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Influence of BII Backbone Substates on DNA Twist: A Unified View and Comparison of Simulation and Experiment for all 136 Distinct Tetranucleotide Sequences Marie Zgarbova, Petr Jurecka, Filip Lankas, Thomas E. Cheatham, Jiri Sponer, and Michal Otyepka J. Chem. Inf. Model., Just Accepted Manuscript • DOI: 10.1021/acs.jcim.6b00621 • Publication Date (Web): 06 Jan 2017 Downloaded from http://pubs.acs.org on January 8, 2017

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Journal of Chemical Information and Modeling

Influence of BII Backbone Substates on DNA Twist: A Unified View and Comparison of Simulation and Experiment for all 136 Distinct Tetranucleotide Sequences

Marie Zgarbová1), Petr Jurečka1),*, Filip Lankaš2), Thomas E. Cheatham, III3), Jiří Šponer4), Michal Otyepka1)

1)

Regional Centre of Advanced Technologies and Materials, Department of Physical Chemistry, Faculty of Science, Palacky University, 17. listopadu 12, 77146 Olomouc, Czech Republic 2)

Laboratory of Informatics and Chemistry, University of Chemistry and Technology Prague, Technická 5, 16628 Prague, Czech Republic

3)

Department of Medicinal Chemistry, University of Utah, 30 South 2000 East, Skaggs 105, Salt Lake City, Utah 84112, United States

4)

Institute of Biophysics, Academy of Sciences of the Czech Republic, Královopolská 135, 61265 Brno, Czech Republic Corresponding author: Petr Jurečka, e-mail: [email protected]

Abstract Reliable representation of the B-DNA base pair step twist is one of the crucial requirements for theoretical modeling of DNA supercoiling and other biologically relevant phenomena in B-DNA. It has long been suspected that the twist is inaccurately described by current empirical force fields. Unfortunately, comparison of simulation results with experiments is not straightforward owing to the presence of BII backbone substates, whose populations may differ between experimental and simulation ensembles. In this work, we provide a comprehensive view of the effect of BII substates on the overall B-DNA helix twist and show how to reliably compare twist values between experiment and simulation in two scenarios. First, for longer DNA segments freely moving in solution we show that sequence-averaged twist can be compared directly between different BI/BII ensembles owing to approximate cancellation of the opposing BII effects. Second, for sequence specific data, such as a particular base pair step or tetranucleotide twist can be compared only for a clearly defined BI/BII backbone conformation. For the purpose of force field testing we designed a compact set of fourteen 22-base pair B-DNA duplexes (Set 14) containing all 136 distinct tetranucleotide sequences and carried out a total of 84 µs of molecular dynamics simulations primarily with the OL15 force field. Our results show that ff99bsc0εζOL1χOL4, parmbsc1 and OL15 force fields model the B-DNA helical twist in good agreement with X-ray and minicircle ligation experiments. The comprehensive understanding obtained of the effect of BII substates on the base pair step geometry should aid meaningful comparisons of various conformational ensembles in future research.

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1. Introduction The geometry of DNA duplexes exhibits notable variations at the level of base pair steps, depending on the sequence of DNA nucleotides. These local variations are conveniently described by sets of helical parameters,1-4 such as twist, slide or shift, and manifest also as variable widths of the major and minor grooves of DNA. The sequence dependence of local variations is often considered a major factor contributing to sequence-dependent interactions of DNA with proteins and drugs. Groove widths and their shape may affect sequence-dependent interactions of DNA with small molecules, drugs or proteins,5, 6,7, 8 along with hydrogen-bonding patterns in B-DNA grooves. For instance, it has been shown that longer sequences of AT pairs without a TA step, called A-tracts, exhibit a significantly narrowed minor groove that may specifically bind certain types of molecules.9,10 Besides the static B-DNA structure, sequence-dependent deformability may also play an important role in molecular recognition processes.11-18 Thus, description and modeling of conformational sequencedependent features of DNA are important for understanding and predicting DNA’s biological functions. Experimental studies of local conformational variations have mainly been based on plentiful X-ray data. Helical parameters as well as deformation properties (see, e.g., refs 2,19,20) have been studied and their effects on important biological processes, such as nucleosome positioning, have been inferred.21 However, it has been argued that the results based on X-ray data obtained for short oligonucleotides may be strongly influenced by packing effects22,23 and biased toward sequences occurring more frequently in the databases. In addition, backbone conformational substates (e.g., BI/BII) may be assigned incorrectly in some structures owing to low resolution and intrinsic conformational heterogeneity of B-DNA24,25 and presence of cations may induce conformational heterogeneity.26 For studies of the biological roles of DNA and comparison with molecular modeling, it is desirable to accurately know the DNA structure in solution. Unfortunately, although NMR experiments are widely used for DNA structure determination and hundreds of NMR structures are available in the NDB database, they mostly lack the detail and precision of X-ray structures. It is extremely demanding to measure backbone angles and base pair step parameters with an accuracy of a few degrees by NMR experiments. Although elaborate NMR studies exist (see, e.g., ref. 27), high accuracy is still very hard to achieve for more than a handful of structures. However, combination of NMR with other techniques may increase the reliability of the data.28 When considering helical twist, another important source of information is experiments based on measuring the ligation probability of small DNA minicircles,29,30 which are very sensitive to twist values and are perhaps our best reference for the sequence averaged twist. Also sequence dependent information can be partially extracted from these experiments,30 although with lower accuracy due to noise in the fitted data. The lack of highly accurate and unbiased experimental data is one of the main obstacles restricting quantitative description of the sequence-dependent properties of B-DNA. It also hampers assessment and calibration of the empirical force fields for modeling and simulations. Theoretical studies represent an important complement to experiment. Molecular dynamics (MD) simulations allow studies in solution, free of the packing effects that can compromise X-ray data. Any sequence can be studied, and thus there is no bias toward more frequent sequences as in the X-ray databases. Recently, advances in GPU computing have made microsecond timescales available, enabling extensive sampling31 and high statistical power. For instance, helical twist in the inner base pair steps of the Drew-Dickerson dodecamer (DDD) can be determined with a statistical precision 2 ACS Paragon Plus Environment

