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Molecular dynamics study of the effect of ion concentration on the B-DNA, Z-DNA and DNA-daunomycin complex
Journal of Molecular Structure (Theo&em), 257 (1992) 33-47 Elsevier Science Publishers B.V., Amsterdam
33
Molecular dynamics study of the effect of ion concentration on the B-DNA, Z-DNA and DNA-daunomycin complex Mee Young Song and Mu Shik Jhon Department of Chemistry and Center for Molecular Science, Korea Advanced Institute of Science and Technology, P.O. Box 150, Cheong Ryang Ri, Seoul (South Korea) (Received 11 March 1991; in final form 11 September 1991)
Molecular Dynamics simulations were used to study the internal mobility of B-DNA double helix, Z-DNA double helix, and B-DNA complex with daunomycin, an anthracycline antibiotic, as a function of sodium ion concentration. The simulations were carried out during 100 ps by using Amber force field in DISCOVER force field libraries. The helix form of DNA was preserved throughout the simulations. The results show that the mobility of B-DNA increases as the sodium ion concentration increases, but that of Z-DNA decreases. The motions of the B-DNA complex with daunomycin are smaller than those of B-DNA without daunomycin. The presence of daunomycin stabilizes B-DNA double helix. The transition from Z-DNA complex to B-DNA complex with daunomycin was investigated.
INTRODUCTION
Molecular Dynamics (MD) simulations can provide a phase-space trajectory (the position and velocities as a function of time) for a system equilibrated at a given temperature from which the dynamical properties can be obtained. Z-DNA presents a landscape entirely different from the usual B-form [l3 ], allowing the opportunity for differential recognition and action by proteins which interact with DNA. In addition to the differences in the static structures of B-DNA and Z-DNA, each structure has a characteristic flexibility and mobility determined by the potential energy restraints associated with it. The flexibility of DNA has been shown to be important for interaction with proteins, drugs, and other ligands [4-61. The mobility of DNA has been studied Correspondence to: Dr. Mu Shik Jhon, Department of Chemistry and Center for Molecular Science, Korea Advanced Institute of Science and Technology, P.O. Box 150, Cheong Ryang Ri, Seoul, South Korea.
0166-1280/92/$05.00
0 1992 Elsevier Science Publishers B.V. All rights reserved.
34
by the theoreticalMD simulationand normal mode analysis [ 7-91 in addition to a varietyof experimentaltechniques [ 10-121. Because of the difficultiesin accuratelyrepresentingthe electrostaticeffect in polyanion nucleic acid, fewer app~cationsof dynamicsimulationhave been made to nucleic acids than to proteins. Tidor et al. [7] have performed harmonic normal mode analysis and 60 ps of MD simulation on d(CpGpCpGpCpG)2. The magnitude of the motion shows general accord with X-ray experimental results. Levitt [8] has performed an extensive simulations on dynamics with 90 ps DNA study of d t~Gp~GpApAp~~~GpCpG)~ and dA,,=dT,,. The overallbendingand
(a)
(b)
35
Fig. 1 (opposite and above). Stereoviews of initial structures used in minimization: (b),Zform; (c),B-DNAcomplexwithdaunomycin (D:daunomycin).
(a), B form;
TABLE 1 R.m.s. deviation (A) of all atoms between minimized and initial structures Ion concentration
B form
z form
Free 3.5 M
0.469 0.460
0.441 0.382
twisting of these large helices were analyzed. The fluctuations in hydrogen bonds, torsional angles, and their correlated motion were characterized. Daunomycin is a DNA-binding drug [ 13-171, which exhibits anti-tumor activity. In solution it has been shown that the binding of daunomycin to DNA probably involves three distinct steps, on the basis of the results of equilibrium binding and kinetic studies [ 151. The first step is a rapid external binding, followed by intercalation, which is then followed by conformational changes of either the drug, the DNA, or both. In addition, high-resolution NMR studies have performed on the binding of daunomycin to DNA [ 18,191. Comparative studies show that daunomycin stabilizes right-handed DNA more effectively than ethidium and proflavin [ 201. The local mobility of the complex between daunomycin and the polynucleotide d (CpGpTpApCpG )2has been determined by anisotropy refinement of single crystal X-ray diffraction data [ 211. In general, daunomycin inhibits the formation of Z-DNA and interacts preferentially with B-DNA. Daunomycin has also been shown to have a potent effect on the equilibrium between right-handed B-DNA and left-handed Z-DNA [22]. The
36 TABLE 2a Torsional angles (deg) along the backbone and glycosyl angles (deg) of B-DNA Ion
Torsion angle8 ix
B
Y
6
E
-60.02 ( - 71.47 - 60.38 (- 73.92 -41 -63
179.35 174.51 179.13 176.59 136 171
55.95 60.97 55.48 53.94 38 54
135.05 124.27 131.14 132.65 139 123
-
Free 3.5 M B-DNA’ B-DNAd
178.49 170.28 174.83 174.81 133 169
c
x
- 108.41 - 99.46 - 103.66 -90.27 -157 - 108
- 109.87 - 116.06)b -111.95 - 117.53)b - 102 -117
“Torsion angles along the backbone of the polynucleotide are defined as P-05’-C5’-C4’-C3’03’ -P and K is the glycosyl angle. “This work. %rom ref. 2. dFrom DISCOVER package published by BIOSYM of HagIer et al. TABLE 2b Torsion angles (deg) along the backbone and glycosyl angles (deg) of Z-DNA Ion
Residue
Free
dC
3.5 M
t:: (dG dC
Z-DNAb
(% (dG dC dG
Torsion angle a
B
Y
6
e
4.
