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B-DNA’s BII Conformer Substate Population Increases with Decreasing Water Activity. 1. A Molecular Dynamics Study of d(CGCGAATTCGCG)2 Rudolf H. Winger, Klaus R. Liedl,* Arthur Pichler, Andreas Hallbrucker, and Erwin Mayer Institute of General, Inorganic and Theoretical Chemistry, UniVersity of Innsbruck, Innrain 52a, A-6020 Innsbruck, Austria ReceiVed: May 18, 2000; In Final Form: August 24, 2000
The BII substate of the biologically active B-DNA family is characterized by a different phosphate backbone geometry. In contrast to the current belief, the BII substate of the d(CGCGAATTCGCG)2 dodecamer is showns both experimentally and by molecular dynamics simulationssto be significantly populated at water activities in the range of those in the cell nucleus. Three systems with different water contents show an increase in BI h BII transitions as the water activity decreases. In addition, the transition mechanism is investigated and the sequence dependence of these interconversion processes in the form of pyrimidine-purine interconversion dominance is demonstrated.
Introduction The B-DNA backbone, above all the polar phosphate group, is a major player in the interaction processes involved in ligand and protein recognition, interaction, and binding.1-13 Pabo and Sauer14 wrote about the phosphate’s “integral role in site-specific recognition”. Thus, aspects of the phosphate geometry as the BI h BII interconverts, where the PO2- group switches from its central position toward the minor groove,15 should have pronounced effects on those vital interactions. The corresponding backbone angles describing this phosphate flip are the and ζ angles: For BI, and ζ derived from X-ray structures are between 120° to 210° (trans) and 235° to 295° (gauche-), respectively; for BII, the angle lies between 210° and 300° (gauche-), and ζ is between 150° and 210° (trans).16,17 The analysis of these B-DNA conformer substates has only recently become feasible through improvements in X-ray crystallography (higher resolution),18 in NMR spectroscopy (increased field strengths and sensitivity),18 in vibrational spectroscopy (quenching and subsequent relaxation of substate populations),19,20 and in molecular dynamics techniques (improved force fields21 and inclusion of long-range electrostatic interactions by means of Ewald summation).22,23 Therefore, investigations focusing on the properties of these substates in solution, contrary to the mere geometric findings in X-ray structures, NMR experiments or simulations24-29 are rather new (experimental and modeling studies are described in refs 1920 and ref 15, 30, and 31, respectively). The theoretical approach is ideally suited to create a model for this substate transition because the interconversion occurs at the nanosecond to the subnanosecond time scale,32-34 which is readily accessible by current molecular dynamics (MD) simulation techniques.35-37 In recent years, the importance of B-DNA’s BII substate and the BI h BII interconversion has been regarded as rather limited due to its small concentration in solution,18 except for its involvement in base-pair mispairing.38-40 Recently, experimental evidence for a significant BII population in salmon DNA in solution was first demonstrated by Ru¨disser et al.19 NMR studies by Lefebvre et al.41 also supported the occurrence of BI h BII * Corresponding author. Fax: +43/512/507/5144. E-mail: Klaus.Liedl@ uibk.ac.at.