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better than 0.2 degrees from a 1 µs simulation.32 Effects of nearest neighbors and conformational polymorphism in B-DNA have been extensively investigated by the ABC consortium.33-37 It has been shown that backbone conformational substates can influence several helical parameters.35,36,38 Thermodynamics of the effect of a BII conformational substate on a phosphate that is in the 3’direction from the investigated base pair step has been studied in detail.36 Using simulations of an extended set of B-DNA duplexes containing all 136 unique tetranucleotides, it was demonstrated that nearest neighbor effects are sizeable34,38 and it was suggested that next-nearest-neighbor effects should also not be ignored.38 Effect of sequence on the deformability of a DNA helix was extensively studied13,19,39,40 and its consequences on indirect DNA readout were discussed.14-18 Lately, tetranucleotide sequence effects in B-DNA on a µs time scale have been extensively studied by Pasi et al.35 In the present work, we present a comparative study of all 136 unique tetranucleotide steps using MD simulations with our latest OL15 force field. For the purpose of comparison, simulations were also carried out using other AMBER family force fields, namely parmbsc0, ff99bsc0εζOL1χOL4 and parmbsc1. We designed a new set of 14 22-base pair DNA sequences that contains all 136 unique tetranucleotides (Set 14). Our set represents a more compact alternative to previously published sets16, 33 as it contains only 14 sequences instead of previously published 3933 or 136 sequences16 and can be simulated with significantly less computational effort (see below). Our main focus was on the base pair step twist, which is one of the most discussed characteristics of B-DNA double helical structures. There has been a long lasting discussion regarding the accuracy of the twist in simulated DNA duplexes. The early ff94 version of the Cornell et al. force field underestimated the twist considerably, and this, in part, motivated the ff98 correction,41 which helped to increase twist values. In contrast, two subsequent modifications, ff9942 and parmbsc0,43 had only slight effects on twisting. Based on DNA minicircle ligation experiments, it has been argued that twist values were still underestimated in parmbsc0.44 A further increase of twist was achieved by our χOL4 correction of the glycosidic potential introduced in 201145 and the ff99bsc0χOL4 potential was shown to improve description of DNA supercoiling.46,47 In 2013, we published another refinement of the ff99 force field by considering the ε/ζ potential,32 which improves the twist behavior and balance of BI/BII backbone substates. Finally, in 2015 we refined the β potential, which is crucial better description of Z-DNA and G-DNA and also further increases population of BII backbone substates.48 All these refinements (ff99 + bsc0 + χOL4 + εζOL1 + β OL1) are now available as the OL15 force field.48 It should be noted that although OL15 performs well for B-DNA (see also below), it does not predict ADNA as the most stable form in low water activity environment (for more detail see Ref. 48). Concerning CHARMM force fields, CHARMM 36,49 which is characterized by improved description of the BI/BII equilibrium provides too low average helical twist31 and is not considered in our testing. Because both the ff99bsc0εζOL1χOL4 and OL15 force fields increase helical twist with respect to the previous variants,31,48 we focus on their further testing in this study. Along with the base pair step twist, we also investigated the populations of BI and BII backbone substates because they are strongly coupled with DNA twisting.50 BI/BII substates are defined by the backbone angles ε and ζ, which can adopt two distinct combinations, ε/ζ = t/g- (BI) and g-/t (BII). It has been shown that the presence of BII substates can influence helical twist. If one of the two phosphates within a base pair step (denoted as inner phosphates in the following) in the BII conformation (BI.BII or BII.BI) increases its helical twist with respect to the canonical BI.BI 3 ACS Paragon Plus Environment


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conformation.51,52 When both phosphates of the given step are in the BII conformation (BII.BII), the helical twist is even higher.53 Recently, it has been reported that the conformation of 3’- phosphates adjacent to a given step (denoted as 3’-outer phosphates in the following) also matters.38,54 In particular, it has been observed that the BII conformation on the flanking 3’-phosphates strongly decreases the twist of the upstream step. As we will show below, conformations of not only the inner and 3’-outer phosphates, but also the 5’-outer phosphates can influence twist of the central step. Because BI/BII populations are strongly sequence dependent53,55 and differ in solution compared to crystal structures, comparison of twist values obtained from these two ensembles is not straightforward. It should also be noted that BII backbone substates may sometimes be incorrectly assigned in X-ray structures, as discussed in detail in Ref. 24. Moreover, different force fields generate different populations of the BII substates. Compared to bsc0, in which the BII percentage is clearly underestimated, the ff99bsc0εζOL1χOL4, OL15, recently introduced parmbsc156 and also latest versions of the CHARMM family, CHARMM3649 and polarizable Drude-2013 CHARMM force field,57 provide much higher BII populations more closely matched to those from experiment.28,58,59 However, differences between force fields are still relatively large and, in addition, the dependence on ion type and ionic concentrations seems not to be captured correctly by the non-polarizable models.60 Clearly, influence of the populations of the BII substates must be taken into consideration when comparing the base pair step twist obtained from different simulations and experiments. In this work, we conducted a comprehensive analysis of the effect of BII substates on the overall BDNA helix twist and show how to reliably compare twist values between experiment and simulation, both for the sequence averaged twist and sequence specific data for a particular base pair step or tetranucleotide. This allowed straightforward comparison of X-ray or other experimental data with those obtained from MD simulations in solution. Our MD simulation results were primarily obtained with the OL15 force field and were extensively compared with experiments and several other force fields.

