Conformational dynamics of the human propeller telomeric DNA quadruplex on a microsecond time scale.

11.3 MB·English

by  Islam, Barira

Published online 4 January 2013 Nucleic Acids Research, 2013, Vol. 41, No. 4 2723–2735
doi:10.1093/nar/gks1331
Conformational dynamics of the human propeller
telomeric DNA quadruplex on a microsecond
time scale
Barira Islam1, Miriam Sgobba1, Charlie Laughton2, Modesto Orozco3, Jiri Sponer4,5,
Stephen Neidle6 and Shozeb Haider1,*
1Centre for Cancer Research and Cell Biology, Queen’s University of Belfast, Belfast BT9 7BL, UK, 2School of
Pharmacy, Nottingham University, University Park, Nottingham NG7 2RD, UK, 3Institute of Research in
Biomedicine, Barcelona 08028, Spain, 4Institute of Biophysics, Academy of Sciences of the Czech Republic,
Kralovoplka 135, Brno 612 65, Czech Republic, 5Central European Institute of Technology, Campus Bohunice,
Kamenice 5, Brno 625 00, Czech Republic and 6University College London, School of Pharmacy,
Brunswick Square, London WC1N 1AX, UK
Received November 8, 2012; Revised November 26, 2012; Accepted November 27, 2012
ABSTRACT INTRODUCTION
The human telomeric DNA sequence with four Telomeric DNA, containing guanine rich tandemrepeats,
repeats can fold into a parallel-stranded propeller- is found at the end of all eukaryotic species, and is
associated with a number of specialized proteins,
type topology. NMR structures solved under
forming the telomere. Its function is to protect chromo-
molecular crowding experiments correlate with the
somal DNA from degradation, end-to-end fusions and
crystal structures found with crystal-packing inter-
recombination (1). Telomeres shorten progressively due
actions that are effectively equivalent to molecular
to the end-replication problem, a consequence of the
crowding. This topology has beenused for rational-
inability of DNA polymerases to fully replicate the
izationofliganddesignandoccursexperimentallyin 30-ends of chromosomal DNA (2,3). Human telomeric
anumberofcomplexeswithadiversityofligands,at DNA consists of repeats of the hexanucleotide sequence
least in the crystalline state. Although G-quartet d(TTAGGG) . A minimum of four repeats can fold into
n
stems have been well characterized, the inter- four-stranded intramolecular structures termed G-quad-
actions of the TTA loop with the G-quartets are ruplexes (4). These structures have been shown to play a
much less defined. To better understand the con- role in telomere regulation and therefore have been
formational variability and structural dynamics of proposedasapotentialtargetfortherapeuticintervention
the propeller-type topology, we performed molecu- by small molecule ligands (4,5).
lar dynamics simulations in explicit solvent up to The folding and formation of human G-quadruplex
structures have been studied by a variety of biophysical
1.5ks. The analysis provides a detailed atomistic
and chemical probe methods (6–9). The central unit of
account of the dynamic nature of the TTA loops
G-quadruplexes is a hydrogen-bonded array of four
highlighting their interactions with the G-quartets
guanine bases stacked on top of another to form the
including formation of an A:A base pair, triad,
G-quartet stem. The highly electronegative channel
pentad and hexad. The results present a threshold
running along the axis of the stem is stabilized by mono-
in quadruplex simulations, with regards to under-
valent cations (10). The TTA linker sequence is arranged
standing the flexible nature of the sugar-phosphate
diagonally, external to the helical-like stacks of the
backbone in formation of unusual architecture G-quartet stem, forming the loops. At least six intramo-
within the topology. Furthermore, this study lecular G-quadruplex structures with different topologies,
stresses the importance of simulation time in formed from the human telomeric sequence, have been
sampling conformational space for this topology. reported to date, including the basket, chair, 3+1
*To whom correspondence should be addressed. Tel:[phone]; Fax:[phone]; Email: [email protected]
(cid:2)TheAuthor(s)2013.PublishedbyOxfordUniversityPress.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.org/licenses/by-nc/3.0/),which
permitsnon-commercialreuse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.Forcommercialre-use,pleasecontact
[email protected].
