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Origins of Conformational Differences between Cis and Trans DNA Adducts Derived from Enantiomeric anti-Benzo[a]Pyrene Diol Epoxides Xiao-Ming Xie, Nicholas E. Geacintov,* and Suse Broyde* Chemistry and Biology Departments, New York University, New York, New York 10003 Received February 5, 1999
The two enantiomeric metabolites of the carcinogen precursor benzo[a]pyrene, (+)- and (-)anti-BPDE [(7R,8S)-dihydroxy-(9S,10R)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene and the corresponding 7S,8R,9R,10S enantiomer, respectively], bind predominantly to the exocyclic amino groups of dG residues in double-stranded DNA by either cis or trans addition to yield four stereoisomerically distinct [BP]-N2-dG adducts. Both the 10S (+)-trans and 10R (-)-trans adducts assume minor groove conformations in normal, full duplexes, but with opposite 5′ or 3′ orientations, respectively, relative to the modified strand. In contrast, the 10R (+)-cis and 10S (-)-cis adducts assume oppositely oriented base-displaced intercalative conformations in normal duplexes, with the inserted pyrenyl residues pointing toward the major groove in the (+)-cis isomer and toward the minor groove in the (-)-cis isomer. A BPDE-modified nucleoside is a small system which can be studied by computational methods with a very thorough survey of the potential energy surface. To investigate conformational differences between cis and trans adducts, and to elucidate origins governing the opposite orientations of these (+)- and (-)-diol epoxide adducts, we have carried out extensive investigations of the (+)- and (-)-trans-antiand (+)- and (-)-cis-anti-[BP]-N2-dG deoxynucleoside adduct pairs. We report results for the (+)- and (-)-cis-anti pair, and compare them with the (+)- and (-)-trans-anti adducts. We created 373 248 different conformers for each adduct, which uniformly sampled at 5° intervals the possible rotamers about three flexible torsion angles governing base (χ) and carcinogen (R′ and β′) orientations, and computed each of their energies. The potential energy surface of the molecule was then mapped from these results. While four potential energy wells or structural domains are found for the (+)-trans adduct and four for the (-)-trans adduct, only two of these four domains are favored for each of the two cis adducts. In both cis and trans adducts, the (+)/(-) pairs of each structural domain are nearly mirror images. The most favored of the domains in both cis and trans adducts is observed experimentally in the duplexes containing each of these [BP]-N2-dG lesions. The opposite orientations in both cis and trans adducts stem from steric crowding at the benzylic ring, engendered when a (+) stereoisomer is rotated into the analogous conformation of its (-) partner, and vice versa. Furthermore, the key role of the difference in absolute configuration between trans and cis adducts at the hydroxyls of C9 and C8 in governing conformational preferences and flexibility is delineated. Cis adducts are less conformationally flexible than trans adducts because they are inherently more sterically crowded, with C9-OH and C8-OH on the same side of the benzylic ring as guanine and sugar, while they are on the opposite side of the benzylic ring in the trans adducts. Consequently, the cis adducts inherently favor less the minor groove position adopted by trans adducts in DNA duplexes because the C9-OH and C8-OH are directed inward into the minor groove in the cis adducts. In the trans adducts, the C9-OH and C8-OH are directed outward, away from the interior of the minor groove. Observed differential processing of these four adducts by replication, repair, and transcription enzymes may well stem from their differing conformational preferences.
Introduction The stereochemical properties of ligands that form noncovalent complexes or covalent adducts with DNA have profound effects on their biological and biochemical functions. The absolute configurations of different substituents about chiral carbon atoms can lead to strikingly different conformations when these stereoisomeric compounds bind to DNA. A remarkable example of such * To whom correspondence should be addressed. N.E.G.: phone, (212) 998-8407; fax, (212) 998-8421; e-mail,
[email protected]. S.B.: phone, (212) 998-8231; fax, (212) 995-4015; e-mail,
[email protected].
stereochemical effects is seen in the pronounced differences in the four stereoisomeric adducts that are formed when the two enantiomeric metabolites of the carcinogen precursor benzo[a]pyrene, (+)- and (-)-anti-BPDE,1 bind covalently to deoxyguanosine residues in DNA. Both BPDE enantiomers bind predominantly to the exocyclic amino groups of dG residues in double-stranded DNA (1, 2) via either cis or trans addition to yield the four stereoisomerically distinct BP-N2-dG adducts shown 1 Abbreviations: (+)- and (-)-anti-BPDE, (7R,8S)-dihydroxy-(9S,10R)epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene and the corresponding 7S,8R, 9R,10S enantiomer, respectively.