2. Methods Starting structures were prepared using the nucgen61 module of AMBER. A list of all sequences of the test set (Set 14) is given in Table S1 of the Supporting Information. MD simulations were performed with 150 mM KCl using Dang62 parameters and the SPC/E63 water model, consistent with ABC protocol.38 All simulations were carried out using the PMEMD code from the AMBER 1464,65 program suites under NPT conditions (1 bar, 298 K) with default temperature and pressure settings (tautp = 1.0 ps, taup = 1.0 ps), a 9 Å direct space non-bonded cutoff and SHAKE applied to bonds to hydrogen atoms with default tolerance (1.10−5 Å). Hydrogen mass repartitioning and a 4 fs time step has been applied.66 Although it has been shown that the hydrogen mass repartitioning and a 4 fs time step trajectories exhibited no significant difference in kinetics and thermodynamics when compared to the conventional 2 fs time step, we performed additional detailed analyses of 1 µs DDD simulations ascertaining that the 4 fs time step does not introduce any perceptible differences in B-DNA’s geometry characteristics (Supporting Information, Table S2 and Figures S1-S6). The non-bonded pair list was updated every 25 steps. PME was used with default grid settings (1 Å) and default tolerance (1.10−5). Coordinates were stored every 10 ps. PMEMD for CUDA was used for all simulations.67 Initial equilibration was performed as described elsewhere.68 Force-field variants tested in this work are 4 ACS Paragon Plus Environment

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shown in Table 1. For the sake of brevity, in the following discussion, we abbreviate the original force field names parmbsc0, parmbsc1 and ff99bsc0εζOL1χOL4 as bsc0, bsc1 and εζOL1χOL4, respectively. The total simulation time reported in this work was 84 µs.

Table 1. List of tested force fields and dihedral modifications. Force Field

Abbreviation

Modifications

parmbsc0 ff99bsc0εζOL1χOL4 parmbsc1 ff99bsc0β OL1εζOL1χOL4

bsc0 εζOL1χOL4 bsc1 OL15

ff9942 + α/γ43 ff9942 + α/γ43 + ε/ζ32 + χ45 ff9942 + α/γ43 + ε/ζ/χ/P56 ff9942 + α/γ43 + β48 + ε/ζ32 + χ45

Simulation time (µs) 1 1 1 3

Trajectories were analyzed using the cpptraj69 module of AMBER. Structural parameters were calculated by the same approach as used in the software package 3DNA.3 BI and BII backbone substates, defined by ε/ζ = t/g- (BI) and ε/ζ = g-/t (BII), were distinguished by the ε-ζ difference (ε-ζ < 0 (BI), ε-ζ > 0 (BII)). Precision of the mean of twist values from MD simulations was characterized by confidence intervals (CIs) at the 95% confidence level calculated as 1.96*SEM (standard error of the mean), which were estimated using uncorrelated samples from the trajectories. Based on analysis of autocorrelation functions, all base pair step parameters were considered uncorrelated after 1 ns. Averages over the whole Set 14 were calculated such that each of the 136 unique tetranucleotides contributed only once to the average. Values for ten unique base pair steps were calculated by averaging the average tetranucleotide twists over all flanking sequences (10 or 16 values, depending on the step). X-ray data for the protein-DNA complexes were obtained from the 3DNA Landscapes database (http://3dnascapes.rutgers.edu/). On-line filters provided by the databases were applied to choose B-type structures with resolution better than 2.5 Å (list of structures in Table S3), and only B-DNA segments (more than 50% in the B-form) were retained. Z-DNA and deformed TATA box structures were removed manually. NMR structures of naked B-DNA were obtained from the same database (Table S3). Non-canonical pairs (other than Watson-Crick), modified bases and structures with bound ligand were not considered. Naked X-ray structures were taken from Dans et al. (Z-DNA structures were removed).70 Twist values deviating by more than 2 standard deviations (SD) from the average (for each base pair step separately) were not included in our analysis.

3. Results and Discussion Test Set The test set (Set 14) containing all 136 unique tetranucleotides was built using 14 duplexes, each 22 nucleotides long (Supporting Information, Table S1). All duplexes were capped with a GCGC sequence at both ends. The GC base pair step at the end was chosen based on our previous study68 showing that it is a particularly stable closing step that can be used to avoid excessive fraying. Fraying may be 5 ACS Paragon Plus Environment


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either natural,71 or a force field artifact, e.g., as reported for a CG step,54,68 and may affect helical parameters up to the third base pair step from the terminus.32,54 Steps further away from the terminus are usually relatively unaffected. In our test set, the termini showed only minor fraying, with canonical pairing accounting for 81.4%, 94.2%, 97.2%, and 98.5% of the simulation time with bsc0, εζOL1χOL4, bsc1, and OL15, respectively (see Supporting Information, Figure S7). The four terminal base pair steps of the GCGCN sequence (GC, CG, GC, CN) were excluded from our analyses and only the 14 inner base pairs were considered, from which 11 tetranucleotides could be formed. Thus, our set contains 11 x 14 = 154 tetranucleotides, of which 136 are unique. The 18 redundant steps were not included in our analyses (see Table S1). Although our sequences are longer than those in the ABC set (22 bp vs. 18 bp), there are only 14 sequences altogether, as compared to 39, resulting in about 50% saving in computational effort. The RMSD of the simulated sequences (Supporting Information, Figure S8 and Table S4) indicated that the simulations were stable. The RMSD over the inner 14 base pairs calculated with respect to the average MD structure was on average 3.77, 2.55, 2.57 and 2.37 Å for bsc0, εζOL1χOL4, bsc1 and OL15, respectively.

Convergence of Helical Parameters Base pair step parameters in B-DNA duplexes are coupled with populations of the BI/BII backbone substates, which makes their convergence relatively slow. Whereas 100 ns simulations cannot be considered converged, it has been suggested that distributions are converged after about 500 ns for DNA 18-mers.70 We have previously shown that base pair step twist can be obtained with a statistical precision of about ±0.2 degree at the 95% confidence level for the three internal steps of the 1 µs DDD simulation32 (i.e., excluding four terminal steps at each end, as in the present work). Similar results were reported by Dršata et al.72 In the present work, we assessed convergence of the B-DNA 22-mers by evaluating the CI of the mean at the 95% confidence level (assuming Student’s tdistribution) using uncorrelated samples. Average twist values along with their CIs are shown in Table S5 for one of our test 22-mers and for 50, 100, 500, 1000 and 3000 ns long simulations, both for unfiltered simulations containing a mixture of BI and BII substates and for a pure BI substate (i.e., with BII substates on the inner and outer phosphates filtered off). For comparison, we also show the standard deviations of twist. Note that whereas the standard deviations were relatively large, from 3 to 9 degrees, the CIs of the average were relatively narrow, typically below 0.3° for a 1 µs simulation. Thus, the average can be determined relatively accurately, at least for simulations of 500 ns and longer. This allows for accurate comparisons between different force fields, making even small differences statistically significant.

