WDS'06 Proceedings of Contributed Papers, Part III, 64 69, 2006. ISBN 80-86732-86-X MATFYZPRESS Quantum Chemistry Study of NMR Parameters of cis Watson-Crick/Sugar Edge RNA Base Pair Family Z. Vokáčová Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo square 2, 166 10 Prague 6, Czech Republic Abstract. Ribonucleic acid (RNA) non-watson-crick base pairs show an astonishing variability of base-pairing. Quantum chemistry study of nuclear magnetic resonance (NMR) parameters of eight members of the cis Watson-Crick/Sugar edge family of binding patterns in RNA molecules was provided by mean of calculation of indirect spin-spin constants with the CP-DFT method. RNA bases interact via hydrogen bond and correspondence of local geometry parameters with the NMR parameters of three different hydrogen-bonding patterns in one sugar-base and two base-base contacts. Each H-bond pattern has its own set of representative J-coupling which can be used for the identification of particular RNA bonding pattern via NMR spectroscopy. Introduction DNA and RNA molecules play essential role in many biological processes. While standard type of base pairing, the Watson-Crick (WC) base pairing, is dominant in the DNAs, RNA molecules exhibit a large variability of base-pairing patterns. WC base pair interaction in RNAs represent only 50%, in addition many of the RNA base-pairing families have no counterparts in DNA. A role of the non-wc base pairs in RNA is fundamental since they participate in folding and stabilization of RNA tertiary structure. Detection of binding motives in rather complicated and highly variable RNA macromolecules can help in better understanding of chemical processes of nucleic acids. Each nucleobase possesses three edges shown in Figure 1: WC edge, Hoogsteen edge (for purines) or CH edge (for pyrimidines), and Sugar edge (SE). A given edge of one nucleobase can in principle interact with one of the three edges of a second nucleobase. This interaction can be either cis or trans with respect to sugar moiety. According to Leontis and Westhof (Leontis et al., 2001), all possible combinations lead to twelve families of distinct geometry patterns. Leontis et al. also formulated an isostericity principle, which tells that any base of pair can be replaced by another one with similar (isosteric) shape without loss of RNA folding and function. Present study deals with the analysis of NMR spectroscopy parameters in the cis-wc/se class of RNA base pairs. There are in total sixteen possible members of this family. Thirteen of them have already been found in RNA molecules. This fact makes the cis-wc/se base pair family one of the most biologically important family for RNA base pairing. The cis-wc/se family consists of four isosteric subfamilies. Two of them are presented here subfamily with Adenosine in the WC position, and subfamily with Cytosine in the WC position. All members of two subfamilies have been already found by X-ray spectroscopy in crystal structures of RNAs. Figure 1. Classification of the interaction edges in purine and pyrimidine nucleobases (Leontis et al., 2001). 64
Base pairs interact via hydrogen bonds (H-bond). H-bonds play important role in stabilization the threedimensional structure of nucleic acids. The H-bond X-H Y is a bond between the hydrogen atom H which is covalently bonded to the strongly electronegative atom X and the atom Y which possesses lone pair of electrons. The length of the H-bond is distance between atoms H and Y, and angle of the H-bond is angle X-H-Y. In this study we correlate the mentioned geometry parameters with the calculated values of indirect spin-spin couplings n (J-couplings). According to the IUPAC recommendations, J should be used for the nuclear spin-spin