- 141.73 72.26 - 143.48 76.31 - 138.68 72.96 - 155.27 73.43 - 146 92
173.08 171.87 171.15 166.59 171.19 173.48 171.23 162.75 164 136
59.00 74.26 55.62 71.69 55.71 75.40 54.21 30.88 66 38
140.96 100.79 141.96 147.16 138.85 98.48 142.28 107.74 147 139
- 87.76 -96.72 - 51.55 - 141.60 - 88.85 - 97.76 - 84.47 - 165.62 -100 - 133
71.88 57.69 96.49 91.01 70.93 57.77 83.33 63.67 74 55
x 59.47 - 158.26 51.56)” - 150.75)” 63.07 - 156.63 61.33)’ - 177.27)a 32 -118
“This work. bFrom ref. 24.
pathway for the B-DNA to Z-DNA transition has been examined in detail in two studies. In the first, the Watson-Crick hydrogen bonds are broken, the bases slide out into the solvent, they flip separately, and then slide back into the stack of bases, where the hydrogen bonds are reformed [ 231. In the second mechanism, the Watson-Crick base pair geometry is retained throughout the transition, and flipping is done within a cavity formed when the base pairs above and below the flipping pair are separated by about 14A.The first mechanism is &&ally unfeasible, whereas the second mechanism, in which the
37
8_,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,~ 60
50
100
150
200
Atom
250
300
350
number
Fig. 2. R.m.s. motions of atoms (8) as a function of atom number in simulation: (a), B form; (b), Z form. TABLE 3 R.m.s. deviation (A) of all atoms between dynamic and minimized structures Ion concentration
B-DNA
Z-DNA
B-DNA complex
Free 3.5 M
1.793 3.320
3.403 2.143
1.287 2.282
base pairs remain hydrogen bonded, is sterically feasible and would account for the observed cooperativity of the transition [ 241. Therefore, the transition probably occurs in two steps: first, formation of the cavity; secondly, the base pair flip. The aims of this study include (1) a comparison of the mobilities of B- and Z-DNA and the B-DNA complex with daunomycin, as a function of sodium ion concentration, (2) determination of the differences between the mobilities of B- and Z-DNA at the same sodium ion concentration, (3) determinationslbf
38
the differences between the mobilities of B-DNA and the B-DNA complex with daunomycin at the same sodium ion concentration, and (4) an investigation of the transition between the Z-form and the B-form of the complex with daunomycin, by using the r.m.s. minimization technique. MODEL COMPOUND
Both the B-form and Z-form of a DNA double helix with the alternating sequence d(CpGpCpGpCpG), were considered. Each end of the phosphate
(a)
free
(a 1 3.5
F&3.
M
free
lb)
(b)
3.5
M
Fig. 3 (opposite and above), Stereoviews of dynamic DNA structure: (a), B form (free, ionic strength of 3.5 M); (b) Z form (free, ionic strength of 3.5 M).
group of the nucleotide was terminated by a hydrogen atom. The necessary hydrogen-atom positions were generated using standard bond lengths and angles. The atomic coordinates of B-DNA were obtained from the cylindrical coordinates provided by Arnott et al. following their refined X-ray diffraction study [ 25 ] and those of Z-DNA were obtained from the cartesian coordinates provided by Wang et al. [ 261. The structure of daunomycin was obtained from the X-ray coordinates
[27,2$] oThe moieculrxx structure of a B-DNA-daunomycin compbx was construc&d from the X-ray data and minimized by the conjugate gradient method. Figure8J(a), 1 (b) and 1 (c) show tha initial conformations of the B and 2 forms and ofthe ~-D~A~~orn~c~ complex,
The MD simuktio~ metbodolagy is described &ewhere [295, Alt energy ~~rn~~t~on~ dy~~~~ c~cu~at~o~~ and thek an&&sis, were done w&b the corn~~~r p~~ ~~~~~~ pu~~~~ by ~~~~ of Hagler et &I.The force field parameters ofDNA and daunomycin used in the MSCOVER,pmgrm were developed by Kollman sxld co-workem [ 301. To &bowthe ion cancentration effect, the shielding by counteriona is given app~~mate~y by the condensation modef f31,32] md counterioxxs were not ~nc~~d%~eq&cit& ~b~~fo~~ the charge on ea& ~b~p~te gro* was Paz_ C&y %&uced with ~~c~e~~g sod&n ion ~n~~t~~t~on. Solvex& modules were not explicitly included in the calculations, but the effect of solvent was
Atom
numbsr
TABLE 4 Torsion angles (deg) along backbone and glycosyl angles (deg) of the minimized and dynamic BDNA complex with daunomycin Ion
Residue
Torsion angle a
Free
Free”
3.5M
3.5 M”
B
Y
6
Cl G2 c3 G4 c5 G6
-81.68 100.54 116.70 - 63.95 -68.21
- 178.57 -88.99 177.55 170.89 179.71
57.56 48.88 - 175.08 - 166.67 72.61 52.91
144.06 139.61 142.91 74.01 153.77 130.67
Cl G2 c3 G4 c5 G6
-81.80 109.10 169.12 - 107.73 - 79.20
174.33 - 128.37 173.75 79.02 177.90
50.88 65.19 - 173.28 - 163.59 150.24 55.14
Cl G2 c3 G4 c5 G6
- 78.74 99.81 116.87 - 63.62 - 68.44
- 177.45 - 82.82 177.91 171.16 179.28
- 67.97
178.32 169.84 173.24 171.55 179.35
Cl G2 c3 G4 c5 G6
65.29 - 7.04 - 79.93 - 72.67
Y
E
X
4
154.03 - 70.31 -52.20 - 94.87 -97.75
-81.31 163.52 -97.16 - 108.07 176.85 - 103.50
178.41 156.32 154.93 174.87 114.55
- 154.04 - 74.97 - 146.75 - 74.27 - 107.55
- 64.08 166.65 -99.43 - 141.97 174.34 - 107.83
174.40 164.36 147.62 177.19 110.08
- 153.43 - 70.39 -51.83 - 94.23 - 97.90
- 84.06 162.80 - 90.03 - 108.53 173.34 - 103.78
- 122.89 - 167.61 - 82.27 - 164.21 - 118.41
- 147.20 - 82.86 - 105.08 -83.21 - 95.25
- 77.49 162.96 - 98.39 - 102.48 179.25 - 110.19
-
173.93 164.80 147.96 176.54 110.66
157.06 154.78 150.64 100.19 126.91 140.66
-
56.12 46.64 - 175.88 - 165.86 71.69 53.56
141.15 139.07 135.25 74.56 153.35 128.61
-
44.42 62.00 - 163.40 -63.13 56.42 59.81
85.67 79.09 156.04 100.54 143.10 112.68
-
-
“This work.