Effect of the BII Backbone Conformation on Base Pair Twist Six phosphates nearest to a given base pair step are shown in Figure 1. Here, we refer to them as inner and outer phosphates: the inner phosphates are located inside the base pair step and the outer ones outside, directed toward the subsequent bases in a sequence. Outer phosphates may be either on the 3’-end of the nucleotide constituting the central base pair step (3’-outer) or on the 5’-end (5’outer). Where necessary, the Crick strand is denoted by an additional prime (inner’, 3’-outer and 5’-


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outer’) and the Watson strand is unprimed. BI/BII backbone substates are assigned to the phosphate nearest to the respective ε/ζ conformation.

Figure 1. Schematic of a base pair step in context of its flanking bases. The conformation (BI/BII) of the six closest phosphates can influence the twist between the central base pairs. The six phosphates closest to the base pair step are denoted as inner, 3’-outer and 5’-outer.

As discussed above, it has long been recognized that the presence of a BII substate on an inner phosphate strongly increases the helical twist of the base pair step.52,55 In contrast, when a BII substate is present on a 3’-outer phosphate, the twist is strongly reduced.54 The effect of the remaining 5’-outer phosphates has not been studied before. As we will show below also the 5’-outer phosphates affect the twist of the central step. Consequently, when there is a mixture of BI and BII substates, such as in MD simulation ensembles or experimental database analyses, a mixture of Gaussian distributions of twist results, because each combination of the BI/BII conformations on the six mentioned phosphates generates different distribution of twist on the central base pair step.54,70 In some cases, the nature of the mixture may lead to a bimodal character of the twist distribution. An example of a mixture distribution and its decomposition into distributions arising from different BI/BII substates are shown in Figure 2 for a GCGC tetranucleotide in MD simulation with the OL15 force field.



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Figure 2. Mixture distribution of twist due to presence of BI/BII substates in CpG step of the GCGC tetranucleotide. (A) Total distribution (black curve) and distributions of substates populated by more than 4% of the total population (colored curves). (B) Influence of the inner BII phosphates on twist (all outer phosphates are in BI). (C) Influence of the outer BII phosphates on twist (all remaining phosphates are in BI). Curves corresponding to all six phosphates in BI are dashed. Inner phosphates are abbreviated as “i” and outer as “3o” or “5o”. For instance, BII(i,5o’) denotes that there are two BII substates, on an inner phosphate of the Watson strand and on a 5’-outer phosphate of the Crick strand).

To analyze the mixture distribution further, we examined the effect solely due to the inner BII conformations while all the outer phosphates were in the BI conformation (Figure 2 B). As can be 8 ACS Paragon Plus Environment

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seen, when all phosphates were in a defined state, the distribution of the helical twist was unimodal and approximately Gaussian. The BI.BI curve (dashed), corresponding to both inner phosphates (as well as all outer phosphates) in BI, is shown for reference. When one of the inner phosphates was in BII, the twist distribution shifted to higher values by roughly +4°. When both phosphates were in BII (BII.BII), the twist shifted even higher (by about +8°). The effect of the 3’-outer or 5’-outer phosphates is displayed in Figure 2 C, which shows that the 3’-outer phosphates shifted the twist distribution toward lower twist by about -6° when one of the 3’-outer phosphates was in BII and by about -12° when both were in BII. Interestingly, the 5’-outer phosphates also caused a shift of the distributions. However, they did so by a lesser extent, by about +1.5° for one BII and 3° when both 5’outer phosphates were in BII. Thus, all six nearest phosphates were found to influence the twist of the central step, with the effect of the 3’-outer and inner phosphates being dominant and that of the 5’-outer phosphate being smaller. Similarly, we analyzed the effect of the BII substates on the base pair step twist of all 136 unique tetranucleotides (Figure S9 of the Supporting Information). Changes (differences) of twist due to a BII conformation with respect to a pure BI conformation for all tetranucleotides are shown in Figure S10 of the Supporting Information. The general features were the same as described above, although the magnitudes of the differences were dependent on the sequence. Average differences in twist induced by the BII substates on various phosphates are shown in Table 2 calculated over all 136 unique tetranucleotides from OL15 MD simulations. Results for other force fields were similar in nature (not shown). Overall, the 3’-outer phosphates provided the largest (negative) contribution to twist, whereas the inner phosphates increased twists, but significantly less in absolute value. The effect of the 5’-phosphates was much smaller, though still measurable. The twist changes were roughly additive. Thus, for two BII conformations of the same type, the twist change was about twice as large.

Table 2. Changes in twist induced by BII substates on various phosphates. All remaining phosphates are in BI. Averaged over all 136 unique tetranucleotides for OL15.

Phosphate Inner 3’-outer 5’-outer Sum

Twist change one BII two BII (BI.BII or BII.BI) (BII.BII) 4.0 7.7 -5.7 -12.2 1.5 3.6 -0.2 -0.9

Comparing Sequence Averaged Twist Next, we consider the effect of a BII conformation on the overall twist of a longer DNA segment. In a longer sequence, each phosphate will be inner to some step and 3’- and 5’-outer to neighboring steps. From Table 2, it can be seen that a BII conformation will increase the twist in the base pair step in which it occurs on an inner phosphate, but at the same time decreases or increases the twist in 9 ACS Paragon Plus Environment


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neighboring steps, for which it corresponds to a 3’-outer phosphate or 5’-outer phosphate, respectively. Thus, the BII substate causes only a very small overall twist change of the DNA segment (last row of Table 2). Note that the values in Table 2 correspond to a given phosphate being 100% of time in the BII conformation, thus in a real ensemble the average changes of twist will be smaller due to lower BII populations. Therefore, the presence of BII substates should lead to only a very small overall change of the B-DNA duplex twist when compared to the same duplex in a pure BI substate. This is in fact rather intuitive when we consider that a longer duplex cannot change its overall geometry quickly enough to accommodate the short-lived BII substates stochastically appearing at different places of the duplex. Instead, when a BII substate emerges at a certain place, it generates twist changes of both signs in its neighborhood that approximately cancel out. This could be demonstrated by our Set 14, where the average twist in the presence of BII substates (full unfiltered trajectory) was very close to the twist of the pure BI substate (trajectory filtered for BI on all inner and 3’- and 5’-outer phosphates) (Table 3). The most important consequence of this is that the overall average twist (twist averaged over a relatively long DNA sequence) is almost unaffected by the population of BII substates. Thus, it is directly comparable between experiment and simulation in solution, assuming that other conditions are the same.