transitions in oligonucleotides. Our findings, however, shed new light on the significance of these conformers in biological systems. We now show that the BI-BII ratio depends strongly on the water content, that is, at low water activitiessas they can be assumed in the cell nuclei42sthe BII substate population even seems to dominate. In this study, which is followed by the experimental evidence that the BI-BII ratio is a function of the Γ-value (i.e., water molecules per nucleotide), we analyze by means of molecular dynamics simulation techniques the striking BI h BII interconversion dependence of the d(CGCGAATTCGCG)2 dodecamer on concentration. A schematic diagram of the EcoRI dodecamer is shown in Figure 1. Methods To take advantage of the findings of previous extensive simulations,28,43-45 protocols employed therein were directly adapted for our needs. The canonical B form46,47 of the DNA oligonucleotide d(CGCGAATTCGCG)2 was used as a starting point. Each strand contains 11 PO4- anions and is terminated with OH groups. Electroneutrality of the system was achieved by adding 22 Na+ counterions using the CION program of the AMBER48 package. Subsequently, solvation of the DNA with TIP3P49 Monte Carlo water boxes was carried out. System A (described in ref 31) was hydrated by a 12-Å thick solvent shell; for system B, a 2.2-Å thick hydration layer in all directions was used, and in system C, the water box extended the DNA for 1.3 Å in the x direction and 1.2 Å in both the y and z directions. The resulting box for system A measured 68 × 45 × 45 Å3 and contained 3784 water molecules (Γ ≈ 158); the box for system B measured 49 × 26 × 25 Å3 and contained 491 water molecules (Γ ≈ 20.5), and for system C, the 46 × 23 × 23 Å3 box contained 316 water molecules (Γ ≈ 13.2). As the number of counterions is constant for all three simulations, the concentration of Na+ increases accordingly with decreasing box sizes. The simulations were carried out using the AMBER 4.148 suite of programs, employing the all atom force field21 for the DNA and counterions. To include long-range electrostatic interactions, an Ewald summation, which is implemented in the
10.1021/jp001842n CCC: $19.00 © 2000 American Chemical Society Published on Web 11/04/2000
11350 J. Phys. Chem. B, Vol. 104, No. 47, 2000
Figure 1. Schematic diagram of the EcoRI dodecamer. The phosphate residues are numbered according to Kopka et al.57
AMBER program48 in the form of the so-called particle mesh Ewald method,22,23 was employed. Minimizations were carried out, starting with 500 steps and harmonic restraints of 25 kcal mol-1 Å-2 on DNA and counterion positions. During the following five 100-step minimizations, the restraints on the counterions were relaxed faster than on the DNA duplex. Finally, 500 steps of unrestrained minimization were carried out. For the equilibration, a similar procedure was applied. After heating the constant volume system for 10 ps from 50 to 300 K and keeping the DNA and ion positions constant, the harmonic restraints were reduced throughout the following 25 ps, faster on the counterions than on the oligonucleotide. Finally, 5 ps of unrestrained constant-volume simulation was carried out before the simulation was switched to constant-temperature and constant-pressure MD. The temperature bath coupling was achieved by the Berendsen algorithm.50 General simulation parameters were kept constant during the whole simulation: 2 fs time step, SHAKE constraints of 0.00005 Å on all bonds involving hydrogen atoms, 9-Å nonbonded cutoff, and 0.00001 convergence criterion for the Ewald part of the nonbonded interactions. The structural information was collected every 50 steps (0.1 ps). Results General Characteristics of the Simulated Systems. System A, simulated for 3 ns, was extensively described in ref 31 and is only used as a reference in this study. Systems B and C, due to the reduced number of solvent molecules, allowed a simulation time up to 10 ns. Investigation of the corresponding energy curves (not shown) and rmsd (root-mean-square deviation) plots (Figure 2) show that the systems are well-equilibrated around 1000 ps.
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Figure 2. Rmsd development of the systems B (top) and C (bottom): Root-mean-square deviation of the simulated structures with respect to the canonical B-DNA starting structure (dark line in the lower part of the graphs), with respect to canonical A-DNA (line in the upper part of the graphs), and with respect to the X-ray structure (light gray line mostly hidden by rms to B-DNA in the lower part of the graphs).
The energetics, due to the careful setup procedure, converge rapidly to an equilibrium value around which the system oscillates. The rmsd development of both systems is analyzed with respect to their starting structure (canonical B-DNA46), with respect to canonical A-DNA,46 and with respect to their X-ray structure.29 Both the stability of the overall DNA structure as well as the predominant occurrence in the B form for all systems are shown in Figure 2. For system B, the rmsd with respect to A-DNA is about 5.5 Å, whereas the deviation with respect to B-DNA and the X-ray structure oscillates around 2.5 Å. For the less hydrated C system, the rmsd with respect to the starting B-DNA is even less (around 2.0 Å), and the difference from the canonical A-DNA is even larger (around 7.0 Å). This is consistent with recent experiments,51 which showed that the nonoriented d(CGCGAATTCGCG)2 dodecamer persists in the B form even at low water activity. Another way to assess the reliability of these highly concentrated simulations (ion concentrations of 0.3, 1.7, and 2.2 mol/L, respectively) is to analyze the base-pair parameters. To get a graphical representation of these DNA characteristics, the Molecular Dynamics Toolchest was used.52,53 A direct comparison between the complete set of all helical parameters (shift, slide, rise, tilt, roll, twist, shear, stretch, stagger, buckle, propeller twist, opening, x displacement, y displacement, inclination, and tip) for the differently hydrated systems exhibits no drastic differences, thus confirming the reliability of simulations of such highly concentrated systems.
