Journal of Biomolecular Structure and Dynamics, 1997, 15, 619-624.
Quantum-chemical ab initio study on the adenine-difluorotoluene complex - a mimic for the adenine-thymine base pair
M. Meyer* and J. Sühnel*
Biocomputing, Institut für Molekulare Biotechnologie, Beutenbergstr. 11, D-07745 Jena, Germany
* Correspondence can be addressed to both authors. FAX: +49 3641 65 6210 E-mail:
[email protected];
[email protected]
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Abstract
Recent experiments have shown that difluorotoluene (F), a nonpolar isostere for thymine (T), codes efficiently and specifically for adenine (A) in DNA replication. F has almost the same shape as thymine but it is unable to form conventional hydrogen bonds with adenine. Therefore, it has been claimed that not hydrogen bonding but shape complementary may be important for the selection of the correct bases by DNA-replicating enzymes. In order to gain deeper insight into structure, charge distribution and energetics of the A-F and A-T base pairs we have performed quantum-chemical ab initio and density functional calculations at the HF, MP2 and B3LYP levels. The interaction energy of the A-F complex amounts to -3.8 kcal/mol (MP2) and is thus substantially smaller than typical ab initio interaction energies for WatsonCrick or non-canonical base pairs. The A-T and A-F complexes are planar and their overall geometries are similar (root-mean-square deviation: 0.4 Å). The calculated donor acceptor atom distances in A-T are in good agreement with the experimental mean values obtained from an analysis of 21 high resolution DNA structures. However, A-F shows a base pair opening as compared to A-T. Even though the interaction energy in the A-F base pair is small, the distances for the N6-H...F and N1...H-C3 contacts are still below the sum of the van-der-Waals radii, which means that the interaction is not governed by van-der-Waals forces alone. If the experimental findings can be confirmed, then our results indicate that DNA polymerase is able to retain high fidelity with base pairs of much smaller interaction energies than found for the conventional Watson-Crick and non-canonical base pairs.
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Introduction
Recently difluorotoluene (F) has been placed as a nonpolar isostere of thymine (T) opposite to adenine (A) within DNA (1). Such a substitution destabilizes the DNA helix by approximately 4-5 kcal/mol relative to T at the same position. According to NMR studies F cannot form hydrogen bonded complexes with adenine derivatives in chloroform (2). However, replacement of T by F had no measurable effect on DNA synthesis. These findings have led to the conclusion that not hydrogen bonding but shape complementarity may be the key to faithful copying of DNA. In view of the fact that hydrogen bonding is usually considered as a major determinant both of biopolymer structures and of DNA replication this would be an extremely important new finding.
Ab initio quantum-chemical calculations with inclusion of electron correlation have recently provided a relatively consistent picture on base pair interaction energies and geometries (3). We have therefore performed calculations of this type for the A-F and A-T base pairs. This can lead to a more detailed information on structure, charge distribution and energetics of the base pairs as compared to the simple isosterism concept. In addition, the results obtained have been related to geometrical data for the A-T pair derived from an analysis of a set of experimental high resolution DNA structures and to NMR results for the A-F pair.
Methods
Ab initio calculations at the HF/6-31G(dp) (4,5) and density functional (DFT) studies at the
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B3LYP/6-31G(dp) (6,7) levels were carried out to determine the complex structures with the programs GAMESS (8) and GAUSSIAN94 (9). Energy minima of the optimized complexes have been verified at the Hartree-Fock level by a calculation of the Hessian. The interaction energies were corrected for the basis set superposition error (BSSE) by the standard counterpoise method approach (10). Ab initio interaction energies were evaluated using the HF/6-31G(dp)-optimized geometries but taking into account electron correlation by means of second order Møller-Plesset perturbation theory (MP2/6-31G(dp)//HF/6-31G(dp)). The deformation energy EDEF is defined as the energy difference between the geometry optimized monomers and the structures of the monomers adopted in the complex. Finally, the changes in the zero point vibrational energies ∆ZPE upon complex formation have been computed at the HF/6-31G(dp) level. The interaction energy of the base pairs taking into account electron correlation at the MP2 level is called total stabilization energy ET=EMP2+EDEF. Finally, this energy can be corrected for the zero point energy changes by E0=ET+∆ZPE.
The Gauge Independent Atomic Orbital (GIAO) method (12) has been used to estimate the change of NMR shifts upon complex formation using the HF/6-311+G(2d2p) basis augmented with diffuse functions and geometries from HF/6-31G(dp) optimizations.