Table 3. Comparison of twist for the whole 1 µs MD simulations (mixture of BI and BII substates, BI+BII) and simulations with BII on all inner and 3’- and 5’-outer phosphates filtered off (BI only). Values are averaged over all 136 unique base pair steps of Set 14. Precision of the mean is characterized by 95% CIs and variation by standard deviations (SD) of the distribution. Experimental data come from this work (X-ray, protein–DNA complexes) and minicircle ligation experiments (ML exp.). Twist Twist BI only BI+BII ± CI (SD) ± CI (SD) bsc0 31.9 ± 0.1 (4.4) 32.1 ± 0.2 (5.6) εζOL1χOL4 34.4 ± 0.2 (4.7) 34.3 ± 0.2 (5.6) bsc1 34.3 ± 0.4 (5.0) 34.3 ± 0.2 (5.6) OL15 34.5 ± 0.2 (4.8) 34.5 ± 0.2 (5.7) X-ray 34.1 (3.4) 34.3 (4.0) ML exp.30 34.3 (0.5)*) *) Average over 10 dinucleotide values in Table S2 of ref. 30 and standard deviation of these 10 values. For precision of these data (typically ± 0.02 in helical repeat, corresponding to an error of ± 0.07 degree in twist) see ref. 30 Force field

Comparison of the sequence averaged twist obtained from our simulations with the X-ray (see below) and minicircle ligation experiments is shown in Table 3. The table also compares results obtained from natural ensembles containing a mixture of BI and BII substates (BI+BII, right column) and results filtered for BI on all inner and 3’- and 5’-outer phosphates (BI only, left column). First, let us note the very good agreement of the unfiltered results including BII conformations (BI+BII) with the “pure BI” results (BI only). This agreement further supports the above discussed notion that the effects of BII substates on helical twist nearly cancel out in longer DNA fragments, even when 10 ACS Paragon Plus Environment

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different BI/BII ensembles are considered. Further, we can see that results obtained with the newer εζOL1χOL4, bsc1 and OL15 force fields agree remarkably well with the minicircle ligation experiments performed in solution (for more detailed comparison see Figure S11 of the Supporting Information).30 It should be noted, however, that the above comparison assumes equal representation of base pair steps and tetranucleotides in different data sets. This assumption is valid when comparing our MD simulations, in which all 136 unique tetranucleotides are equally represented with the X-ray data from protein–DNA complexes, which are also nearly complete. However, sequences considered in the minicircle experiments have different tetranucleotide composition, which may limit accuracy of this comparison. Another potential limitation stems from neglecting dependence of twist on salt (and especially Mg2+) concentration, which is known from experiment (see, e.g., refs 73-78) and also simulations.37, 79 For instance, use of standard T4 DNA ligase reaction buffer (as in ref 30) results in 10 mM MgCl2 concentration in the reaction mixture, which could decrease helical repeat γ by up to 0.09 (corresponding to twist increase of up to 0.3 degrees) as compared to zero MgCl2 concentration (see Figure 6 in ref. 75), depending on concentration of monovalent ions. Thus, considering Mg2+ ions in our simulations could increase the simulated twist by up to 0.3 degrees and move the MD results further away from the experiment. However, even in this case the agreement would remain fairly good. Next, we can see that the results for the protein–DNA complexes gathered in this work agree well with the minicircle ligation experiments performed in solution. This is a very interesting result, because comparison of X-ray and solution experiments is not straightforward as discussed in Introduction. Finally, regarding the force field performance, our data indicate that whereas twist was indeed strongly underestimated by the bsc0 force field, as suspected earlier, it was much improved by all three newer force field variants and is in very good agreement with minicircle ligation experiments.

BI/BII Conformation-Specific Comparisons When considering a specific base pair step or tetranucleotide, it is clear that its twist cannot be directly compared with the twist obtained from a different ensemble. In this case, comparison must be carried out for a defined BI/BII conformational substate, accounting for conformations on all inner and nearest outer phosphates. We started with the twist of the BI conformation, which is dominant for most dinucleotide contexts, and thus contributes most to the ensemble average. Namely, we filtered the MD snapshots to retain only those in which all the inner and the 3’- and 5’-outer phosphates of the given base pair step were in the BI conformation. In the X-ray structures of protein–DNA complexes we filtered only the inner and 3’-outer phosphates, because filtering 5’phosphates would significantly reduce the number of experimental reference data. On several more abundant tetranucleotides in the X-ray database we checked that this approximation has only a minor effect on quality of the comparisons. Note that the effect of the 5’-outer phosphates on twist is relatively small. Data for the bsc0 and OL15 force fields are shown in Figure 3 for each tetranucleotide separately (R is purine and Y pyrimidine) and Figure 4 shows data for ten unique base pair steps averaged over all flanking sequences. The mean of twist was determined very accurately in our MD simulations as the CIs were very narrow, typically below 0.3 degrees (see above and the last column of Table S5 of the Supporting Information), and therefore are not shown in the figure. We, however, show standard deviations which characterize amount of variation of twist (not precision of the mean) and can be compared with standard deviations of the X-ray data. The spread of X-ray data 11 ACS Paragon Plus Environment


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was quite wide, the quality of the data was comparatively lower and there were only a few data points for some tetranucleotides. Also, we should keep in mind that some of the BI/BII conformational substates may be assigned incorrectly in some structures owing to low resolution and intrinsic conformational heterogeneity of B-DNA.24,25 Thus, only averages over the flanking sequence type (RR, YY, RY or YR) are shown for the X-ray data in Figure 3 (black dots) along with the standard deviation. Averages for a particular tetranucleotide are shown only for 22 cases having more than 15 data points and exhibiting good Gaussian distributions.