2724 NucleicAcidsResearch,2013,Vol.41,No.4
hybrid and propeller form (11,12). The elucidation of earlier 15ns MD simulations of the same sequence con-
NMR solution structures reveals that the sequence can firmedthattheG-quartetstemisahighlystablestructure.
switch between parallel, antiparallel and hybrid conform- The simulations revealed some flexibility in the loop
ations depending on the precise sequence, metal ions, region in line with other studies (20,23). However, no
solvent conditions, oligonucleotide concentration and interactions of loops with the G-quartet stem were
possibly other factors (13). noted,anditislikelythatsamplingoftheconformational
The22-mersequence,d[AG (T AG ) ]adoptsadistinct space of the loops was not exhaustive (20). Owing to the
3 2 3 3
topology in concentrated K+solution, as observed in the recent improvements in force fields and wide availability
crystal structure (PDB id:1KF1) (10). The loops are in an of high-end processing power, microsecond (ms) long
open conformation in a propeller-like arrangement, con- simulations of nucleic acids are becoming affordable and
sistent with the folding of a parallel-stranded often dramatically change the picture obtained by shorter
G-quadruplex. The crystal structure contains three simulations and older force fields (24–28).
loops: Loop1 (Thy5-Thy6-Ade7), Loop2 (Thy11-Thy12- In this study, we present a detailed analysis of a con-
Ade13) and Loop3 (Thy17-Thy18-Ade19), which link tinuous 1.5ms long MD simulation of a quadruplex
the bottom (30-end) and the top (50-end) of the formed from the human 22-mer telomeric repeat. The
three-stacked G-quartets. The adenine in each TTA study focuses on the conformational sampling and the
sequence is swung back forming a Thy-Ade-Thy stack dynamic nature of the connecting loops. The folding of
(10). Recently, experiments carried out under molecular the loops external to the G-quartet stem are of particular
crowding conditions have demonstrated that the 22-mer interest, as they present an interface for interactions with
sequence also adopts a similar propeller-type topology in proteins, other nucleic acids and ligands. In addition, the
the (non-crystalline) solution state (12). The environment topologyandtheconformationadoptedbytheloopshave
inside a cell is molecularly crowded owing to macromol- profound consequences for small-molecule drug design
ecularconcentrationofupto40%(w/v)(14).Therefore,it and development. The present simulation represents a
can be speculated that human telomere quadruplexes can threshold in G-quadruplex simulations, with regards to
adopt a propeller-type topology under physiological con- testing the force field for simulating four-stranded struc-
ditions. The biological relevance of this topology can be tures,theroleofcations,dynamicflexibilityandstructural
established from the reports that it greatly decreases variability of parallel stranded propeller-type topology
telomerase activity and processivity and therefore is a po- G-quadruplexes.
tential target for anti-cancer therapy (15). Furthermore,
allG-quadruplexstructuresco-crystallizedincombination
with small molecule ligands/drugs also show propeller- MATERIALS AND METHODS
type topology (16,17). This is unlike the other forms
MD simulations
observed by NMR methods for native telomeric se-
quences, suggesting that the preferred favoured topology The crystal structure of the parallel stranded
for ligand binding is the intramolecular parallel-stranded propeller-type quadruplex formed from the human telo-
propeller-type (18). In the light of these findings, we have mericsequenced[AG (T AG ) ](PDBid:1KF1)wasused
3 2 3 3
used this topology in the present molecular dynamics asthestartingstructureforthesimulation(10).TheX-ray
(MD) simulation and aim to provide, for the first time, structure contains a vertical alignment of K+ions along
an extended analysis of quadruplex loop structural the axis within the electronegative core of the structure.
dynamics on a microsecond timescale. Theions,arrangedinasquareantiprismaticcoordination,
The MD simulations approach has provided much in- aresandwichedbetweenthequartetsandwereretainedin
formation regarding the dynamic behaviour of DNA in positions as in the crystal structure. The MD simulations
solution, and an increasing number of studies have used were carried out using AMBER11 software using the
this approach with quadruplex nucleic acids (12,19–21). Parmbsc0 version of the Cornell et al. force field
Although X-ray crystallography provides detailed (29–31). The structure was explicitly solvated with
atomic-level information of a quasi-static state, general- TIP3P water molecules in a periodic box
ization cannot be made that crystal structures necessarily (65A˚ (cid:2)64A˚ (cid:2)68A˚ ) whose boundaries extended at least
representthemajorconformations.TheX-raystructureof 10A˚ fromanysoluteatom.Additional19K+counterions
the propeller-type quadruplex reveals that although the were added to the system to neutralize the charge on the
dihedral angles for G-quartets are constrained in the quadruplex backbone, whose parameters were adapted
range of B-DNA, the dihedral angles for loops vary from Hazel et al. (32).