A-F was studied both in the Watson-Crick and reverse Watson-Crick orientations (Figure 1). The interaction energies for A-F were compared with data from conventional base pairs, which have been discussed recently in detail by Šponer et al. (3).
Mean values of intermolecular donor acceptor atom distances of A-T pairs have been
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determined from 21 DNA structures with a resolution better than 1.5 Å using the program HBexplore (14) for comparison between experimental and calculated data. The Protein Data Bank codes of the DNA structures are: 151d, 152d, 191d, 1d10, 1d11, 1d15, 1d23, 1d36, 1d49, 1d54, 1d61, 1d78, 1d79, 1da0, 1dn8, 1ims, 224d, 245d, 2des, 3dnb, 5dnb.
Results
The BSSE corrected interaction energies are summarized in Table I and structural parameters are given in Table II. Differences in the intermolecular structure parameters and energies of the standard and reverse A-F and A-T pairs are very small and not further discussed. As observed previously for other base pairs (16) the HF level interaction energy is smaller than the corresponding MP2 interaction energy. The ab initio stabilization energy corrected for electron correlation, deformation and zero point vibration is E0 = -3.0 kcal/mol for A-F and 10.3 kcal/mol for A-T. The latter value is in close agreement with the results obtained by Šponer et al. for A-T (16). The same relation between A-F and A-T is also obtained from the DFT results, Table I. Thus even though different approximations yield slightly different interaction energies the basic result is that the base pair interaction energy of A-F is much smaller than for A-T and for all other base pairs as well (3).
Selected structural parameters for the A-T and A-F complexes are listed in Table II. The structural parameters for the A-T pair agree with the data reported recently at the same HF level (16). For the B3LYP hybrid density functional level the calculated interaction energies are close to those obtained at MP2 level. In general the intermolecular distances determined
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with this method are somewhat shorter as compared to HF optimizations.
Discussion
The results obtained give a clear picture of the differences between the A-F and A-T base pairs, which is independent, at least in a qualitative sense, on the methods and approximation levels adopted. Nevertheless, we discuss briefly a methodological aspect of the calculations first and then proceed with a discussion of the basic results without reiterating statements on the finer details of approximations and methods.
Hybrid density functional methods have been applied frequently as a less computationally demanding alternative to MP2 calculations (15). But the accuracy of B3LYP calculations has been considered as insufficient for base pair interactions (16). Our calculations indicate, however, that for the A-T and A-F pairs interaction energies are in good agreement with MP2 calculations. For A-T we computed EDFT=-14.0 kcal/mol for a structure optimized at the same level, whereas -11.9 kcal/mol have been obtained with the same method at a HF/6-1G(dp) structure (16). This indicates that the DFT interaction energies depend critically on the assumed structures. We have determined average intermolecular donor acceptor atom distances from 62 A-T pairs in 21 high resolution DNA X-ray structures. The results are (standard deviations in parentheses): r(N6-O4) = 2.98 (± 0.14) Å, r(N1-N3)= 2.82(± 0.07) Å, r(C2-O2)=3.52 (±0.14) Å. It turns out that the DFT distances are in excellent agreement with the experimental data, whereas the HF distances are somewhat too long, Table II. Therefore, we have to conclude that, at least for the properties of the hydrogen bonded
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systems studied in this work, density functional calculations give similar or even better results than the conventional ab initio studies.
The interaction energies calculated with different methods and at different levels of approximation vary between -10.2 and -14.0 kcal/mol for A-T and between -2.7 and -3.9 kcal/mol for A-F. This means that the interaction energy for A-F is significantly smaller than for A-T. In a systematic study the smallest ab initio MP2 interaction energy was found for the G-A complex with -9.6 kcal/mol (3). Hence, the A-F interaction energy is smaller than for any other Watson-Crick or non-canonical base pair. Experimentally, it has been found that the substitution of T with F destabilizes the DNA helix by approximately 4-5 kcal/mol which is less than the computed interaction energy difference. Note, however, that a direct comparison of these values makes no sense. As a consequence of the small interaction energy between both components the deformation energy is also smaller than the typical range of 0.6-2.5 kcal/mol calculated for neutral base pairs. (16).