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Figure 3. Base pair step twist for all 136 unique tetranucleotide sequences in the BI conformation (BII excluded) compared with X-ray data from protein–DNA complexes. Ten unique central steps are shown in separate graphs and flanking bases are further subdivided as RR, YY, RY and YR in each of the graphs. Confidence intervals of the MD results are very narrow, typically below 0.3 degrees and are not shown. Shown are standard deviations of the MD and X-ray twist distributions.

First, we compare the latest OL15 refinement with the previously used bsc0 force field. One of the most obvious changes is increased twist obtained with OL15 with respect to bsc0 for the majority of the tetranucleotides. The average increase in twist was also reported in the original paper48, 80 and the strong sequence dependence of the twist increase has already been discussed in one of our previous papers for εζOL1 correction and a limited set of sequences and the effect has been shown to be consistent with results from experiment.32 However, in the original work, it was not clear whether the twist increase was due to a change of BII population, which was significantly higher for εζOL1 and OL15, or whether it was a consequence of a shift in the BI (or possibly BII) geometry. From Figures 3 and 4, which show twist for the BI state only (BII is excluded), it is now clear that the majority of the twist increase is due to a change of geometry of the BI substate, which exhibits on average larger twist with OL15 than with the original bsc0 force field. Overall, the OL15 results are much closer to those from experiment than the bsc0 results.

Figure 4. Base pair step twist for ten unique base pair steps in the BI conformation (BII excluded) averaged over all possible flanking sequences. Gray background indicates standard deviation of the X-ray data.

The performance of the other tested force fields is compared in Figures S12 and S13 of the Supporting Information. Several differences are apparent when comparing OL15 with εζOL1χOL4 and bsc1 in Figure S12. Note that the CIs of the mean are relatively narrow for the MD simulations (see 14 ACS Paragon Plus Environment

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above), thus even small differences are statistically significant. However, the observed differences are not very large or systematic. For instance, the spread of the averages for a given flanking type (RR, YY, RY or YR) of a given base pair step is mostly larger for bsc1, but in some cases, it is larger for εζOL1χOL4 or OL15. The twist values averaged over the flanking sequences (Supporting Information, Figure S13) are usually largest for OL15, but for some steps, they are larger for bsc1. The εζOL1χOL4 force field seems to give results that are on average closer to the X-ray values than those obtained with both bsc1 and OL15, but the differences are again rather small. The order of the effects of the flanking sequences in Figure 3 is mostly similar for the εζOL1χOL4, bsc1, and OL15 force fields. As discussed below, it is not possible to judge which of the force fields is better because the uncertainty in the X-ray data is comparatively large. Thus, we are only able to conclude that compared with bsc0, the latest variants bsc1 and OL15 provide very similar changes of sequence dependent twist values and the εζOL1χOL4 force field, available since 2013, also provides performance very similar to the newer modifications. A key consideration here is the quality of the reference (X-ray) data. In Figures 3 and 4, we show Xray data for the protein-DNA complexes, which may differ from the naked DNA sequences. Unfortunately, X-ray data for naked DNA are very sparse, strongly biased to only a handful of tetranucleotides and completely missing for the vast majority of the 136 possible tetranucleotides. Also the NMR data are very sparse. Comparison of the naked DNA and NMR twist with the appropriately weighed MD results (only the tetranucleotides present in the naked X-ray and NMR data are included in the MD averages; these averages differ from those in Figure 4) is shown in Figure S14 of the Supporting Information. Although average twist of newer force field variants seems to be in a reasonable agreement with the naked X-ray and NMR data, large errors were obtained for certain steps. We would like to emphasize that only very few data points are available for some of the base pair steps in these databases (see Table S6) and this seriously limits the robustness of the estimate of the mean for the naked X-ray and NMR data. Thus the more complete data on the protein–DNA complexes may provide a much more robust reference. Another important consideration is how packing effects influence the X-ray data. As noted in the Introduction, the naked DNAs are quite prone to packing artifacts. In contrast, when DNA interacts with proteins, it is usually the protein that is subjected to most intermolecular contacts between molecules in neighboring cells, not the nucleic acid. Also, proteins provide a more diverse environment compared to DNA structures, which may result in wider distributions of helical parameters but may also reduce the likelihood of systematic biases. Thus, it is often assumed that the packing effects are better averaged out in the protein–DNA complexes,19 thus providing a more reliable reference. Let us now consider the twist of the base pair steps in which some of the phosphates are in a BII substate. Here, we focused on two most important types of BII substates: (i) one BII substate on one of the inner phosphates (BII.BI or BI.BII), which increases the twist, and (ii) one BII substate on one of the 3’-outer phosphates (BII.BI or BI.BII), which decreases the twist of the central step. The twist values for 10 unique base pair steps averaged over flanking sequences obtained from X-ray and MD simulations are compared in Figure 5. We can see that bsc0 and OL15 provide very similar descriptions of the BII substates on the inner phosphates, showing only very small differences between each other (upper traces of Figure 5). Overall, the twist of the inner BII substates seems to be underestimated compared with the X-ray data. This also applies to the other tested force fields 15 ACS Paragon Plus Environment


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(see Supporting Information, Figure S15). From the lower traces of Figure 5, we can see that the OL15 refinement improves upon the bsc0 force field, although it still seems to underestimate the twist of the BII substate on the 3’-outer phosphate. Again, the same applies to the other tested force fields (Supporting Information, Figure S15). Thus, although the new force fields provide overall improved description of both the BII conformational substates, further refinements may be needed.

Figure 5. X-ray and MD twist values for ten unique base pair steps for one BII substate on one of the inner phosphates (all other phosphates in BI, solid lines) and one BII substate on one of the 3’-outer phosphates (all other phosphates in BI, dotted lines) averaged over all flanking sequences. X-ray values are shown in black. For comparison, the gray curve shows the twist for a pure BI substate.