coupling 2 constants through n bonds. Parentheses may be used to indicate the species of nucleic coupled, e.g. J(A,B) is nh the two-bond interaction between nuclear spins of atoms A and B. J is coupling across the H-bond, where 2h index h identifies H-bond between coupled nuclei. In DNA were already measured the J-couplings J(N,N) and 1h J(N,H) which were used the detection of the H-bond N-H N. We calculated also other intermolecular J- couplings ( 1h 3h 2h 2h J(N,H), J(C,N), J(N,N), J(C,H) ) and one intramolecular coupling 1 J(H,N). We will show that these J-couplings be also used for detection of H-bonding patterns in RNAs. Our molecular models X.rY shown in Figure 2 consists of two bases, the base X interact via its WC edge and the base Y (after a dot, with sugar r - ribose) interact via its SE. X and Y can be in principle any purine or pyrimidine nitrogenous basis Adenine (A), Cytosine (C), Guanine (G) or Uracil (U). In this work we selected complexes with Adenine or Cytosine (A.rY or C.rY) in WC position. Monomer units are linked by two H-bonds. One H-bond links sugar and base while the second H-bond links the bases (see in Figure 2). We calculated NMR parameters of these complexes A.rY and C.rY because they exhibit only one of the H-bonding patterns shown in Figure3. The selected subfamilies A.rY and C.rY differ only in C1 -N1/N9 distance. Distance difference is about 1.6 Å. (Table 5 in Leontis et al., 2002). Calculation Method Molecular geometry of all complexes was optimized previously (Šponer et al., 2005). Geometry optimization was carried out at the DFT level of theory with the B3LYP functional and 6-31G** basis set. A.rA A.rC A.rG A.rU C.rA C.rC C.rG C.rU Figure 2. Patterns of RNA base pairs investigated in this work. 65
Figure 3. H-bond patterns and numbering of atoms used in the text. A. Base-base pattern BN-B (via nitrogen N) and sugar-base S-B pattern, B. Base-base pattern BO-B (via oxygen O) and S-B pattern. Dashed lines indicate H-bonding contacts. Indirect NMR spin-spin coupling constants (J-couplings) were calculated using the method of the coupledperturbed density-functional theory (CP-DFT) with B3LYP functional and with the atomic basis called IgloIII, by including the diamagnetic spin orbit (DSO), paramagnetic spin orbit (PSO), Fermi-contact (FC) and spindipolar (SD) terms (Ramsey, 1953; Helgaker et al., 2002). All calculations were done with the Gaussian03 program package. Results and Discussion Sugar-Base H-bond The sugar-to-base contacts occur between hydroxyl group at carbon C2 of sugar and nitrogen N3 of Cytosine of C.rY complex or nitrogen N1 of Adenine of A.rY complex. We calculated two different J-couplings between the atoms of the motif C-O-H N (see Figure 3) - 3h J(C,N) and 1h J(H,N), the values are given in Table 1. Absolute value of the three-bond coupling 3h J(C,N) is smaller or equal to 0.1 Hz in all investigated complexes. All values of the 3h J(C,N) coupling are too small to make any conclusion about its dependence on the structure and thus can be hardly used for structural interpretation of C-O-H N H-bond interaction. Table 1. Geometry parameters of S-B motif in the WC/SE RNA base pairs and calculated J-couplings in Hz (d is distance in Å between atom H and atom N in C-O-H N, and a is angle O-H-N in degree, a COH is angle C-O- H in degree, t COH N is torsion C-O-H N in degree). RNA complex d a a COH t COH N 1h J(H,N ) 3h J(C,N) A.rA 1.78 169.9 109.5 132.8-0.10-1.77 A.rC 1.79 162.7 108.6 103.4-0.10-1.66 A.rG 1.81 167.2 110.7 173.4-0.07-2.05 A.rU 1.78 166.5 108.7 97.4-0.08-1.66 C.rA 1.76 178.5 108.6 141.9-0.06-1.65 C.rC 1.79 169.6 107.3 52.6 0.05-1.67 C.rG 1.82 166.9 110.2 215.8-0.07-1.92 C.rU 1.76 171.7 107.5 39.4 0.04-1.65 Values of the one-bond coupling 1h J(H,N) shown in Table 1 range from -2.0 Hz to -1.6 Hz should correlate with the length of the H-bond and angle C-O-H (Alkorta et al., 2003). This correlation was however not detected in this study since the variation of local geometry parameters is too small. 66