The A-T and A-F base pairs are planar and the root-mean-square deviation of the superimposed atomic coordinates is 0.4 Å, which confirms the geometrical similarity of the two structures. However, when superimposing adenine alone one sees clearly that A-F exhibits a base pair opening as compared to the A-T pair, Fig. 2. The structures of F and T are almost identical except for the CF/CO bond lengths (root-mean-square deviation: 0.1 Å). However, the intermolecular H2…F2 distance (HF: 4.197 Å; DFT: 3.981 Å) in A-F is much larger than the corresponding H2…O2 distance in A-T (HF: 2.961 Å; DFT: 2.801 Å), whereas the H61… O4 (HF: 2.091 Å; DFT: 1.919 Å) and H61… F4 (HF: 2.253; DFT:
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2.089Å) distances are not very different. In addition the N1…H3-N3 hydrogen bond (HF: 1.976 Å; DFT: 1.796 Å) in A-T is replaced be a weaker and longer N1…H3-C3 hydrogen bond (HF: 2.572 Å; DFT: 2.366 Å) in A-F.
The small interaction energy between A and F is also reflected in the electron distribution at the atoms involved in hydrogen bonding and the corresponding charges (Table III). In the A-F pair the H61 charge increases by 0.02 and the decrease at N1 is 0.03, whereas the electron distribution at H2 is not changed at all relative to the uncomplexed A at the HF level. In the A-T pair the H61 charge increases by 0.06 and the acceptor atom N1 charge is reduced by 0.11. For H2 a small increase of 0.02 may be caused by a long distant interaction with the T oxygen atom. The Coulomb interaction energy between the hydrogen atoms and the corresponding oxygen acceptor atoms O2 and O4 in A-T should exceed the ones between hydrogen and fluorine in A-F pairs as the charges at the T oxygen atoms are larger than the charges of the corresponding F fluorine atoms F2 and F4. The A-T and A-F dipole moments of 2.0 and 1.6 D are not very different.
The NMR shift calculations we have performed are of a very approximate nature because correlation and solvent effects have not been taken into account. The calculated shifts of H61 (A-F: 1.2 ppm, A-T: 3.0 ppm) and N1 (A-F: -3.0, A-T: -12.5 ppm) relative to the isolated A confirm the fact the interaction between A and F is smaller than between A and T. The results are, however, not in line with the experimental finding that F does not affect the chemical shift of H61 in adenine at all.
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The final conclusion of our study is that the A-F base pair has a substantially smaller interaction energy than A-T. Their overall geometry is similar but A-F exhibits a base pair opening. In spite of the smaller interaction energy the computed H61...F4 and N1...H3 contacts are below the sum of the corresponding van-der-Waals radii. This means that the interaction between A and F cannot be understood by van-der-Waals forces alone.
The experimental results by Kool and coworkers (1,2) have led to the suggestion that not hydrogen bonding but shape complementarity is important for DNA replication. Our results clearly show that the overall geometries of the A-T and A-F base pairs are similar and insofar the DNA should indeed not be distorted seriously at the A-F site. If we assume that a van der Waals interaction is characterized by distances between the base pairs corresponding to the sum of van der Waals radii, then the calculated base pair geometries indicate that the interaction cannot be accounted for by a van der Waals interaction alone. The interaction energy is much smaller than for any other Watson-Crick or non-canonical base pair with standard bases but again larger than expected for a van der Waals interaction alone. When discussing interaction energies one should realize, however, that the relevant quantity is the free energy of interaction, which is not available so far. In summary, we want to point out that both geometric and energetic considerations have to be taken into account for an understanding of base pair interactions.
A challenge for future studies with non-standard base pairs is to define in quantitative terms the extent of geometrical distortion and the range of base pair interaction energies the replication machinery is able to cope with. A further interesting question is whether or not
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DNA replication is also unaffected if more than one subsequent A-T pairs are replaced by AF.
Acknowledgements
We thank the Thüringer Ministerium für Wissenschaft, Forschung und Kultur for support. A portion of this work was supported by a European Union Access to Large Scales Facilities grant (ERBCHGECT940062) to EMBL. Finally, we are grateful to H.U. Reißig, Institut für Organische Chemie, Technische Universität Dresden, for directing our attention to this problem.
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S. Moran, R. X.-F. Ren, S. Rumney and E. T. Kool, J. Am. Chem. Soc. 119, 2056 (1997).