fromA-,B-andZ-DNAforms(22).Thesugar-phosphate The protocol for energy minimization and MD in
backbone has considerable potential for conformational explicit solvent was adopted from Haider et al. (20). The
variability within the quadruplex. Thus, although the initialroundofequilibrationwithexplicitsolventandions
G-quartet stem is considered to be well characterized by involved 1000 steps of steepest descent, followed by 1000
experimental and theoretical approaches, conformational stepsofconjugategradientenergyminimization.A300ps
plasticity and structural dynamics of the single-stranded MDequilibrationwasperformedinwhichthequadruplex
loops as well as interactions of loops with the G-quartets was constrained, whereas the solvent and ions were
are much less understood. As loops are likely dynamical, allowed to equilibrate. The system was gently heated
static structures alone cannot provide details of conform- from 0K to 300K with a time step of 0.5 ps. This was
ational transitions occurring within a quadruplex. Our followed by subsequent rounds of MD simulation, at
NucleicAcidsResearch,2013,Vol.41,No.4 2725
constant pressure and 300K for 200ps. The constraints G-quadruplex systems published to date were performed
were gradually relaxed, until no constraints were applied on a time scale from 1 to 100ns (12,20,23,39–42). One of
to the system. The final MD production run was per- the principal results of our work is that such simulations
formedat300Kusingtheparticlemeshewaldsummation are insufficient to properly characterize structural
method with a charge grid spacing of (cid:3)1.0A˚ (33). dynamics of the G-quadruplex loops. As shown in
A cut-off of 10A˚ for non-bonded Lennard–Jones inter- Figure 1, different timescales provide dramatically differ-
actions was used, and the non-bonded pair list updated ent qualitative description of the structural dynamics. In
every 20 steps. The SHAKE algorithm was used to con- fact,weshowthatthefirst(cid:3)300nsofoursimulationgive
strain hydrogen atoms with a tolerance of 0.0005A˚ and a amisleadingpictureoftheloopstructuraldynamics.Only
2fs time step (34). The final production run was carried after this period does the system settle down. In addition,
out without any restraints on the system for a total of althoughtheloopsestablishfewcharacteristicstableinter-
1.5ms.Framescollectedevery2psmadeupthetrajectory actions (mainly Ade1-Ade13 and Thy17-Gua15, see later
and were analysed using the ptraj module in Ambertools in the text), they sample a rich ensemble of transient
1.4 (35). The simulation were run on GPU clusters and geometries. The loop geometry thus cannot be
lasted (cid:3)45 days. characterized by a single structure. Examination of the
trajectory in short bins reveals that the structure can
Clustering adopt stable conformations that can last several nanosec-
onds.Althoughsuchstructuresmightbeeasilyinterpreted
Clustering analysis can detect and classify objects,
as ‘stable’ in short simulations, considerisng them in the
described by structural data, into different groups based
contextofalongmsorlongersimulation,thesemetastable
on structural similarity (36). To identify structural
structures are transient, and some of them can be only
clusters from a trajectory, a root mean squared deviation
marginally populated in sufficiently long simulations. We
(RMSD)parameterwasused,wherethepairwisedistances
havetherefore treated thefirst 300nsas equilibration and
measured as coordinates between structures are defined
carried out all analyses (except when explicitly stated
by a cut-off value reflecting the range of conformations
otherwise) presented later in the text on the trajectory
and their relative populations. The algorithm generates
portion beyond this point.
centroids describing each cluster and then gives an
The average RMSD of the all-atom structure (4.5A˚ ) is
RMSD for each structure in the trajectory with respect
significantlygreaterthanthatofthebackbone(3.4A˚ )and
to each identified cluster. Clustering was carried out after
can be attributed to the wobbling effect of bases and the
removal of the first 300ns, which were attributed to the
flexible nature of the TTA loops (Supplementary Figure
equilibration phase. The frames were extracted at a time
S1). The stacked G-quartet stem is the most stable
interval of 6ps yielding 50000 frames. The MMTSB
toolkitcode(29)wasusedwithacut-offof3.0A˚ . sub-segment of the structure. To highlight regions of
stability and similarity between the crystal and the
simulatedstructureviaMD,wirefigurescolouredbytem-
Data representation
perature factors and RMSF have been illustrated in
The backbone angles a, b, g, e, d, z and the glycosidic Figure 2. The stable (blue) central G-quartets are
torsion angle (cid:2) were monitored using the ptraj module surrounded by more mobile TTA loops (red). Plotting
of AMBER (35). Deoxyribose puckering pseudorotation RMSF values, calculated from the simulation and
angles (p) and amplitudes (A) have been determined fol- comparing with the B-factors in the crystal structure,
lowing the definitions of Altona and Sundaralingam (37), highlights similar thermal fluctuations.