Absolute value of the 1h J(H,N) coupling increases little with the increase of torsion angle C-O-H N over whole wide torsion angle distribution that ranges from 40 to 220. Although both complexes A.rX and C.rX have similar dependence on torsion angle C-O-H N, they differ a little in inclination of dependence. The curve is steeper for A.rX complexes dependence what is the most evident in region about 200. Variation of the calculated 1h J(H,N) coupling is small, only about 0.5 Hz and more accurately information about the dependence of the coupling on the local geometry parameters it is necessity to use more complexes for calculation with more different structure. Base-Base H-bond: BN-B pattern The binding motif which involves the amino group of the base interacting via the WC edge and the nitrogen N3 of the purine base interacting via SE edge is shown in Figure 3A. For the H-bond pattern N H-N we can consider three different J-couplings, 2h J(N,N), 1h J(N,H) and 1 J(H,N). The coupling 1 J(H,N) will be discussed in separate paragraph because it is present also in the BO-B base-base motif discussed in the following paragraph. Calculated values of the J-couplings and geometry parameters are shown in Table 2. Calculated value of the 2h J(N,N) coupling ranges from 2.3 Hz to 2.6 Hz. Although the variation of the coupling is relatively small we can speculate about its dependence on local geometry since the smaller values were obtained for the larger distance while opposite holds for the larger values of the coupling. The same trend, decrease of the 2h J(N,N) coupling with the increase of the H-bond length, was also reported in the literature (Alkorta et al., 2003). The 2h J(N,N) coupling measured across the N H-N link in WC DNA base pairs ranges from 5.0 Hz to 8.0 Hz (Pervushin et al., 1998, Wohnert et al., 1999) but in this case the interaction involves the imino group. The measurement of the 2h J(N,N) coupling involving the amino group performed for the reverse Hoogsteen base pairs (Wohnert et al., 1999, Hennig, 2000) gives smaller value of the coupling, about the 5.5 Hz. It can possible to distinguish amino or imino group according to the 2h J(N,N) coupling. The value of 2.5 Hz was measured for the 2h J(N,N) coupling in the mismatched A-A base pair of DNA molecule (Majumdar et al., 1999). The bonding pattern N H-N in A-A mismatched base pair corresponds fully to the WC/SE BN-B pattern and the experimental couplings measured by Majumdar nicely agree with our calculation. The 2h J(N,N) coupling of 2.5 Hz was also measured by group of Dingley (Dingley et al., 2000) in DNA and since the structure of this DNA molecule is currently not available the measured value of the coupling most probably indicates similar binding pattern as the WC/SE BN-B one that involves the amino group. The 1h J(N,H) coupling calculated in WC/SE BN-B pattern ranges from -1.7 Hz to -2.0 Hz and similarly as for the 2h J(N,N) coupling its larger magnitude was calculated for smaller length of the N H-N H-bond indicating possible increase of the coupling for the closer contacts of the bases in WC/SE base pair. Opposite sign of calculated 1h J(N,H) coupling than experimental J-coupling sign can be explained that sign information of experimental J-coupling wasn t performed because determination of J-coupling sign can be difficult at some time. Magnitude of the 1h J(N,H) coupling measured in DNA canonical WC base pairs ranges from 2.0 Hz to 3.6 Hz (Pervushin et al., 1998, Wohnert