Molecular Dynamics Study of d(CGCGAATTCGCG)2
Figure 3. Analysis of the BI-BII transition processes: The number of base steps that are in substate BII are plotted as a function of time for systems A (short line in the bottom, 500 to 3000 ps), B (line in the middle), and C (line in the top of the figure). To enhance legibility, only every hundredth conformation is depicted.
Base steps Involved in BI-BII Transition Processes. The number of base steps in substate BII at a given time is depicted in Figure 3. Obviously, there is a strong influence of the hydration number on BI h BII transitions because system A (Γ ≈ 158) reaches a maximum of 5 interconverted phosphates, whereas system B (Γ ≈ 20.5) goes up to 9 and system C (Γ ≈ 13.2) has up to 13 simultaneously interconverted base steps. System B is relatively constant and oscillates around an average value of 5.38 interconverted base steps (standard deviation: 1.08). System C has an average of 7.99 simultaneously interconverted phosphates, but visual inspection indicates an increase of the BI h BII transition activity as the simulation proceeds, as well as a higher spread (standard deviation of 2.02). Although the number of base steps in the BII conformation in system C seems to increase in the course of the simulation, and thus this simulation is not fully comparable with system A, there is already a distinct difference among all the systems during the first 3 ns. Comparison of the averages over this period reveals that for system A, 2.3 out of 22 (10%), for system B, 5.1 out of 22 (23%); and for system C, 5.8 out of 22 (26%) are interconverted. For system C, this value grows to one-third (7.99 out of 22 corresponding to a BI/BII ratio of 1.75) if the average from 0.5 to 10.0 ns is used. Analysis of the individual base steps involved reveals, for system B (Figure 4, top), three different types: steps with little BII activity or no interconversion at all, steps more or less constantly in substate BII, and steps with frequent substate transitions. The phosphates more or less permanently in BII are P2 (C-G), P11 (G-C), P17 (G-A), and P23 (G-C). Steps with frequent interconversions include P5 (G-A), P10 (C-G), P14 (C-G), P16 (C-G), and P20 (T-T). All other phosphate groups are predominantly in the conventional BI substate. Thus, 9 steps out of 22 are active in BI h BII substate dynamics. Of these 9 steps, C-G dominates with an occurrence of four, followed by 2 G-C and G-A steps and one T-T step. In terms of purine (R) and pyrimidine (Y) bases, this means that 4 of these steps are Y-R, 2 are R-Y and R-R, respectively, and one is Y-Y. Analysis of the individual base steps for system C (Figure 4, bottom) shows 4 steps more or less permanently in substate BII, namely, P3 (G-C), P14 (C-G), P15 (G-C), and P23 (GC). Phosphates that change frequently between the two substates
J. Phys. Chem. B, Vol. 104, No. 47, 2000 11351
Figure 4. Analysis of the individual base steps in substate BII for systems B (top) and C (bottom) from 500 to 10000 ps: Each base step, which is interconverted at a given time into BII, is marked by a black point/line (for phosphate numbers cf. Figure 1).