10.1021/tx990021a CCC: $18.00 © 1999 American Chemical Society Published on Web 07/01/1999
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Figure 1. (A) Structures of (+)- and (-)-cis-anti- and (+)- and (-)-trans-anti-[BP]-N2-dG nucleoside adducts. Torsion angles χ, R′, and β′ are defined as follows: χ, O4′-C1′-N9-C4; R′, N1-C2-N2-C10(BP); and β′, C2-N2-C10(BP)-C9(BP). (B) NMR solution structures of (+)- and (-)-cis-anti (9,10) and (+)- and (-)-trans-anti[BP]-N2-dG adducts (4,5), in stereoview. The central 5-mer of the 11-mer is shown.
in Figure 1, which manifest differential biological properties (3). Both the 10S (+)-trans and 10R (-)-trans adducts assume minor groove conformations in normal, full duplexes, but with opposite orientations relative to the
modified dG strand; in the (+)-trans adducts, the hydrophobic BP pyrenyl ring system points toward the 5′-end, while in the (-)-trans adduct, it points in the opposite, 3′-direction (4-6). Furthermore, the opposite orientation phenomenon is manifested quite differently in a “dele-
Cis and Trans anti-[BP]-N2-dG Structure Difference
tion” duplex of this adduct, in which there are 11 residues on the modified strand and only 10 on the partner, with the BP-modified dG containing no partner residue (as if the replication machinery had skipped the BP-damaged residue). In this case, the BP residue is intercalated into the helix with displacement of the modified guanine in both (+) and (-) adducts, but in the (+) case, this guanine and its attached BP benzylic ring are positioned on the major groove side of the double helix (7); in the (-) adduct, the displaced guanine and the attached benzylic ring are situated on the opposite, minor groove side of the helix (8). In contrast, the 10R (+)-cis and 10S (-)-cis adducts assume base-displaced intercalative adduct conformations in both normal and deletion duplexes; the modified dG residues and the dC partner bases are displaced out of the double helix interior, and the pyrenyl residues occupy the hydrophobic intercalative binding sites normally occupied by the dG‚dC base pairs. In the (+)-cis and (-)-cis adducts, the pyrenyl residues also point oppositely, toward the major and minor grooves of DNA, respectively (9, 10). Thus, these cis adduct structures in normal duplexes are very similar to the “deletion” duplex structures of the trans adducts with the same absolute configuration at C10, 10S (+)-trans/(-)-cis and 10R (-)trans/(+)-cis. The “deletion” duplex structure for the (+)cis adduct is essentially the same as the normal duplex structure (11), but no “deletion” duplex structure is yet available for the (-)-cis adduct. A key question is why the cis and trans adducts adopt such different conformations in full duplexes in the same sequence context (4, 5, 9, 10), and why trans adducts are more conformationally heterogeneous, manifesting both minor groove and base-displaced intercalation structures, while the observed cis adduct structures are all of the base-displaced intercalation type. Since the absolute configuration at C10 of 10S (+)-trans/(-)-cis and 10R (-)trans/(+)-cis adduct pairs is the same (Figure 1), why are the structural effects associated with the inverted positions of the OH groups at C9 and C8 in these (+) and (-) stereoisomers so profound? Another important issue concerns the origin of the opposite orientation phenomenon. Computational studies first suggested that the BP moieties would assume opposite orientations along the DNA in (+)- and (-)trans-anti-BP-N2-dG adducts (12). Since then, opposite orientations of adducts of (+)/(-) pairs of diol epoxide derivatives of aromatic hydrocarbons have been observed in a number of stereoisomeric adduct pairs, as reviewed in Geacintov et al. (3) and Jerina et al. (13) (4-11, 1422). The opposite orientations are observed regardless of the specific conformation adopted by the aromatic moiety in relation to the DNA (in the major groove, in the minor groove, base-displaced intercalated, or quasi-classically intercalated), of the specific base modified (guanine or adenine), and of the structure of the parent aromatic hydrocarbon; thus, the phenomenon appears to be general. Preliminary modeling efforts (3) suggested that primary steric hindrance between the polynuclear aromatic residue and the covalently linked base and sugar moieties manifested at the level of the modified nucleoside could be responsible for the opposite orientations. A BPDE-modified nucleoside is a small system which can be studied by computational methods with a very thorough survey of the potential energy surface. To investigate conformational differences between cis and
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trans adducts, and to elucidate origins governing the opposite orientations of these (+) and (-) diol epoxide adducts, we have carried out extensive investigations of the (+)- and (-)-trans-anti- and (+)- and (-)-cis-anti[BP]-N2-dG deoxynucleoside adduct pairs (Figure 1). In this work, we report results for the (+)- and (-)-cis-anti pair, and compare them with the (+)- and (-)-trans-anti adducts (23). We created 373 248 different conformers for each adduct, which uniformly sampled the possible rotamers about three flexible torsion angles χ, R′, and β′ (Figure 1) at 5° intervals, and computed each of their energies. The potential energy surface of the molecule with a B-DNA C2′-endo sugar conformation and B-DNA orientation of the C4′-C5′ bond was then mapped from these results. While four potential energy wells or structural domains are found for the (+)-trans adduct and four for the (-)-trans adduct, only two of these four domains are favored for