from the crystal structure of the quadruplex complex. The structure was not subjected to any initial molecular mechanics energy minimisation prior to starting the simulations. A molecular model of the drug was constructed and partial charges calculated semi-empirically using MOPAC in the InsightII software. The AMBER 6.0 suite‡ was used to carry out all molecular dynamics simulations. The force-field parameters for the ligand were extrapolated from the existing values for analogous groups in the AMBER and CFF force fields. The DNA – drug complex was then solvated in a periodic TIP3 water box of dimen˚ £ 50 A ˚ £ 50 A ˚ that extended at least 10 A ˚ sions ,50 A from any solute atom. The X-ray structure had revealed a vertical alignment of consecutive potassium ions along the helical axis within the central core of the complex, between the guanine residues of each G-quartet. The ions were retained in the positions observed in the crystal structure. Additional positively charged potassium counter-ions were included in the system to neutralise the charge on the DNA backbone. These maintained consistency with the crystallisation conditions and hence simulated a uniform Kþ ionic environment. The counter-ions were automatically placed by the LEAP program throughout the water box, at grid points of negative Coulombic potential. The final system had a net zero charge. The resulting complex was then equilibrated with the explicit solvent molecules by 1000 steps of minimisation and 25 ps of molecular dynamics at 300 K. The entire complex was kept constrained, while allowing the ions and the solvent molecules to equilibrate. The complex was then subjected to a series of minimisations and dynamics calculations in which the constraints were gradually relaxed over six successive steps, until no ‡ P. A. Kollman et al. (1999). AMBER 6.0, University of California, San Francisco.
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constraints at all were being applied. The final production run was performed without any restraints on the complex, for 2000 ps and co-ordinates were saved after every 10 ps for analysis of their trajectories. All calculations were carried out with the SANDER module of AMBER 6.0, and with the SHAKE algorithm enabled for ˚ non-bonded Lennard– Jones hydrogen atoms. A 10 A cut-off was used and the non-bonded pairs list updated every 20 steps. The particle-mesh-Ewald summation term was used for all simulations, in order to include all long-range electrostatic interactions in the calculations. ˚ , and the The PME charge grid spacing was , 1.0 A charge grid chosen to be products of the powers two, three and five to ensure efficiency for the fast-Fourier transform calculation. Trajectories were analysed using the CARNAL and RDPARM modules available in the AMBER 6.0 suite.
A G-quadruplex-Ligand Structure
11. 12. 13.
14.
15. 16.
Acknowledgements This work took place at the Institute of Cancer Research, who provided a research studentship to S.M.H. It, together with Cancer Research UK (programme grant SP13894) is thanked for supporting these studies. We are grateful to John Harrison for his synthesis of the acridine ligand, and together with Martin Read and Tony Reszka, for many useful discussions.
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Edited by A. Klug (Received 13 September 2002; received in revised form 14 November 2002; accepted 21 November 2002)