approximated by multiplying the electrostatic energy term by a (l/r) screening function [ 7,29,33-361. The cutoff distance was determined by a 12 ps test run with non-bonded cutoff distances at 10 A, 12 A,15 A,and infinity, respectively. The overall MD simulations were carried out with a cutoff of 12 A. We started with the B- and Z-forms of DNA from the X-ray crystal structure. Energy minimizations using both steepest descent methods and conjugate gradient methods were performed first. The initial structures of the Band Z-forms of DNA and the B-DNA complex with daunomycin were minimized for 4000 cycles. The MD simulations of B-DNA, Z-DNA and B-DNA complex with daunomycin were continued from the minimized structure. The structures were heated to 298 K over a 2 ps time span and then equilibrated. The time step of the dynamics was 0.001 ps. The temperature during the sim-
=: ti-
-
Free
----. 3.5 M
Fig. 5. R.m.s. motions of B-DNA complex with daunomycin (A) as a function of atom number in simulation: (a), t&al atoms; (b), groups.
ulationwas 298 3:5 K and the change in the total energywas < 0.01 kcal mol-’ for each time step. The calculationsof dynamic trajectorywere continued for 100ps. The resultsof 100 ps portions of the trajectorywere averagedover pairs of symmetry-relatedatoms. The transitionpath from Z-DNA complex to B-DNA complex with daunomycin was describedby using template forcing. The initial geometryof the ZDNA-daunomycin complex was determinedby using a minimizationmethod. The minimumstructureof the B-DNA-daunomycin complex is the giventemplate system.In templateforcing,the function which is minimizedis expressed as
(1) where E is the standardpotential energy function, xi the coordinate of the ZDNA complex with daunomycin, ii the corresponding coordinate of the BDNA complex with daunomycin, and IV is the total number of atoms being forced. The effect of templateforcing m~imization is to determinethe amount
43
(a)
rb, Fig. 6. Stereoviews of dynamic structures of B-DNA complex with daunomycin (D : daunomycin ) : (a), free; (b), ionic strength of 3.5 M.
of energy necessary to bring about a better superposition of atomic positions between the minimized system and the template system. The value of K= 400 kcal mol A was chosen, based on previous experience. After the template forcing minimization, MD simulations of the minimized system were begun over 10 pa. Until the optimum r.m.s. value was obtained, the above two processes
44 TABLE 5 Torsion angles (deg) along backbone and glycosyl angles (deg) for the transition of the Z-DNA complex with daunomycin Residue
Torsion angle a
Cl G2 c3 G4 c5 G6
-
77.99 47.30 84.60 50.82 71.71
B
Y
s
t
172.60 165.16 164.88 163.95 171.16
53.06 79.69 60.75 67.96 75.85 76.30
148.86 107.59 150.52 123.93 154.93 123.22
-
179.49 167.98 174.84 169.63 173.95
r
x
- 154.53 -90.46 - 136.34 - 111.44 - 94.56
- 69.75 - 176.90 -84.49 -99.22 - 175.31 -88.80
were repeated. The overall MD simulations and analysis were performed on a Cray-2s/4-128 computer and required about 100 h of Cray time. RESULTS AND DISCUSSION
The effect of ion concentration on the minimization results of the B- and Zforms is summarized in Tables 1, 2a and 2b. The minimized structures are relatively close to the initial structure. They are consistent with the results of Tidor et al. [ 71. The r.m.s. atomic fluctuations for 100 ps of simulations are shown in Figs. 2 (a) and 2 (b) in the absence of ions and at an ionic strength of 3.5 M. The total r.m.s. deviations between minimized and dynamic structures are given in Table 3. In all concentrations, Figs. 2 (a) and 2 (b) indicate that there are generally larger motions in the backbone than in the ribose rings, and larger motions in the ribose rings than in the base pair. These results are qualitatively similar to those found by Tidor et al. [ 71 and Singh et al. [ 361 and follow the same order as motions inferred from X-ray temperature factors. The fluctuation of B-DNA is small in the absence of ions but that of Z-DNA is small at an ionic strength of 3.5 M. These results show that the Z-form of DNA is more stable than B-DNA at higher ionic strengths. The result of dynamic structures are shown in Figs. 3 (a) and 3 (b). Therefore, the B-form is easily transformed to the Z-form of DNA because the B-form has the largest motion at high ionic strengths. In Figs. 4 (a) and 4 (b), we present the r.m.s. motion of the various atoms (sugar-phosphate backbone, purines and pyrimidines), except terminal groups, as a mean value. These results show in detail the motion of the groups at two ion strengths. In Figs. 4 (a) and 4 (b) , the sugar-phosphate backbone shows greater motion in the absence of ions than it does at the ionic strength of 3.5 M, but the base groups show larger movement in the opposite direction.