using the same reference state for P=0.0 degrees. The
torsion, pseudorotation angles and amplitudes were ex- The three loops show non-equivalent dynamics
tracted from the AMBER trajectory and plotted using
MATLAB (cid:3)(2012a,The Mathworks City, USA). The Following the flexibility of the loop structure during the
structural illustrations were prepared using VIDA (open course of the simulation and comparing them with the
eyescientificsoftware,SantaFe,NewMexico),VMD(38) X-ray structure reveals significant conformational
and PyMol (http://www.pymol.org) programs. The clus- changes. However, the loop conformational rearrange-
tering data were plotted using the Xmgrace program ment did not have any great impact on the structure of
(http://plasma-gate.weizmann.ac.il/Grace). the central G-quartet stem. The RMSF analysis indicates
that the mobility of each of the simulated loops is inde-
pendent of one another (Figure 2c and d). Although the
RESULTS three loops might appear at first sight to be equivalent,
they occur in different structural contexts, which make
Structural dynamics on a microsecond timescale differs
their dynamics distinct. The most important factor
dramatically from shorter simulations
leading to non-equivalence of the loop dynamics is the
Thestructuralintegrityofthequadruplexpersistedduring flanking Ade1 nucleotide. Ade13 from loop2 forms
the complete course of the lengthy 1.5ms of MD simula- hydrogen bonds with the flanking Ade1 and end caps
tion.Toanalysetheimportanceofthetimescaleonwhich the quadruplex at one end. This interaction once formed
the propeller-type structures equilibrate and vary, the is retained throughout the simulation. The stable con-
pairwise RMSD were assessed over both short and long formation of Ade13 on top of the terminal quartet
simulation times (Figure 1). Most of the simulations of stretches loop2, and as a result of this arrangement, the
2726 NucleicAcidsResearch,2013,Vol.41,No.4
Figure 1. Conformationaldynamicsofhumanparallelstrandedpropeller-typequadruplexDNAondifferenttimescaleassessedbypairwiseRMSD
matrices. The minimum and maximum was assigned to a linear colour bar to indicate the variation in RMSD. Owing to large sample size, the
trajectory for(a) 0–10ns wassampledatdt=1ps;(b) 0–100nswassampled atdt=10ps; (c) 0–1000nswas sampledatdt=100ps; (d)0–1500ns
wassampledatdt=150ps.Thematrixof10nstrajectoryshowsrelaxationofcoordinatesafterfirst(cid:3)2ns.ThehighRMSD(yellowstripes)in100ns
matrix showed that the trajectory in this duration was still in equilibration. It is evident from matrix (c and d) that the trajectory equilibrates
at (cid:3)300ns. A large conformational transition observed at (cid:3)720ns is owing to the formation of a transient triad.
Thy11-Ade13-Thy12 stacking conformation, as observed proteins and other molecules to interact (44). The
inthecrystalstructure,ispermanentlylost.Basesfromthe backbone conformation of the quadruplex has been
other two loops sample an ensemble of transient loop analysed based on the following degrees of freedom:
geometries, which can be characteristically stable for (cid:2) angle, sugar puckering, a/g and e/z crankshaft
several nanoseconds. For instance, the first thymines in motions.The(cid:2)angledescribestherelativeglycosylorien-
loops 1 and 3 (Thy5 and Thy17) dynamically interact tation and is in either anti (180(cid:4)–360(cid:4)) or syn (0(cid:4)–180(cid:4))
with the middle quartet to form pentad and hexad archi- conformation.Thesugarpuckerdescribestheextenttoby
tecture(seelaterinthetext).Moreover,Ade7alsorotates which the furanose ring in nucleic acids deviates from a
around the (cid:2)-torsion angle to interact via p-p stacking plane. The a and g angles occupy trans (t) and gauche
with Thy6 and via edge-to-face with Thy5. We also (g)±regions, and a/g sampling is useful to validate the
observe in the simulation that the loops interact with the force fields. For example, the a/g crankshaft motions in-
quartets to adopt distinct global geometries, and long definitely trapped in non-canonical regions of a/g con-
simulation times are required to explore the conform- formational space lead to force-field-dependent artefacts
ational space appropriately. (45). The nucleic acid backbone is considered to be in the
B form whentheeand zdifferenceisnegativeandinthe
Sugar pucker and the backbone angles I
B formwhenitispositive(46).Theabove-describedcon-
II
The furanose ring in nucleic acids is conformationally formational classes are typically discussed for B-DNA
flexible (43). The backbone torsion angles and sugar con- duplexes, but they also have relevance to the quadruplex
formations in DNA arrange to present an interface for backbone.Asummaryofconformationalvariabilityofall
NucleicAcidsResearch,2013,Vol.41,No.4 2727
Figure 2. Thermalmobilityintheparallelstrandedpropeller-typequadruplex.Acomparisonofatomicfluctuationsinthe(a)crystalstructureand
(b) MD simulation isrepresented as wireframe. The mobility distribution was calculated using B-factors from the crystal structure(PDB id 1KF1)
and averaged RMSF from the MD simulation. The stable central G-quartets (blue) are surrounded by more mobile loops (red). This is also
illustrated in (c). The mobility of the loops is independent of each other. (d) Loop1 (red) and Loop2 (green) exhibit higher mobility than the
Loop3 (blue).