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M. W. Schmidt, K. K. Baldridge, J. A. Boatz, T. S. Elbert, M. S. Gordon, J. H. Jensen, S. Koseki, S., N. Matsunaga, K. A. Nguyen, S. Su, T. L. Windus, M. Dupuis, and J. A. Montgomery, J. Comp. Chem. 14, 1347 (1993). 10
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M. J. Frisch, G. W. Trucks, H. B. Schlegel, P. M. W. Gill, B. G. Johnson, M. A. Robb, J. R. Cheeseman, T. A. Keith, G. A. Petersson, J. A. Montgomery, K. Raghavachari, M. A. Al-Laham, V. G. Zakrzewski, J. V. Ortiz, J. B. Foresman, J. Cioslowski, B. B. Stefanov, A. Nanayakkara, M. Challacombe, C. Y. Peng, P. Y. Ayala, W. Chen, M. W. Wong, J. L. Andres, E. S. Replogle, R. Gomperts, R. L. Martin, D. J. Fox, J. S. Binkley, D. J. Defrees, J. Baker, J. P. Stewart, M. Head-Gordon, C. Gonzalez and J. A. Pople GAUSSIAN 94, Revision E. 1, Gaussian, Inc., Pittsburgh PA (1995).
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K. Lindauer, C. Bendic and J. Sühnel, Comput. Appl. Biosci. 12, 281 (1996).
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M. J. Frisch, G. W. Trucks and J. R. Cheeseman, in: Theoretical and Computational Chemistry, Vol. 4, J. M. Seminario, Ed., Elsevier, p. 679 (1996).
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P. J. Kraulis, J. Appl. Cryst. 24, 946 (1991).
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Table I. Energies of the A-T, A-F and reverse A-F base pairs. A-T
A-F
A-Freverse
EHF
HF/6-31G(dp)
-10.2
-2.7
-2.6
EMP2
MP2/6-31G(dp)//HF/6-31G(dp)
-12.3
-3.9
-3.9
EDFT
B3LYP/6-31G(dp)
-14.0
-3.3
-3.4
EDEF
HF/6-31G(dp)
0.6
0.1
0.1
-11.7
-3.8
-3.8
1.4
0.8
0.8
-10.3
-3.0
-3.0
ET=EMP2+EDEF ∆EZPE
HF/6-31G(dp)
E0 = ET+ ∆EZPE
1) EHF, EMP2, EDFT - interaction energies at different levels of approximation; EDEF - ab initio deformation energy upon complex formation; ∆ZPE change in zero point energy (scaling factor 0.9); ET- total stabilization energy, Eo - interaction energy taking into account zero point energy.
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Table II.
Structural parameters of the A-T and A-F base pairs
base pair
A-T
A-F
HF1)
DFT2)
VDW3) EXP4)
HF1)
DFT2)
VDW3)
r(H61…O4/F4)/Å
2.091
1.919
2.60
2.253
2.089
2.55
r(N1…H3)/Å
1.976
1.796
2.74
2.572
2.366
2.74
r(H2…O2/F2)/Å
2.961
2.801
2.60
4.197
3.981
2.55
r(N6 O4)/Å
3.085
2.937
-
2.98
3.627
3.440
-
r(N1 N3)/Å
2.989
2.845
-
2.82
3.246
3.098
-
r(C2 O2) /Å
3.774
3.629
-
3.52
4.911
4.580
-
∠(N6 H61 O4/F4)/°
172.7
174.5
-
178.5
177.0
-
∠(H61 O4/F4 C4)/°
126.4
124.5
-
129.7
126.6
-
∠(N1 H3 N3/C3)/°
178.7
179.9
-
166.8
169.0
-
1) Hartree-Fock (HF/6-31G(dp)) 2) Density functional theory (B3LYP/6-31G(dp)) 3) Sum of van der Waals radii adopting values of 1.2, 1.35, 1.4, 1.54 Å for H, F, O, N. 4) Mean values of distances derived from a high resolution set of 21 DNA structures.
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Table III. Selected atomic charges of monomers and base pairs from a Mulliken population analysis (HF/6-31G(dp))
A
F
T
A-F
A-T
H61
0.32
0.34
0.38
N1
-0.67
-0.70
-0.78
H2
0.16
0.16
0.18
0.24
0.43
H3
0.19
0.35
F2
-0.39
-0.39
F4
-0.39
-0.40
O2
-0.60
-0.61
O4
-0.58
-0.64
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Figure captions
Fig. 1 Structure formulas of the A-F and reverse A-F base pairs. Fig. 2 Superposition of the A-T and A-F base pairs (A-T: bright; A-F: dark). a. Superposition of the complete complexes. b. Superposition of A alone. This figure has been drawn with MOLSCRIPT (17).
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H N6 H61
N
N
N1
F
Me
H3
N H2
F
A−F
H N6 H61
N
N
N1
F
H3
N H2
F
Me
A − F reverse
A
F/T