45
The mobility of the sugar-phosphate backbone in the DNA-daunomycin complex is generally similar to the results for the B and 2 forms of DNA. There are definite differences in the area where the drug molecule intercalates into the DNA. The DNA sugar-phosphate backbone maintains a fairly uniform range of conformations throughout the molecule which has been perturbed by the rather complex daunomycin molecule. The average torsion angles along the DNA backbone are: CX=-71”; /3=176”; y=57”; r&142”; E=-173’; r= -98”;x= - 95 O.The minimized and dynamic structures are compared with experimental data and shown in Table 4. The phosphate groups on either side of the daunomycin have different conformations. This is associated with the asymmetric shape of the daunomycin molecule. Daunomycin shows smaller movement in the planar ring system than in the amino sugar group. These results are in good agreement with previous experimental results [ 221. In Figs. 5 (a) and 5 (b), the r.m.s. fluctuations are increased with increasing ion concentration. At the same ion concentration, the r.m.s. movement of the B-DNA complex with daunomycin is smaller than that of B-DNA. Therefore, the structures of B-DNA are stabilized by intercalating daunomycin at the same ion concentration. The binding of daunomycin inhibits the transition from the B-form to other helix forms (especially the formation of the Z-form) at high ion concentrations. In Figs. 6 (a) and 6 (b) the results of dynamics simulations for the B-DNA complex with daunomycin are shown. We investigated the transition from the Z-form to the B-form of the DNA complex with daunomycin in the absence of ions. R.m.s. minimization of distances and torsional restraints taken from the minimized B-form of DNA complex was used. The atomic r.m.s. difference between the B-DNA complex and the Z-DNA complex with daunomycin was reduced from 8.64 to 1.18 (A). In spite of the existence of an external force, hydrogen bonding of the base pairs persisted during the transition. These results are consistent with the proposed mechanism in which the Watson-Crick base pair geometry is retained throughout the transition [ 241. Owing to the presence of the drug, intercalating cavities between base pairs were formed, which was followed by a conformational change of DNA with a flexible backbone. The average torsional angles along the DNA backbone are listed in Table 5. It can be seen that they fall in the region corresponding to the conformations of the B-DNA complex with daunomycin. In conclusion, the mobilities of B-DNA, Z-DNA and the B-DNA complex with daunomycin were calculated by using MD simulation techniques. The results show the relatively small mobility of B-DNA at lower ion concentrations and the relatively small mobility of Z-DNA at higher ion concentrations. B-DNA showed small atomic fluctuations with intercalated daunomycin in the absence of sodium ions. Daunomycin interacts preferentially with B-DNA, so B-DNA is stabilized by intercalating daunomycin and inhibits the formation of Z-DNA at higher ion concentrations.
46 ACKNOWLEDGMENTS
The authors express appreciation for financial support to the Korea Science and Engineering Foundation and the Korea Research Center for Theoretical Physics and Chemistry.
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A.H.J. Wang, G.J. Quigley, F.J. Kolpak, J.L. Crawford, J.H. van Boom, G. van der Mare1and A. Rich, Nature, 282 (1979) 680. S. Arnott, R. Chandrasekaran, D.L. BirdsaIl, A.G.W. Leslie and R.L. Ratcliff, Nature, 283 (1980) 743. D.J. Pate&L.L. Canuel and F.M. Pohi, Proc. Natl. Acad. Sci. USA, 76 (1979) 2508. C.A. Frederik, J. Grable, M. Melia, C. Samudzi, L. JenJacobsen, B.C. Wang, P. Greene, H. Boyer and J.M. Rosenberg, Nature, 309 (1984) 327. R. Kim, P. Modrich and S.-H. Kim, Nucleic Acids Res., 12 (1984) 7285. A.H.-J. Wang, G. Ughetto, G.J. Quigley, T. Hakashima, G. van der Marel, J.H. van Boom and A. Rich, Science, 225 (1984) 1115. B. Tidor, K.K. Irikura, B.R. Brooks and M. Karplus, J. Biomol. Struct. Dyn., 1 (1983) 231. M. Levitt, Cold Spring Harbor Symp. Quant. Biol., 47 (1983) 251. S.C. Harvey, M. Parbhakaran, B. Mao and J.A. McCammon, Science, 223 (1984) 1189. M.E. Hogan and 0. Jardetzky, Biochemistry, 19 (1980) 3460. D.P. MilIar, Rd. Robbins and A.H. Zewail, Proc. NatI. Acad. Sci. USA, 77 (1980) 5593. T.J. Thomas and V.A. Bloomfield, Nucleic Acids Res., 11 (1983) 1919. J.B. Chaires, Biochemistry, 22 (1983) 4204. J.B. Chair-es,N. Dattagupta and D.M. Crothers, Biochemistry, 21 (1982) 3933. J.B. Chaires, N. Dattagupta and D.M. Crothers, Biochemistry, 24 (1985) 260. T.W. Plumbridge and J.R. Brown, B&him. Biophys. Acta, 563 (1979) 181. J.B. Chaires, K.R. Fox, J.E. Herrera, M. Britt and M.J. Waring, Biochemistry, 26 (1987) 8227. D.R. Phillips and G.C.K. Roberta, Biochemistry, 19 (1980) 4795. J.M. Neumann, J.A. CarailIes, M. Herve, S. Tran-Dinh, B. Langlois d’Estaintot, T. HuynhDinh and J. Igolen, FEBS Lett., 182 (1985) 360. J.B. Chaires, Biochemistry, 25 (1986) 8436. A.H.