nucleotides is presented in Figure 3, and descriptive (48). The (cid:2) angle switches back to an anti conformation
analysis is presented in the Supplementary section. ((cid:3)470ns), which is accompanied by a change in sugar
pucker from C20-endo to C10-exo. The (cid:2)-angle then oscil-
Terminal capping by A:A pair and A:A:A triad lates between anti and syn positions during the latter
course of the simulation (Figure 4a). The flexibility of
The 50 adenine (Ade1) in the crystal structure protrudes
the sugar phosphate backbone allows Ade1 to form p-p
awayfromthecoreintothesolvent(Figure2a).Itadopts
stacking interactions with guanines on the terminal
ananticonformationanddoesnotinteractwithanyother
quartet. Stacking is observed with the adjacent Gua2 or
part of the quadruplex. In our previous work, we had
across with the neighbouring Gua20. The hydrogen bond
removed this nucleotide assuming its conformation to be
between 50-OH-N3 is lost when Ade1 is in anti conform-
a crystallization artefact that might affect the stability of
ation. We observe that owing to stochastic backbone
the quadruplex (20). An NMR solution structure of a
movements, Ade1 can flip back into the solvent.
parallel-stranded quadruplex formed from human telo-
meric DNA sequence has subsequently been determined However, this is an unfavourable and only transient
under molecular crowding conditions (12). It adopts the event, as Ade1 subsequently flips back to stack on the
same fold as the X-ray structure; however, the 50 adenine G-quartet stem. Ade13 also moves to the top of the
is stacked on the adjacent terminal G-quartet. Based on quartet to form reverse Watson–Crick hydrogen bonds
thisobservation,wedecidedtoretainAde1inthestarting with Ade1 (Figure 4d). The syn conformation of Ade1
structure.Withinthefirstfewnanosecondsoftheproduc- and anti conformation of Ade13 facilitates head-to-head
tionrun,theglycosidic(cid:2)angleofAde1changedfromanti base pairing between Ade1 and Ade13 (Supplementary
to syn conformation. This leads to formation of an intra- Figure S10a). Ade7 from Loop1 also comes in-plane
molecular hydrogen bond between the 50-OH group and with the Ade1:Ade13 pair to form an A:A:A triad
N3. An analogous hydrogen bond has been observed in (Figure 3e). It bonds to Ade1 by side-by-side
experimental structures that have a guanine nucleotide at trans-sheared A:A pairing, which appears to be the only
50 termini in a syn conformation (47). Such an intramo- plausible configuration for the A:A:A structure observed
lecular hydrogen bond has also been shown to strongly in the simulation (49). Quadruplex loops are highly
stabilize the syn conformation of the first guanine in the flexible structures, and the p-stacking interactions of
G-quartetstemsintheabsenceofanyupstreamnucleotide Ade7 with the first quartet are accompanied by
2728 NucleicAcidsResearch,2013,Vol.41,No.4
Figure 3. Conformational variability in the backbone of the parallel stranded propeller-type quadruplex. The arrows indicate similar values for
different bases.
stereochemically permissible distortions in the backbone are in plane with the quartet. In the crystal structure,
angles of the loop residues. To achieve the chain Thy17 forms p-p interactions with Ade19. Early during
turn-back, the g torsion angle changes from g+to trans, the simulation, the p-stack with Thy17 is lost, and
allowing the sheared A:A bonding to take place Ade19 forms equivalent interactions with Thy18. Thy17
(Supplementary Figure S7) (50). moves in-plane with the central quartet owing to changes
in the backbone torsion angles, to form a pentad
Formation of a pentad and hexad (Figure 5c). The first indication of Thy5 and Thy17 inter-
Analysis of the trajectory also reveals that Thy5 and actions with the middle quartet occurs at (cid:3)100ns,
Thy17 interact with the quartets resulting in diverse strongly suggesting that these features are not observable
quadruplex geometries. These thymine bases bury their in short simulations. During the course of the simulation
methyl groups into the groove, as the O4 and imino while Thy17 makes a pentad with the middle quartet,
hydrogen atoms of Thy5 and Thy17 hydrogen bond Thy5 vacillates to interact with G-quartet stem in
with the N2 amino hydrogen and N3 atom of Gua3 and various ways. When Thy5 approaches the plane of the
Gua15(Figure5aandb).However,apentadandhexadis middle quartet, and both Thy5 and Thy17 are simultan-
onlyformedwhenbothO4andN3atomsofThy5/Thy17 eously aligned with the middle quartet, a planar
are hydrogen bonded to a guanine from the middle T:(GGGG):T hexad is formed (Figure 5d). This motif is
quartet. These interactions ensure that the thymine bases maintained by simultaneous hydrogen bonding of Thy5
NucleicAcidsResearch,2013,Vol.41,No.4 2729
Figure 4. TimedevelopmentofA:AbasepairandA:A:Atriadontheterminalquadruplex.Scattergramofglycosidicchiangleversussugarpucker
angleof(a)Ade1,(b)Ade7and(c)Ade13.(d)ThesynconformationofAde1andanticonformationofAde13facilitatesreverseWatsonandCrick
A:A base pairing on terminal quartet. (e) Ade7 flips to syn conformation and interacts with Ade1 to form A1:A7:A13 triad. The K+ ion is
represented as purple sphere.