are P2 (C-G), P4(C-G), P10 (C-G), P12(C-G), P17 (GA), and P24 (C-G). Modest transition activity is found for P8 (T-T), P11 (G-C), P18 (A-A), and P21 (T-C). Thus, 14 steps out of 22 show at least modest BI-BII substate dynamics, and out of those 14, 9 phosphates are predominantly in BII. With respect to sequence, that means that 6 steps are C-G, 4 steps are G-C and 1 step is G-A, T-T, A-A, and T-C or, in terms of R and Y, 6 steps are Y-R, 4 steps are R-Y, and 2 steps are R-R and Y-Y. Mechanism of the Substate Interconversion. The mechanism of the substate interconversion is already well-investigated for “standard” conditions;15,30,31,54 for highly concentrated systems, there exists only a solid-state NMR investigation55 and no atomic description of the transition process. Thus, we analyzed one interconversion for both systems B and C. In the case of system B, base step G4pA5 was chosen. The corresponding phosphate P5 is mainly in the BII state, but between 1ns and 2ns, P5 interconverts back to BI twice. To control the mechanism, backbone angles and inter base-pair and axis basepair parameters as well as the hydration were analyzed (Figure 5). The backbone angles with the biggest change are the and ζ angles. Angles R and β, which are also correlated to the interconversion, show only slight changes in the interconverted regions. Changes for the other backbone parameters are restricted to the 5′-terminal base (i.e., P(G4) and δ (G4)) for the first interconversion around 1300 ps, but they are clearly visible for both steps around 1900 ps. The hydration shell of the ionic phosphate shows the previously described decrease of the number of water hydrogens closer than 2.2 Å for the BII state. As far as the inter base-pair parameters are concerned, both shift and roll exhibit lower values in the BII state. However, for the short interconversion to BI at 1300 ps, the changes are smaller than for the transition at 1900 ps. The same is true for the axis base-pair parameter x displacement of step G4:C21(XDP(G4)), which shows higher changes for the second, longer interconversion around 1900 ps. The axis base-pair parameters for A5:T20 do not change significantly. In the case of system C, base step G10pC11 was chosen. The corresponding phosphate P11 exhibits one BI f BII
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Figure 5. Backbone angles, hydration of phosphate group, inter basepair parameters shift (SHF), slide (SLD), roll (ROL), and twist (TWS) and axis base-pair parameters x displacement (XDP) and tip (TIP) of step G4:C21pA5:T20. Hydration of the phosphate oxygen is represented by the number of water hydrogens closer than 2.2 Å. It should be noted that this step is mostly in the BII conformation and interconverts around 1350 and 1930 ps.
transition during the interval from 1000 to 2000 ps. To control the mechanism backbone angles, inter base pair and axis base pair, as well as the hydration, were analyzed (Figure 6). Indicators for the interconversion are the angles and ζ, accompanied by β and a bit more slowly by R. Changes for the other backbone parameters are restricted to the 5′-terminal base (i.e., P(G10) and δ (G10)). The hydration shell of the ionic phosphate shows the previously described decrease of the number of water hydrogens closer than 2.2 Å for the BII state starting at around 1700 ps. Due to the high counterion concentration and small water activity, other minima are observed around 1000-1150, at 1320 and at 1680 ps. As far as the inter base-pair parameters are concerned, there is only a minimal change for both shift and roll toward lower values in the BII state. The same is true for the axis base-pair parameter x displacement of step G10:C15(XDP(G10)), which shows only slightly higher values in the BII state. Discussion The influence of the hydration level on DNA dynamics has been investigated by solid-state NMR,55,56 but no analysis on B-DNA substates was carried out. Starting from molecular dynamics simulations, Figure 3 clearly demonstrates the de-
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Figure 6. Backbone angles, hydration of phosphate group, inter basepair parameters shift (SHF), slide (SLD), roll (ROL), and twist (TWS) and axis base-pair parameters x displacement (XDP) and tip (TIP) of step G10:C15pC11:G14. Hydration of the phosphate oxygen is represented by the number of water hydrogens closer than 2.2 Å. This step interconverts into the BII conformation at about 1720 ps.