Although the main focus of this work was on the base pair step twist of B-DNA, we also briefly compared the remaining base pair step parameters for the bsc0, εζOL1χOL4, OL15 and bsc1 force fields. Histograms for 10 unique base pair steps are shown in Figure S16 of the Supporting Information. The distributions were found to be very similar for rise and twist for all force fields. In the case of roll, εζOL1χOL4, bsc1 and OL15 provided a small average decrease for TA, TG and CG steps, whereas other steps were almost unaffected. Differences in distribution shape were observed for the base pair shift - the original bsc0 distributions were unimodal and Gaussian, εζOL1χOL4, bsc1 and OL15 distributions were a mixture of Gaussian distributions and, again, all newer force fields were very similar. Regarding slide, εζOL1χOL4, bsc1 and OL15 showed increased average slide by a similar magnitude. Clearly, the newer force fields showed moderately altered twist distributions. More detailed analysis of geometrical parameters of several specific sequences may be found in Zgarbová et al.48 and also Galindo et al.31 Overall, changes in the base pair step parameters in the newer force fields were mostly small, with twist being the most notably affected parameter. Most of the described nuances could be explained by the effect of BII substates and the fact that the tested force fields differed in their BII propensities. Effect of BII substates on various phosphates (inner, 3’- and 5’outer) is shown in Figure S17 of Supporting Information for OL15 force field. The most pronounced 16 ACS Paragon Plus Environment

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effects are those of BII present on the inner and 3’-outer phosphates on shift and slide, which were described earlier by Pasi et al.35 Effect of BII conformation on 5’-outer phosphate on slide and shift and also on the remaining base pair step parameters is relatively small and probably less important than its effect on twist analyzed above. Additional analyses showed that the effects of BII substates on roll and X-displacement predicted by OL15 agree well with the experimental TRX scale12 (Table S7 and Figure S18) and also influence of BII on groove width exhibited expected trends8 (Figure S19). For more details see text under Figures S18 and S19 in Supporting Information.

BII Populations BII populations have long been known to be underestimated by the ff99 and bsc0 force fields.49 Since BII substates seem to play an important role in DNA – protein recognition (see, e.g., ref 81), improvement in the description of the BI/BII equilibrium is desirable. This was the main aim of our εζOL1 correction,32 which was indeed found to increase the average BII populations and also improve its sequence dependence, but leaving, however, the BII population still somewhat low.32, 82 Interestingly, further improvement was achieved by using the β OL1 correction, which was primarily aimed at removing problems specific to Z-DNA,48 completing the OL15 force field. The BII content averaged over the 136 unique tetranucleotides of Set 14 calculated in this work was 8.4, 17.0, 22.7 and 23.0 % for bsc0, εζOL1β OL1, bsc1 and OL15, respectively. Here, we analyze the sequence dependence of the BII populations for OL15 and compare them with other available force field variants (Figures 6 and 7 and Supporting Information, Figures S20 and S21). First, we observed that OL15 strongly increased average populations of the BII substate with respect to bsc0 in many steps, whereas it left it almost unchanged in others. As discussed earlier,32, 82 this is desirable and in reasonable agreement with the experimentally observed sequence dependence of BII populations.28, 55 From Figure 6, it can be seen that the BII propensity is strongly predetermined by the nature of the central base step (see also Figure 7 showing results for central steps averaged over all possible flanking sequences): TT, CT, AT, GT and TC steps show only very low BII propensities, whereas GA, AA, GG, CG, CA and AG steps show the largest BII propensities. These results agree with the previous bsc0 simulations35 and are in a reasonable agreement with findings of Heddi et al.12 Although overall BI/BII propensities are influenced by water rearrangements,83, 84 their sequence dependence may be rationalized by considering differences in the extent of base stacking in BI and BII substates on the inner phosphates.85, 86 In addition, BII propensities are also strongly influenced by the nature of the flanking bases, as noted before for the bsc0 force field35 and also observed experimentally.55 Importantly, all newer force field variants (Supporting Information, Figure S20) yielded very similar sequence dependence both with respect to the central step and the flanking bases, increasing the trustworthiness of the results.



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Figure 6. BII populations for bsc0 and OL15 in 256 tetranucleotide sequences. 16 central steps are shown in separate graphs and flanking bases are further subdivided into RR, YY, RY and YR groups in each graph.


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Figure 7. BII populations in 16 base pair steps averaged over all possible flanking sequences for the bsc0 and OL15 force fields.

Conclusions Comparison of B-DNA structural descriptors is complicated owing to the existence of conformational ensembles of BI/BII conformations. This usually precludes direct comparison of structural parameters obtained from different ensembles, such as MD simulations and experimental databases. In this work, we conducted a detailed analysis of the effect of BII conformations on twist in individual tetranucleotides as well as longer DNA sequences. We show that the helical twist of a base pair step is influenced by BII substates appearing not only on the inner and 3’-outer phosphates, as reported previously, but also by the BII substates on the 5’-outer phosphates. However, when twist values are averaged over a longer freely moving duplex, these effects nearly cancel out. This allows meaningful comparison of sequence averaged twist between different BI/BII ensembles, such as those obtained from X-ray data, minicircle ligation experiments or MD simulations. In contrast, when considering sequence specific data for a particular base pair step or tetranucleotide, comparisons have to be carried out for a clearly defined conformational substate, such as a pure BI conformation, or a clearly defined BI/BII combination. Such comparisons were carried for several recent AMBER force field variants and X-ray data. For this purpose, we designed a set of fourteen 22-base pair B-DNA duplexes that contains all 136 unique tetranucleotides (Set 14). Our results indicate that the OL15 refinement provides a good agreement of average helical twist with both X-ray and minicircle ligation experiments and improved description of sequence dependence of helical twist compared to the previous parmbsc0 force field. Similar improvements for B-DNA were also obtained with the ff99bsc0εζOL1χOL4 force field. Thus, the simulations performed with this variant (available since 2013) are of comparable quality as those obtained with OL15. Interestingly, the parmbsc1 refinement published recently showed very similar behavior for B-DNA, despite the fact that torsion potential modifications in parmbsc1 differ. Overall, the OL15, bsc1 or 19 ACS Paragon Plus Environment


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ff99bsc0εζOL1χOL4 modifications are recommended over bsc0 when seeking accurate description of twist and BI/BII conformational equilibria in B-DNA.

ACKNOWLEDGEMENTS This work was supported by grant 14-29874P (M.Z.) from the Grant Agency of the Czech Republic. Further funding was provided by project LO1305 of the Ministry of Education, Youth and Sports of the Czech Republic (M.Z., P.J., M.O.).