Figure 5. Representation of pentad and hexad alignment. The minimum distance plot of (a) Thy5 and Gua3 and (b) Thy17 and Gua15 from the
middle quartet. A pentad is formed when either Thy5 or Thy17 is in plane with the quartet and within hydrogen bonding distance. A hexad is
formedwhenbothThy5andThy17simultaneouslyalignwiththemiddlequartetandformhydrogenbonds.Structuralrepresentationof(c)Thy17
interactionthroughitsWatson–CrickfaceviashearedhydrogenbondswithGua15toformpentadwithmiddlequartet.(d)Concurrentalignmentof
Thy5andThy17withmiddlequartetthroughWatson–CrickfaceformsaT:(GGGG):Thexad.Thefirst300nsarehighlightedasayellowbox,and
an arbitrary line at 3.5A˚ is drawn to highlight hydrogen bond formation. The K+ion is represented as purple sphere.
2730 NucleicAcidsResearch,2013,Vol.41,No.4
with Gua3 and Thy17 with Gua15, which is sampled for Clustering
(cid:3)3% over the equilibrated trajectory.
Clustering analysis aims to identify the main substates
sampled during the simulation. A 3.0A˚ RMSD cut-off
Role of ions and hydration in maintaining the structure identified six clusters (Figure 7). The top three clusters
of the quadruplex account for (cid:3)86% of the sampled conformational space.
This study finds that K+ions remain positioned between Thesimulationishighlyheterogeneous,anddiversestruc-
thestackedG-quartetsthroughoutthecourseofthesimu- turalscaffoldsareobserved.Inthepinkcluster,p-stacking
lation. There was no exchange of cations between the interactions dominate in loop1 and loop3. This cluster
G-quartet stem and bulk solvent as observed in other appears (cid:3)300ns and then again towards the end of the
quadruplex systems (19,41,42). The formation of the simulation at (cid:3)1450ns. The conformation of loop1 and
Ade1:Ade13 base pair is briefly coupled by the position loop3, observed in this pink cluster (at 1481ns), closely
of a K+ion (from bulk) between them. Ade1 and Ade 13 resembles the loops of the X-ray/NMR structures
interact with the ion by forming water bridges with the (Figure 8). The red cluster briefly appears within the
hydrationshellaroundtheK+ion.WhenAde13iswithin pink cluster, when the p-stacking in loop1 is lost. Thy6
bonding distance with Ade1, the K+ ion leaves, and a faces outward into the solvent. However, this is a
stable Ade1 and Ade13 base pair is formed short-lived conformation, and the loops reform their
(Supplementary Figure S11). Additional K+ ions stack, Thy:Ade:Thy stacking geometry quickly. A stable A:A
albeit briefly, between the base pair and the top quartet basepairandapentad(Thy17)contributetotheconform-
and also above the A:A base pair (Figure 6a and b). ation adopted in the green cluster. Ade7 in loop1 is pos-
Similar role of stabilization by ion has also been itioned towards the solvent, similar to Ade1 in the crystal
proposed by Maiti and co-workers (40). Analysis of the structure. The jump observed at (cid:3)720ns, in the orange
trajectoryalsorevealsthepresenceofK+ioncoordination cluster, is a result of the formation of the A:A:A triad
in the loops, which occurs primarily via phosphate on top of the terminal quartet. The position of Ade7
backbone atoms and solvent molecules. The dynamic stretches and constrains loop1. It places Thy6 in plane
movement of the backbone phosphate atoms results in with the middle quartet. However, the distortions in the
the generation of a localized electronegative sink. phosphate backbone prevent Thy6 to form hydrogen
TheloopcapturesaK+iontocompensatefortheelectro- bonds with the middle quartet. Meanwhile, Thy17 inter-
static repulsion (Figure 6c). Similar stabilization of the actswiththemiddlequartettoformapentad.Theorange
loopbyacationhasbeenobservedinthecrystalstructure cluster reappears at (cid:3)1240ns, highlighting a similar con-
of the cKit-1 quadruplex (51). An extensive spine of hy- strained conformation of loop1. The black cluster is
dration is also observed, similar to those in the crystal defined by the formation of A:A base pair and a pentad
structuresofquadruplexes(Figure6d)(51,52).Theexten- (Thy17) along with other p-stacking arrangements within
sive solvent network mediates interactions between bases the loops. In the blue cluster, the interactions of loop3
from the loop and the G-quartet stem. For example, we with the quartet are lost, and the bases orient towards
observe that water bridges mediate the binding of Thy17 the solvent. The structures observed in the orange and
toGua15untilThy17comesintodirecthydrogenbonding blue clusters are transient conformations that lie in the
with Gua15 (Supplementary Figure S12). more stable black cluster.