-J. Wang, G. Ughetto, G.J. Quigley and A. Rich, Biochemistry, 26 (1987) 1152. J.B. Chaires, Biochemistry, 24 (1986) 7479. W.K. Olson, A.R. Srinavasan, N.I. Markey and V.N. BaIaji, Cold Spring Harbor Symp. Quant. Biol., 47 (1983) 229. S.C. Harvey, Nucleic Acids Res., 11(14) (1983) 4867. S.P. Arnott, J.C. Smith and R. Chandrasekaran, CRC Handbook of Biochemistry and Molecular Biology, 3rd edn., Vol. II, CRC Press, Ohio, 1976, p. 411. A.H.J. Wang, G.J. Quigley and F.J. Kolpak, Science, 211 (1980) 171. S. Neidle and G.L. Taylor, Biochim. Biophys. Acta, 479 (1977) 450. G.J. Quigley, A.H.J. Wang, G. Ughetto, G. van der Marel, J.H. van Boom and A. Rich, Proc. Natl. Acad. Sci. USA, 77 (1980) 7204. B.R. Brooks, R.E. Bruccoleri, B.D. Olafson, D.J. States, S. Swaminathan and M. Karplus, J. Comput. Chem., 4 (1983) 187. S. Weiner, P.A. Kolhnan, D.T. Nguyen andD. Case, J. Comput. Chem., 7(2) (1986) 230.
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33
Molecular dynamics study of the effect of ion concentration on the B-DNA, Z-DNA and DNA-daunomycin complex Mee Young Song and Mu Shik Jhon Department of Chemistry and Center for Molecular Science, Korea Advanced Institute of Science and Technology, P.O. Box 150, Cheong Ryang Ri, Seoul (South Korea) (Received 11 March 1991; in final form 11 September 1991)
Molecular Dynamics simulations were used to study the internal mobility of B-DNA double helix, Z-DNA double helix, and B-DNA complex with daunomycin, an anthracycline antibiotic, as a function of sodium ion concentration. The simulations were carried out during 100 ps by using Amber force field in DISCOVER force field libraries. The helix form of DNA was preserved throughout the simulations. The results show that the mobility of B-DNA increases as the sodium ion concentration increases, but that of Z-DNA decreases. The motions of the B-DNA complex with daunomycin are smaller than those of B-DNA without daunomycin. The presence of daunomycin stabilizes B-DNA double helix. The transition from Z-DNA complex to B-DNA complex with daunomycin was investigated.
INTRODUCTION
Molecular Dynamics (MD) simulations can provide a phase-space trajectory (the position and velocities as a function of time) for a system equilibrated at a given temperature from which the dynamical properties can be obtained. Z-DNA presents a landscape entirely different from the usual B-form [l3 ], allowing the opportunity for differential recognition and action by proteins which interact with DNA. In addition to the differences in the static structures of B-DNA and Z-DNA, each structure has a characteristic flexibility and mobility determined by the potential energy restraints associated with it. The flexibility of DNA has been shown to be important for interaction with proteins, drugs, and other ligands [4-61. The mobility of DNA has been studied Correspondence to: Dr. Mu Shik Jhon, Department of Chemistry and Center for Molecular Science, Korea Advanced Institute of Science and Technology, P.O. Box 150, Cheong Ryang Ri, Seoul, South Korea.
0166-1280/92/$05.00
0 1992 Elsevier Science Publishers B.V. All rights reserved.
34
by the theoreticalMD simulationand normal mode analysis [ 7-91 in addition to a varietyof experimentaltechniques [ 10-121. Because of the difficultiesin accuratelyrepresentingthe electrostaticeffect in polyanion nucleic acid, fewer app~cationsof dynamicsimulationhave been made to nucleic acids than to proteins. Tidor et al. [7] have performed harmonic normal mode analysis and 60 ps of MD simulation on d(CpGpCpGpCpG)2. The magnitude of the motion shows general accord with X-ray experimental results. Levitt [8] has performed an extensive simulations on dynamics with 90 ps DNA study of d t~Gp~GpApAp~~~GpCpG)~ and dA,,=dT,,. The overallbendingand
(a)
(b)
35
Fig. 1 (opposite and above). Stereoviews of initial structures used in minimization: (b),Zform; (c),B-DNAcomplexwithdaunomycin (D:daunomycin).