et al., 1999) what agrees with our calculation. Possible explanation for somewhat smaller values of the 1h J(N,H) coupling calculated in WC/SE base pairs compared to the experiment in WC base pairs is probably due to the relatively large twist of base pairs in WC/SE RNA which is known to disfavour the intermolecular spin-spin interaction (see the values of p-p torsion angle in Table 2). Table 2. Geometry parameters of the BN-B motif in WC/SE RNA base pairs and calculated J-couplings in Hz (d is distance in Å between atom N and atom H in N H-N, a is angle N-H-N in degree, d NH is length in Å of covalent bond between N and H atoms of amino group, p-p is twist of bases X and Y in X.rY torsion angle in degree of atoms C4(pur)-N3(pur) in base Y and N4(pyr)-N3(pyr) or N6(pur)-N1(pur) in the base X). RNA complex d a d NH p-p 2h J(N,H) 1h J(N,N) 1 J(H,N) A.rA 2.03 159.3 1.02 300.4 2.53-1.99 66.57 A.rG 2.12 149.9 1.02 291.8 2.38-1.75 66.45 C.rA 2.05 154.6 1.02 314.5 2.46-1.85 67.73 C.rG 2.13 149.0 1.02 288.9 2.35-1.71 65.52 Base-Base H-bond: BO-B pattern This X.rY base-base interaction shown in Figure 3B links pyrimidine base Y with C=O termini the other base via C=O H-N H-bond. While Y base interact via SE edge the X base interacts via WC edge involving H6- N6 or H4-N4 for X base corresponding to Adenine or Cytosine, respectively. 67
Magnitude of both calculated 2h J(C,H) and 3h J(C,N) couplings across the H-bond shown in Table 3 are smaller then 1 Hz. Although both calculated couplings vary only a little somewhat larger values, maximally by 0.3 Hz, were obtained for the geometries with shorter distance, by 0.15 Å, between oxygen and hydrogen of the C=O H-N link. This finding indicates similar dependence of both couplings on length of H-bond as was discussed for 2h J(N,N) and 2h J(N,H) couplings in the previous paragraph. Magnitude of both calculated couplings of this H-bond pattern fit nicely the interval 0.1-0.9 Hz measured in Guanosine quartets (Dingley et al., 2000) and in human ubiquitin (Cordier et al., 1999). Table 3. Geometry parameters of BO-B motif in WC/SE RNA base pairs and calculated J-couplings in Hz (d is distance in Å between atom O and atom H in C=O H-N, a is angle O-H-N in degree, d NH is length in Å of covalent bond between N and H, t CO HN is torsion C=O H-N in degree, p-p is twist of bases X and Y in X.rY - torsion angle in degree of atoms N1(pyr)-C2(pyr) in base Y and N4(pyr)-N3(pyr) or N6(pur)-N1(pur) in base X). RNA d a d complex NH p-p t CO HN d 2h CH J(C,H) 3h J(C,N) 1 J(H,N) A.rC 1.81 177.2 1.02 354.8 83.3 2.88-0.79 0.78 69.50 A.rU 1.92 167.5 1.02 337.8 279.3 3.09-0.64 0.72 68.56 C.rC 1.82 169.6 1.02 350.2 290.6 2.91-0.73 0.83 68.90 C.rU 1.95 159.7 1.02 344.7 268.3 3.08-0.53 0.61 68.92 One-bond coupling 1 J(H,N) of BN-B and BO-N patterns The one-bond 1 J(H,N) coupling was calculated between covalently bound nitrogen and hydrogen of the amino group of the purine or pyrimidine base of both base to base contacts shown in Figure 3A or 3B, respectively. Two different H-bond patterns are significant for the investigated complexes, the N H-N motif and the C=O H-N motif occurring for the purine or pyrimidine base interacting via SE edge, respectively. Calculated value of the 1 J(H,N) coupling shown in Table 2 and Table 3 ranges from 65 Hz to 70 Hz. Since the inter-atomic distance between nitrogen and hydrogen of the N-H bond is 1.02 Å in all base pairs the variation of the calculated coupling should be correlated with the length of the H-bond. The H-bond length ranges from 1.81 to 1.95 Å in the N H-N motif while for the motif C=O H-N it was from 2.03 to 2.13 Å what corresponds to the larger (68.5 69.5 Hz) respective smaller (65.5 67.7 Hz) value of the coupling as shown in Figure 4. It