Figure 6. Roleofbulkwaterandionsinstabilizationofthetopology.AdditionalK+ionspositioned(a)betweentheterminalquartetandtheA:A
diadand(b)ontopoftheA:Adiad.Thisisanalogoustocationbondingbetweenthequartets.(c)K+ioncoordinationwiththebackboneatomsand
water molecules. Ion capture in the loops occurs when an electronegative sink is generated owing to the close proximity of phosphate backbone
atoms. The ion helps in stabilizing the inter-phosphate repulsion. (d) Spine of hydration as observed in a snapshot during MD simulation. The
hydration networks are similar to those observedin quadruplexcrystal structures. The K+ionisrepresented aspurple sphere, whereas the solvent
(water) is coloured red.
NucleicAcidsResearch,2013,Vol.41,No.4 2731
Figure 7. Ensembleofconformationsidentifiedviaclusteringanalysis.RMSD-basedclustering,withacut-offof3.0A˚ ,overequilibriatedtrajectory
identified six clusters. Cartoon representations of top and side view of cluster centres are illustrated. The orange and blue clusters are transitional
substates observed within the black cluster, whereas the red cluster appears within the pink cluster.
Comparison with experimental data
The present analysis focuses on the maximally sampled
base pairing events that modify the topology of the
quadruplex DNA. Many of these conformations are un-
precedentedwithregardtothesimulationofintramolecu-
lar quadruplexes. An important point is that the
simulation, despite showing a wide range of loop
geometries, is capable of spontaneously sampling loop
structures that are similar to those in the currently avail-
able atomistic experiments. Clustering analysis helped in
identifying a sub-state (at 1481ns) where the conform-
ation of loop1 and loop3 closely resembles the NMR
and crystal structure. A superimposition of the identified
conformationwithNMRandcrystalstructuresyieldedan
RMSD of <2.0A˚ (Figure 8). Importantly, the individual
loops 1 and 3 occasionally also transiently sample such
geometry in the other parts of the trajectory, which indi-
cates that the experimental loop geometry, although not
being themost stable one,is within thelow-energy region
ofthesimulation.Loop2isnotsamplingthisgeometry,as
it is constrained by the A1:A13 base pairing. This,
however, does not a priori mean a disagreement with ex-
periment. Although A1 in the X-ray structure is involved Figure 8. Comparison of the loops in the (a) simulated structure
(pink) with the X-ray (blue) and the NMR structure (green). The
in crystal contacts, there is a significant non-equivalence
superimposition highlights the similarity between the structures from
between the NMR and simulated sequences, namely, in
(b) loop1 and (c) Loop3. The snapshot from the simulation was
the NMR experiment, the flanking sequence is 50-TA- extracted at 1481ns.
2732 NucleicAcidsResearch,2013,Vol.41,No.4
instead of 50-A-. There is thus no free adenine 50-OH difficult to fully capture by available experimental
group in the NMR structure so that the intramolecular methods. In addition, we show that long simulations are
hydrogenbondstabilizingtheadenineinthesynconform- needed to characterize the quadruplex DNA loop
ation cannot form. Owing to this, the adenine in the dynamics sufficiently exhaustively and without a visible
NMR structure is in a high-anti (cid:2) region, which is bias from the starting structure. Considering just the
common in DNA. first 300ns (which is still a much longer trajectory than
Our study further reports non-canonical base pairing. themajorityofthoseanalysedinprecedingstudies)would
The A:A pair formed by Ade1 and Ade13 has a Watson– be, regarding the loop dynamics, rather misleading.