(a), B form;
TABLE 1 R.m.s. deviation (A) of all atoms between minimized and initial structures Ion concentration
B form
z form
Free 3.5 M
0.469 0.460
0.441 0.382
twisting of these large helices were analyzed. The fluctuations in hydrogen bonds, torsional angles, and their correlated motion were characterized. Daunomycin is a DNA-binding drug [ 13-171, which exhibits anti-tumor activity. In solution it has been shown that the binding of daunomycin to DNA probably involves three distinct steps, on the basis of the results of equilibrium binding and kinetic studies [ 151. The first step is a rapid external binding, followed by intercalation, which is then followed by conformational changes of either the drug, the DNA, or both. In addition, high-resolution NMR studies have performed on the binding of daunomycin to DNA [ 18,191. Comparative studies show that daunomycin stabilizes right-handed DNA more effectively than ethidium and proflavin [ 201. The local mobility of the complex between daunomycin and the polynucleotide d (CpGpTpApCpG )2has been determined by anisotropy refinement of single crystal X-ray diffraction data [ 211. In general, daunomycin inhibits the formation of Z-DNA and interacts preferentially with B-DNA. Daunomycin has also been shown to have a potent effect on the equilibrium between right-handed B-DNA and left-handed Z-DNA [22]. The
36 TABLE 2a Torsional angles (deg) along the backbone and glycosyl angles (deg) of B-DNA Ion
Torsion angle8 ix
B
Y
6
E
-60.02 ( - 71.47 - 60.38 (- 73.92 -41 -63
179.35 174.51 179.13 176.59 136 171
55.95 60.97 55.48 53.94 38 54
135.05 124.27 131.14 132.65 139 123
-
Free 3.5 M B-DNA’ B-DNAd
178.49 170.28 174.83 174.81 133 169
c
x
- 108.41 - 99.46 - 103.66 -90.27 -157 - 108
- 109.87 - 116.06)b -111.95 - 117.53)b - 102 -117
“Torsion angles along the backbone of the polynucleotide are defined as P-05’-C5’-C4’-C3’03’ -P and K is the glycosyl angle. “This work. %rom ref. 2. dFrom DISCOVER package published by BIOSYM of HagIer et al. TABLE 2b Torsion angles (deg) along the backbone and glycosyl angles (deg) of Z-DNA Ion
Residue
Free
dC
3.5 M
t:: (dG dC
Z-DNAb
(% (dG dC dG
Torsion angle a
B
Y
6
e
4.
- 141.73 72.26 - 143.48 76.31 - 138.68 72.96 - 155.27 73.43 - 146 92
173.08 171.87 171.15 166.59 171.19 173.48 171.23 162.75 164 136
59.00 74.26 55.62 71.69 55.71 75.40 54.21 30.88 66 38
140.96 100.79 141.96 147.16 138.85 98.48 142.28 107.74 147 139
- 87.76 -96.72 - 51.55 - 141.60 - 88.85 - 97.76 - 84.47 - 165.62 -100 - 133
71.88 57.69 96.49 91.01 70.93 57.77 83.33 63.67 74 55
x 59.47 - 158.26 51.56)” - 150.75)” 63.07 - 156.63 61.33)’ - 177.27)a 32 -118
“This work. bFrom ref. 24.
pathway for the B-DNA to Z-DNA transition has been examined in detail in two studies. In the first, the Watson-Crick hydrogen bonds are broken, the bases slide out into the solvent, they flip separately, and then slide back into the stack of bases, where the hydrogen bonds are reformed [ 231. In the second mechanism, the Watson-Crick base pair geometry is retained throughout the transition, and flipping is done within a cavity formed when the base pairs above and below the flipping pair are separated by about 14A.The first mechanism is &&ally unfeasible, whereas the second mechanism, in which the
37
8_,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,~ 60
50
100
150
200
Atom
250
300
350
number
Fig. 2. R.m.s. motions of atoms (8) as a function of atom number in simulation: (a), B form; (b), Z form. TABLE 3 R.m.s. deviation (A) of all atoms between dynamic and minimized structures Ion concentration
B-DNA
Z-DNA
B-DNA complex
Free 3.5 M
1.793 3.320
3.403 2.143
1.287 2.282
base pairs remain hydrogen bonded, is sterically feasible and would account for the observed cooperativity of the transition [ 241. Therefore, the transition probably occurs in two steps: first, formation of the cavity; secondly, the base pair flip. The aims of this study include (1) a comparison of the mobilities of B- and Z-DNA and the B-DNA complex with daunomycin, as a function of sodium ion concentration, (2) determination of the differences between the mobilities of B- and Z-DNA at the same sodium ion concentration, (3) determinationslbf
38
the differences between the mobilities of B-DNA and the B-DNA complex with daunomycin at the same sodium ion concentration, and (4) an investigation of the transition between the Z-form and the B-form of the complex with daunomycin, by using the r.m.s. minimization technique. MODEL COMPOUND
Both the B-form and Z-form of a DNA double helix with the alternating sequence d(CpGpCpGpCpG), were considered. Each end of the phosphate
(a)
free
(a 1 3.5
F&3.