pendence of the BI h BII transition processes on the concentration. This is in agreement with our experimental data, where we use FTIR techniques to determine the BII content of the EcoRI dodecamer and show that the BII substate population is higher in unoriented films than in the crystal20 and that the BI/BII ratio is sequence-dependent. The significance of enhanced BII substate population at the low water activity shown in this study should not be underestimated, given the three facts that phosphates are important interaction partners in binding processes, that the water content in nucleosomes is rather low, and that the phosphate orientation changes upon BI h BII interconversion. Comparison of the results with respect to sequence dependence reveals that there seems to be no general rule for certain base steps to interconvert or to be interconverted, except that the terminal residues show higher substate transition activities than the central AATT tract. However, analysis with respect to general base sequences reveals common features in agreement with earlier studies.30 For all three systems, the BI-BII transition is dominated by C-G steps (A, 4; B, 4; C, 6), followed by G-C (A, 2; B, 2; and C, 4), G-A, and T-T. In terms of base type, this means that Y-R is more active than R-Y. This can be explained by the intra-strand component of the stacking energy, which for C-A and C-G steps is weaker compared to other dinucleotide steps.30
Molecular Dynamics Study of d(CGCGAATTCGCG)2 The mechanism of B-substate interconversion is in agreement with previous studies,31 both for the changes in the corresponding parameters as well as for the time scales involved. However, the changes in some parameters are less distinct, which could result from the weaker hydration shell. Acknowledgment. We are grateful for financial support from the Austrian Science Foundation (Project No. P13845-TPH). We would like to thank R. Lavery for supplying us with the program Curves and G. Ravishanker for the program MDTC. References and Notes (1) Suck, D.; Lahm, A.; Oefner, C. Nature 1988, 332, 464-468. (2) Otwinowski, Z.; Schevitz, R. W.; Zhang, R. G.; Lawson, C. L.; Joachimiak, A.; Marmorstein, R. Q.; Luisi, B. F.; Sigler, P. B. Nature 1988, 335, 321-329. (3) Wolberger, C.; Dong, Y. C.; Ptashne, M.; Harrison, S. C. Nature 1988, 335, 789-795. (4) Jordan, S. R.; Pabo, C. O. Science 1988, 242, 893-899. (5) Carrondo, M. A.; Coll, M.; Aymami, J.; Wang, A. H.; van der Marel, G. A.; van Boom, J. H.; Rich, A. Biochemistry 1989, 28, 78497859. (6) Luisi, B. F.; Xu, W. X.; Otwinowski, Z.; Freedman, L. P.; Yamamoto, K. R.; Sigler, P. B. Nature 1991, 352, 497-505. (7) Somers, W. S.; Phillips, S. E. Nature 1992, 359, 387-393. (8) Beamer, L. J.; Pabo, C. O. J. Mol. Biol. 1992, 227, 177-196. (9) Beese, L. S.; Derbyshire, V.; Steitz, T. A. Science 1993, 260, 352355. (10) Shakked, Z.; Guzikevich-Guerstein, G.; Frolow, F.; Rabinovich, D.; Joachimiak, A.; Sigler, P. B. Nature 1994, 368, 469-473. (11) Luger, K.; Ma¨der, A. W.; Richmond, R. K.; Sargent, D. F.; Richmond, T. J. Nature 1997, 389, 251-260. (12) Becker, S.; Groner, B.; Mller, C. W. Nature 1998, 394, 145-151. (13) Chen, Y.-Q.; Gosh, S.; Gosh, G. Nat. Struct. Biol. 1998, 5, 6773. (14) Pabo, C. O.; Sauer, R. T. Annu. ReV. Biochem. 1992, 61, 10531095. (15) Hartmann, B.; Piazzola, D.; Lavery, R. Nucleic Acids Res. 1993, 21, 561-568. (16) Schneider, B.; Neidle, S.; Berman, H. M. Biopolymers 1997, 42, 113-124. (17) Berman, H. M. Biopolymers 1997, 44, 23-44. (18) Hartmann, B.; Lavery, R. Q. ReV. Biophys. 1996, 29, 309-368. (19) Ru¨disser, S.; Hallbrucker, A.; Mayer, E. J. Am. Chem. Soc. 1997, 119, 12251-12256. (20) Pichler, A.; Ru¨disser, S.; Mitterbo¨ck, M.; Huber, C. G.; Winger, R. H.; Liedl, K. R.; Hallbrucker, A.; Mayer, E. Biophys. J. 1999, 77, 398409. (21) Cornell, W. D.; Cieplak, P.; Bayly, C. I.; Gould, I. R.; Merz, K. M., Jr.; Ferguson, D. M.; Spellmeyer, D. C.; Fox, T.; Caldwell, J. W.; Kollman, P. A. J. Am. Chem. Soc. 1995, 117, 5179-5197. (22) York, D. M.; Wlodawer, A.; Pedersen, L. G.; Darden, T. A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 8715-8718. (23) Cheatham, T.; Miller, J.; Fox, T.; Darden, T.; Kollman, P. J. Am. Chem. Soc. 1995, 117, 4193-4194. (24) Gupta, G.; Bansal, M.; Sasiskharan, V. Proc. Natl. Acad. Sci. U.S.A. 1980, 77, 6486-6490. (25) Prive´, G. G.; Heinemann, U.; Chandrasegaran, S.; Kan, L.-S.; Kopka, M. L.; Dickerson, R. E. Science 1987, 238, 498-504.
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