Crick N1 hydrogen bond, which has been reported in Oneofthemostinterestingstructuralobservationshere
RNA structures such as those in PDB structures id is end capping of a quadruplex with the terminal adenine
1LNG and 1HR2 (53,54). Interactions of Ade7 with the base.Suchinteractionhasalsobeenobservedinaguanine
backbone of Ade1 are typical of interactions commonly andadenine-richquadruplex(63).StackingofaG-quartet
observed in RNA structures (55). Theoretically, an A:A by a Ade(anti):Ade(syn) base pair is also evident in this
pair with Watson–Crick hydrogen bonding can form an structure as well as in another bimolecular quadruplex
A:A:AtriadonlythroughtransshearedHoogsteenbonds (57,63). Interaction of a pyrimidine base with an
(49). This is the same architecture that is observed in the Ade(syn):A(anti) base pair to form a triad has also been
present simulation. A comparable structure with an reported(63).However,tothebestofourknowledge,this
A:A:A triad has also been observed in the 30S ribosomal is the first observation of a stable terminal A:A base pair
subunit (PDB id: 1FJG) (56). The T:(GGGG):T hexad, and an A:A:A triad in a quadruplex simulation. The
which forms briefly when Thy5 and Thy17 are simultan- A:A:A triad with similar bond directionality has been
eouslyalignedwiththemiddlequartet,hasbeenobserved observed in an experimental RNA structure (64). The
in the structure of a bimolecular quadruplex (PDB id current basis for quadruplex-targeted drug design is in
1XCE) (57). The p-stacking interactions within the loops large part that end stacking on G-quadruplexes is the
have been observed in our previous simulation of principalmodeofinteractionforextendedheteroaromatic
quadruplex multimers and are consistent with several ex- ligands(65).TheA:Astacksinoursimulationcorroborate
perimental observations of loop geometries including withthefactthatp-stackinginteractionsovertheterminal
those seen in quadruplex-ligand interactions (20,58,59). quartet stabilize the quadruplex topology. Also, the A:A
The frequent sampling of stacking interactions between pairing on the terminal quartet provides a large surface
Ade-Thy and Thy-Thy observed in the loops suggests area for ligand binding. This enables molecules whose
thattheseinteractionscontributetostabilizingquadruplex chromophores have a larger surface area than, for
geometry. These interactions have been evident in several example, a diamido-acridine moiety, to bind to the ends
other previous simulations of quadruplex (60). The a/g of a quadruplex (22). It is, however, fair to admit that in
torsion angles of the loop bases observed in the present oursimulationstoacertainextent,theA1:A13interaction
workarealsoinagreementwiththea/ganglesasobserved can be facilitated by the specific 50-A1 single-nucleotide
end capping, which supports the A1 syn conformation
in the crystal structures (22).
via the 50-end-specific 50-OH...N3 intramolecular
H-bond. This adenine is involved in crystal packing in
the X-ray structure, and in the NMR structure, it is not
DISCUSSION AND CONCLUSIONS
preceeded by any nucleotide so that the intramolecular
The present study provides an unprecedented dynamic hydrogen bond cannot be formed.
atomistic picture of the propeller-type loops, covering While exploring the conformational flexibility of the
1.5ms time window with ps-scale time resolution. It is TTA loop, neither the pairing of the guanines nor
fair to admit that the dynamics (mainly balance between the stacking geometry of the quartets was altered by the
differentsubstates)maybeaffectedbytheapproximations linking topology of the external loops. We observe that
of the simulation force field, as single-stranded nucleic although the G-quartet stem of the quadruplex remains
acids topologies are specifically challenging for force- rigid, the bases in the loops can form base-pairing align-
field description (23,61,62). This may contribute to the ments that further stabilize the quadruplex. Higher-order
fact that the experimental geometry of the loops, albeit pairingalignments suchasapentad,ahexadoraheptad,
sampled in the simulations transiently, is not the resulting from interactions of adenines with a G-quartet,
dominant simulation sub-state. Obviously, also the simu- have been observed in the 93del aptamer and other
lation time scale of the present study is still far from full quadruplex-forming structures (47,66–68). These align-
convergence; therefore, we cannot claim that we see ‘cor- ments are formed when the major groove edge of
rect’populationsofthesubstates.Forboththesereasons, adenine forms sheared trans Hoogsteen bonds with
the relative balance of different structures seen in the guanine in a G-quartet (47,63). Hexad alignment with
simulation should not be over-interpreted, although we thymine as the interacting base has been reported in the
suggest that the simulation likely samples relevant NMR solution structure of the bimolecular quadruplex
substates. However, a clear result of our simulation is d(GCGGTTGGAT) (57). This structure involves the
that the conformational space of the loops is rich and Watson–Crick face of a thymine forming sheared
more dynamic than we have assumed to date. The simu- hydrogen bonds with a guanine in quartet. This inter-
lationresultssuggestthattheloopsmayexistasadynamic action effectively perturbs the quadruplex shape from a
continuum of interconverting substates, which would be circular towards an expanded toroid architecture (57).