M
free
lb)
(b)
3.5
M
Fig. 3 (opposite and above), Stereoviews of dynamic DNA structure: (a), B form (free, ionic strength of 3.5 M); (b) Z form (free, ionic strength of 3.5 M).
group of the nucleotide was terminated by a hydrogen atom. The necessary hydrogen-atom positions were generated using standard bond lengths and angles. The atomic coordinates of B-DNA were obtained from the cylindrical coordinates provided by Arnott et al. following their refined X-ray diffraction study [ 25 ] and those of Z-DNA were obtained from the cartesian coordinates provided by Wang et al. [ 261. The structure of daunomycin was obtained from the X-ray coordinates
[27,2$] oThe moieculrxx structure of a B-DNA-daunomycin compbx was construc&d from the X-ray data and minimized by the conjugate gradient method. Figure8J(a), 1 (b) and 1 (c) show tha initial conformations of the B and 2 forms and ofthe ~-D~A~~orn~c~ complex,
The MD simuktio~ metbodolagy is described &ewhere [295, Alt energy ~~rn~~t~on~ dy~~~~ c~cu~at~o~~ and thek an&&sis, were done w&b the corn~~~r p~~ ~~~~~~ pu~~~~ by ~~~~ of Hagler et &I.The force field parameters ofDNA and daunomycin used in the MSCOVER,pmgrm were developed by Kollman sxld co-workem [ 301. To &bowthe ion cancentration effect, the shielding by counteriona is given app~~mate~y by the condensation modef f31,32] md counterioxxs were not ~nc~~d%~eq&cit& ~b~~fo~~ the charge on ea& ~b~p~te gro* was Paz_ C&y %&uced with ~~c~e~~g sod&n ion ~n~~t~~t~on. Solvex& modules were not explicitly included in the calculations, but the effect of solvent was
Atom
numbsr
TABLE 4 Torsion angles (deg) along backbone and glycosyl angles (deg) of the minimized and dynamic BDNA complex with daunomycin Ion
Residue
Torsion angle a
Free
Free”
3.5M
3.5 M”
B
Y
6
Cl G2 c3 G4 c5 G6
-81.68 100.54 116.70 - 63.95 -68.21
- 178.57 -88.99 177.55 170.89 179.71
57.56 48.88 - 175.08 - 166.67 72.61 52.91
144.06 139.61 142.91 74.01 153.77 130.67
Cl G2 c3 G4 c5 G6
-81.80 109.10 169.12 - 107.73 - 79.20
174.33 - 128.37 173.75 79.02 177.90
50.88 65.19 - 173.28 - 163.59 150.24 55.14
Cl G2 c3 G4 c5 G6
- 78.74 99.81 116.87 - 63.62 - 68.44
- 177.45 - 82.82 177.91 171.16 179.28
- 67.97
178.32 169.84 173.24 171.55 179.35
Cl G2 c3 G4 c5 G6
65.29 - 7.04 - 79.93 - 72.67
Y
E
X
4
154.03 - 70.31 -52.20 - 94.87 -97.75
-81.31 163.52 -97.16 - 108.07 176.85 - 103.50
178.41 156.32 154.93 174.87 114.55
- 154.04 - 74.97 - 146.75 - 74.27 - 107.55
- 64.08 166.65 -99.43 - 141.97 174.34 - 107.83
174.40 164.36 147.62 177.19 110.08
- 153.43 - 70.39 -51.83 - 94.23 - 97.90
- 84.06 162.80 - 90.03 - 108.53 173.34 - 103.78
- 122.89 - 167.61 - 82.27 - 164.21 - 118.41
- 147.20 - 82.86 - 105.08 -83.21 - 95.25
- 77.49 162.96 - 98.39 - 102.48 179.25 - 110.19
-
173.93 164.80 147.96 176.54 110.66
157.06 154.78 150.64 100.19 126.91 140.66
-
56.12 46.64 - 175.88 - 165.86 71.69 53.56
141.15 139.07 135.25 74.56 153.35 128.61
-
44.42 62.00 - 163.40 -63.13 56.42 59.81
85.67 79.09 156.04 100.54 143.10 112.68
-
-
“This work.
approximated by multiplying the electrostatic energy term by a (l/r) screening function [ 7,29,33-361. The cutoff distance was determined by a 12 ps test run with non-bonded cutoff distances at 10 A, 12 A,15 A,and infinity, respectively. The overall MD simulations were carried out with a cutoff of 12 A. We started with the B- and Z-forms of DNA from the X-ray crystal structure. Energy minimizations using both steepest descent methods and conjugate gradient methods were performed first. The initial structures of the Band Z-forms of DNA and the B-DNA complex with daunomycin were minimized for 4000 cycles. The MD simulations of B-DNA, Z-DNA and B-DNA complex with daunomycin were continued from the minimized structure. The structures were heated to 298 K over a 2 ps time span and then equilibrated. The time step of the dynamics was 0.001 ps. The temperature during the sim-
=: ti-
-
Free
----. 3.5 M
Fig. 5. R.m.s. motions of B-DNA complex with daunomycin (A) as a function of atom number in simulation: (a), t&al atoms; (b), groups.
ulationwas 298 3:5 K and the change in the total energywas < 0.01 kcal mol-’ for each time step. The calculationsof dynamic trajectorywere continued for 100ps. The resultsof 100 ps portions of the trajectorywere averagedover pairs of symmetry-relatedatoms. The transitionpath from Z-DNA complex to B-DNA complex with daunomycin was describedby using template forcing. The initial geometryof the ZDNA-daunomycin complex was determinedby using a minimizationmethod. The minimumstructureof the B-DNA-daunomycin complex is the giventemplate system.In templateforcing,the function which is minimizedis expressed as
(1) where E is the standardpotential energy function, xi the coordinate of the ZDNA complex with daunomycin, ii the corresponding coordinate of the BDNA complex with daunomycin, and IV is the total number of atoms being forced. The effect of templateforcing m~imization is to determinethe amount
43
(a)
rb, Fig. 6. Stereoviews of dynamic structures of B-DNA complex with daunomycin (D : daunomycin ) : (a), free; (b), ionic strength of 3.5 M.