Supporting Information. Sequences in Set 14, twist distributions for all 136 unique base pair steps, distributions of the remaining base pair step parameters, effect of BII on X-displacement, roll and groove widths, and on the remaining base pair step parameters, data for all tested force fields and a comparison of 2fs vs 4 fs DDD simulations.

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34. Dixit, S. B.; Beveridge, D. L.; Case, D. A.; Cheatham, T. E., 3rd; Giudice, E.; Lankas, F.; Lavery, R.; Maddocks, J. H.; Osman, R.; Sklenar, H.; Thayer, K. M.; Varnai, P. Molecular Dynamics Simulations of the 136 Unique Tetranucleotide Sequences of DNA Oligonucleotides. II: Sequence Context Effects on the Dynamical Structures of the 10 Unique Dinucleotide Steps. Biophys. J. 2005, 89, 3721-3740. 35. Pasi, M.; Maddocks, J. H.; Beveridge, D.; Bishop, T. C.; Case, D. A.; Cheatham, T., 3rd; Dans, P. D.; Jayaram, B.; Lankas, F.; Laughton, C.; Mitchell, J.; Osman, R.; Orozco, M.; Perez, A.; Petkeviciute, D.; Spackova, N.; Sponer, J.; Zakrzewska, K.; Lavery, R. Muabc: A Systematic Microsecond Molecular Dynamics Study of Tetranucleotide Sequence Effects in B-DNA. Nucleic Acids Res. 2014, 42, 12272-12283. 36. Dans, P. D.; Faustino, I.; Battistini, F.; Zakrzewska, K.; Lavery, R.; Orozco, M. Unraveling the Sequence-Dependent Polymorphic Behavior of d(CpG) Steps in B-DNA. Nucleic Acids Res. 2014, 42, 11304-11320. 37. Dans, P. D.; Danilane, L.; Ivani, I.; Drasata, T.; Lankas, F.; Hospital, A.; Walther, J.; Pujagut, R. I.; Battistini, F.; Gelpi, J. L.; Lavery, R.; Orozco, M. Long-Timescale Dynamics of the Drew-Dickerson Dodecamer. Nucleic Acids Res. 2016, 44, 4052-4066. 38. Lavery, R.; Zakrzewska, K.; Beveridge, D.; Bishop, T. C.; Case, D. A.; Cheatham, T., 3rd; Dixit, S.; Jayaram, B.; Lankas, F.; Laughton, C.; Maddocks, J. H.; Michon, A.; Osman, R.; Orozco, M.; Perez, A.; Singh, T.; Spackova, N.; Sponer, J. A Systematic Molecular Dynamics Study of NearestNeighbor Effects on Base Pair and Base Pair Step Conformations and Fluctuations in B-DNA. Nucleic Acids Res. 2010, 38, 299-313. 39. Lankas, F.; Sponer, J.; Langowski, J.; Cheatham, T. E., 3rd DNA Basepair Step Deformability Inferred from Molecular Dynamics Simulations. Biophys. J. 2003, 85, 2872-2883. 40. Lankas, F.; Sponer, J.; Langowski, J.; Cheatham, T. E. DNA Deformability at the Base Pair Level. J. Am. Chem. Soc. 2004, 126, 4124-4125. 41. Cheatham, T. E.; Cieplak, P.; Kollman, P. A. A Modified Version of the Cornell et al. Force Field with Improved Sugar Pucker Phases and Helical Repeat. J. Biomol. Struct. Dyn. 1999, 16, 845862. 42. Wang, J. M.; Cieplak, P.; Kollman, P. A. How Well Does a Restrained Electrostatic Potential (RESP) Model Perform in Calculating Conformational Energies of Organic and Biological Molecules? J. Comput. Chem. 2000, 21, 1049-1074. 43. Perez, A.; Marchan, I.; Svozil, D.; Sponer, J.; Cheatham, T. E.; Laughton, C. A.; Orozco, M. Refinenement of the Amber Force Field for Nucleic Acids: Improving the Description of Alpha/Gamma Conformers. Biophys. J. 2007, 92, 3817-3829. 44. Bates, A. D.; Noy, A.; Piperakis, M. M.; Harris, S. A.; Maxwell, A. Small DNA Circles as Probes of DNA Topology. Biochem. Soc. Trans. 2013, 41, 565-570. 45. Krepl, M.; Zgarbova, M.; Stadlbauer, P.; Otyepka, M.; Banas, P.; Koca, J.; Cheatham, T. E., 3rd; Jurecka, P.; Sponer, J. Reference Simulations of Noncanonical Nucleic Acids with Different Chi Variants of the Amber Force Field: Quadruplex DNA, Quadruplex RNA and Z-DNA. J. Chem. Theory Comput. 2012, 8, 2506-2520. 46. Sutthibutpong, T.; Harris, S. A.; Noy, A. Comparison of Molecular Contours for Measuring Writhe in Atomistic Supercoiled DNA. J. Chem. Theory Comput. 2015, 11, 2768-2775. 47. Irobalieva, R. N.; Fogg, J. M.; Catanese, D. J., Jr.; Sutthibutpong, T.; Chen, M.; Barker, A. K.; Ludtke, S. J.; Harris, S. A.; Schmid, M. F.; Chiu, W.; Zechiedrich, L. Structural Diversity of Supercoiled DNA. Nat. Commun. 2015, 6, 8440. 48. Zgarbova, M.; Sponer, J.; Otyepka, M.; Cheatham, T. E., 3rd; Galindo-Murillo, R.; Jurecka, P. Refinement of the Sugar-Phosphate Backbone Torsion Beta for Amber Force Fields Improves the Description of Z- and B-DNA. J. Chem. Theory Comput. 2015, 11, 5723-5736. 49. Hart, K.; Foloppe, N.; Baker, C. M.; Denning, E. J.; Nilsson, L.; MacKerell, A. D. Optimization of the Charmm Additive Force Field for DNA: Improved Treatment of the BI/BII Conformational Equilibrium. J. Chem. Theory Comput. 2012, 8, 348-362. 22 ACS Paragon Plus Environment

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Influence of BII Backbone Substates on DNA Twist ... - ACS Publications

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