is therefore possible to group the calculated couplings into two groups according to the base type, purine or pyrimidine, involved in pairing via SE position. Our calculated data corresponds well to the range of 67.5-89.5 Hz measured in WC DNA base pairs (Alkorta, 2003). Figure 4. Dependence of 1 J(H,N) coupling on length of the H-bond (Distance R) calculated in two H-bond patterns - in BN-B pattern with N H-N binding motif and in BO-B pattern with C=O H-N binding motif. Conclusion We have carried out the calculation of NMR spin-spin coupling constants in eight members of the cis- WC/SE base pair family of RNA molecules. Geometry parameters of three different H-bonding patterns, two base to base and one base to sugar, were correlated with the values of six trans H-bond J-couplings and one intra-complex J-coupling. Each H-bond pattern has its own set of representative J-couplings which can be used 68
for the identification of local binding motif via NMR measurement. The magnitude of the calculated coupling constants is sensitive to the local geometry of the H-bond and the increase of coupling magnitude in many cases correlate with the decrease of H-bond length. This trend is the most obvious for the intramolecular 1 J(H,N) coupling. All calculated couplings shows good agreement with number of available experimental data. Set of specific J-couplings can be used for the identification of particular H-bond motif. Acknowledgments. This work was supported by the Grant Agency of the Czech Republic No.203/05/0388. References Alkorta, I. and Elguero J., Review on DFT and Ab Initio Calculations of Scalar Coupling Constants, Int. J. Mol. Sci., 4, 64-92, 2003 Cordier, F. and Grzesiek, S., Direct Observation of Hydrogen Bonds in Proteins by Interresidue 3h J NC Scalar Couplings, J. Am. Chem. Soc, 121, 1601-1602, 1999 Dingley, A.J., Masse, J.E., Feigon, J. and Grzesiek, S., Characterization of the Hydrogen Bond Network in Guanosine Quartets by Internucleotide 3h J NC and 2h J NN Scalar Couplings, J. Biomol. NMR, 16, 279-289, 2000 Helgaker, T. and Pecul, M., In calculation of NMR and EPR properties, edited by M. Kaupp, Wiley 2002 Hennig, M. and Williamson, J.R., Detection of N-H N Hydrogen Bonding in RNA via Scalar Coupling in the Absence of Observable Imino Proton Resonance, NAR, No 7, Vol. 28, 1585-1593, 2000 Leontis, N.B., Stombaugh, J., Westhof, E., The non-watson-crick Base Pairs and their Associated Isostericity Matrices, NAR, No.16, Vol.30, 3497-3531, 2002 Leontis, N.B. and Westhof, E., Geometric Nomenclature and Classification of RNA Base Pairs, RNA, 7, 499-512, 2001 Majumdar, A., Kettani, A. and Skripkin, E., Observation and Measurement of Internucleotide 2 J NN Coupling Constants between 15 N Nuclei with Widely Separated Chemical Shifts, J. Biomol. NMR, 14, 67-70, 1999 Pervushin, K., Ono, A., Fernandéz, C., Szyperski, T., Kainosho, M. and Wuthrich, K., NMR Scalar Coupling across Watson-Crick Base Pair Hydrogen Bonds in DNA Observed by Transverse Relaxation-optimized Spectroscopy, Proc. Natl. Sci. USA, 95, 14147-14151, 1998 Ramsey, N. F., Phys. Rev., 91,303, 1953 Šponer, J.E., Špačková, N., Kulhánek, P., Leszczynski, J., and Šponer, J., Non-Watson-Crick Base Pairing in RNA. Quantum Chemical Analysis of the cis Watson-Crick/Sugar Edge Base Pair Family, J. Phys. Chem. A, 109, 2292-2301, 2005 Wohnert, J., Dingley, A., Stoldt, M., Gorlach, M., Grzesiek, S. and Brown, L.R., Direct Identification of NH N Hydrogen Bonds in Non-canonical Base Pairs of RNA by NMR Spectroscopy, NAR, 27, No. 15,3104-3110, 1999 Yang, H., Jossinet, F., Leontis, N., Chen, L., Westbrook, J., Berman, H., Westhof, E., Tools for the Automatic Identification and Classification of RNA Base Pairs, NAR, No.13, Vol.31, 3450-3460, 2002 69