of energy necessary to bring about a better superposition of atomic positions between the minimized system and the template system. The value of K= 400 kcal mol A was chosen, based on previous experience. After the template forcing minimization, MD simulations of the minimized system were begun over 10 pa. Until the optimum r.m.s. value was obtained, the above two processes
44 TABLE 5 Torsion angles (deg) along backbone and glycosyl angles (deg) for the transition of the Z-DNA complex with daunomycin Residue
Torsion angle a
Cl G2 c3 G4 c5 G6
-
77.99 47.30 84.60 50.82 71.71
B
Y
s
t
172.60 165.16 164.88 163.95 171.16
53.06 79.69 60.75 67.96 75.85 76.30
148.86 107.59 150.52 123.93 154.93 123.22
-
179.49 167.98 174.84 169.63 173.95
r
x
- 154.53 -90.46 - 136.34 - 111.44 - 94.56
- 69.75 - 176.90 -84.49 -99.22 - 175.31 -88.80
were repeated. The overall MD simulations and analysis were performed on a Cray-2s/4-128 computer and required about 100 h of Cray time. RESULTS AND DISCUSSION
The effect of ion concentration on the minimization results of the B- and Zforms is summarized in Tables 1, 2a and 2b. The minimized structures are relatively close to the initial structure. They are consistent with the results of Tidor et al. [ 71. The r.m.s. atomic fluctuations for 100 ps of simulations are shown in Figs. 2 (a) and 2 (b) in the absence of ions and at an ionic strength of 3.5 M. The total r.m.s. deviations between minimized and dynamic structures are given in Table 3. In all concentrations, Figs. 2 (a) and 2 (b) indicate that there are generally larger motions in the backbone than in the ribose rings, and larger motions in the ribose rings than in the base pair. These results are qualitatively similar to those found by Tidor et al. [ 71 and Singh et al. [ 361 and follow the same order as motions inferred from X-ray temperature factors. The fluctuation of B-DNA is small in the absence of ions but that of Z-DNA is small at an ionic strength of 3.5 M. These results show that the Z-form of DNA is more stable than B-DNA at higher ionic strengths. The result of dynamic structures are shown in Figs. 3 (a) and 3 (b). Therefore, the B-form is easily transformed to the Z-form of DNA because the B-form has the largest motion at high ionic strengths. In Figs. 4 (a) and 4 (b), we present the r.m.s. motion of the various atoms (sugar-phosphate backbone, purines and pyrimidines), except terminal groups, as a mean value. These results show in detail the motion of the groups at two ion strengths. In Figs. 4 (a) and 4 (b) , the sugar-phosphate backbone shows greater motion in the absence of ions than it does at the ionic strength of 3.5 M, but the base groups show larger movement in the opposite direction.
45
The mobility of the sugar-phosphate backbone in the DNA-daunomycin complex is generally similar to the results for the B and 2 forms of DNA. There are definite differences in the area where the drug molecule intercalates into the DNA. The DNA sugar-phosphate backbone maintains a fairly uniform range of conformations throughout the molecule which has been perturbed by the rather complex daunomycin molecule. The average torsion angles along the DNA backbone are: CX=-71”; /3=176”; y=57”; r&142”; E=-173’; r= -98”;x= - 95 O.The minimized and dynamic structures are compared with experimental data and shown in Table 4. The phosphate groups on either side of the daunomycin have different conformations. This is associated with the asymmetric shape of the daunomycin molecule. Daunomycin shows smaller movement in the planar ring system than in the amino sugar group. These results are in good agreement with previous experimental results [ 221. In Figs. 5 (a) and 5 (b), the r.m.s. fluctuations are increased with increasing ion concentration. At the same ion concentration, the r.m.s. movement of the B-DNA complex with daunomycin is smaller than that of B-DNA. Therefore, the structures of B-DNA are stabilized by intercalating daunomycin at the same ion concentration. The binding of daunomycin inhibits the transition from the B-form to other helix forms (especially the formation of the Z-form) at high ion concentrations. In Figs. 6 (a) and 6 (b) the results of dynamics simulations for the B-DNA complex with daunomycin are shown. We investigated the transition from the Z-form to the B-form of the DNA complex with daunomycin in the absence of ions. R.m.s. minimization of distances and torsional restraints taken from the minimized B-form of DNA complex was used. The atomic r.m.s. difference between the B-DNA complex and the Z-DNA complex with daunomycin was reduced from 8.64 to 1.18 (A). In spite of the existence of an external force, hydrogen bonding of the base pairs persisted during the transition. These results are consistent with the proposed mechanism in which the Watson-Crick base pair geometry is retained throughout the transition [ 241. Owing to the presence of the drug, intercalating cavities between base pairs were formed, which was followed by a conformational change of DNA with a flexible backbone. The average torsional angles along the DNA backbone are listed in Table 5. It can be seen that they fall in the region corresponding to the conformations of the B-DNA complex with daunomycin. In conclusion, the mobilities of B-DNA, Z-DNA and the B-DNA complex with daunomycin were calculated by using MD simulation techniques. The results show the relatively small mobility of B-DNA at lower ion concentrations and the relatively small mobility of Z-DNA at higher ion concentrations. B-DNA showed small atomic fluctuations with intercalated daunomycin in the absence of sodium ions. Daunomycin interacts preferentially with B-DNA, so B-DNA is stabilized by intercalating daunomycin and inhibits the formation of Z-DNA at higher ion concentrations.
46 ACKNOWLEDGMENTS
The authors express appreciation for financial support to the Korea Science and Engineering Foundation and the Korea Research Center for Theoretical Physics and Chemistry.
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