each of the two cis adducts. In each of these two domains, the structure of the 10R (+)-cis adduct is like that of the 10R (-)-trans adduct, and the structure of the 10S (-)-cis adduct is like that of the 10S (+)-trans adduct. In both cis and trans adducts, the (+)/(-) pairs of each structural domain are nearly mirror images, with the mirror image symmetry broken by the sugar and its attached C4′-C5′ group. The most favored of the domains in both cis and trans adducts is observed experimentally in the duplexes containing each of these [BP]-N2-dG lesions (4, 5, 7-11). Our results reveal that the opposite orientations in both cis and trans adducts stem from steric crowding at the benzylic ring, engendered when a (+) stereoisomer is rotated into the analogous conformation of its (-) partner, and vice versa. Limited conformational flexibility in the torsion angle β′ about the BP(C10)-N2(dG) covalent bond closest to the bulky BP moiety at the linkage site to guanine plays an important part in governing the energetically most favored orientations. Furthermore, the key role of the difference in absolute configuration between trans and cis adducts at the hydroxyls of C9 and C8 in governing conformational preferences and flexibility is delineated. Cis adducts are less conformationally flexible than trans adducts because they are inherently more sterically crowded, with C9OH and C8-OH on the same side of the benzylic ring as guanine and sugar. In contrast, C9-OH and C8-OH are on the opposite side of the benzylic ring, more distant from guanine and sugar in the trans adducts. Consequently, it is shown here that the cis adducts inherently favor less the minor groove position adopted by trans adducts in DNA duplexes because the C9-OH and C8OH are directed inward into the minor groove in the cis case. In the trans adduct case, the C9-OH and C8-OH are directed outward, away from the interior of the minor groove.
Methods Creating Starting Conformations. Coordinates of the high-resolution NMR solution structures of the (+)- and (-)cis-anti-[BP]-N2-dG adducts in a DNA duplex 11-mer (9, 10) were employed to create the modified nucleoside adducts by excising the [BP]-N2-dG adduct, including the sugar with O3′ and O5′, using the program INSIGHTII from MSI, Inc., which was also used to draw the three-dimensional structures. Hydrogens were added to O3′ and O5′ to produce modified
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nucleosides. Benzylic ring conformations were kept in the H7, H8, H10 pseudoequatorial, H9 pseudoaxial orientations of the NMR solution structures, with small differences between the (+)- and (-)-cis case retained. Starting structures for the energy calculations were then created with a torsion driver program which rotated torsion angles χ, R′, and β′ (Figure 1) to selected values and then computed the coordinates of the resulting structures. The starting structures uniformly surveyed the potential energy surface of the molecule with conformers in which χ, R′, and β′ sampled their 360° conformation space at 5° intervals, in combination, giving a total of 373 248 [(360/5 ) 72)3] conformers for each adduct. The C4′-C5′ bond and the sugar pucker were kept in the conformation they had adopted in the B-DNA duplex from which the nucleosides had been excised. Values of the sugar pseudorotation parameter P (24) are 191° and 173°, in the normal C2′-endo domain of B-DNA, for the (+) and (-) adducts, respectively. The C3′-C4′-C5′-O5′ torsion angles are normal B-DNA values of 35° and 55°, respectively, in the (+) and (-) adducts. Energy Computation. Energies of each of the 373 248 structures for each adduct were computed with the molecular mechanics program AMBER 4.0 (25). Since there are no negatively charged phosphates in the nucleoside, Na+ counterions were not needed. A sigmoidal distance-dependent dielectric function (26) which has been demonstrated to be a suitable treatment for the dielectric constant (27) was employed in the Coulombic term of the force field, to model the dielectric effects of solvent water. Parameters added to the AMBER 4.0 force field for the [BP]-N2-dG adducts are the same as those used in earlier work (12), except that new partial charges were computed with Gaussian 92 at the STO-3G basis set level which is compatible with the rest of the AMBER 4.0 force field. Partial charges, atom types, and topology information are given in Table S1 of the Supporting Information. Computation of Statistical Weights, Thermodynamic Quantities, and Energy Maps. The statistical weight, Pi, of each conformer was computed from the relationship
Pi )
e-∆Ei/RT N
∑e
-∆Ei/RT
i)1
where ∆Ei is the relative energy of a given conformer in kilocalories per mole with respect to the lowest-energy structure, R is the universal gas constant (1.987 × 10-3 kcal/mol-deg), T ) 300 K, and N is 373 248, the total number of conformers for each adduct. The energies were used to construct both three-dimensional energy surfaces as a function of the χ, R′, and β′ torsion angles and two-dimensional energy contour slices through them; slices were made at selected χ values as a function of R′ and β′. The program TECPLOT was used for this purpose. These plots revealed four low-energy wells or domains for the trans adducts, two of which are also low-energy for the cis adducts. The other two are present as higher-energy wells for the cis adducts (see Figure 3 and Figures S1 and S2 of the Supporting Information). This allowed for the grouping of the full data set into four regions, each of which encompassed one of these four low-energy domains: region 1, χ ) 50 ( 50°, R′ ) 0-100° and 285-355°, β′ for (+) adduct ) 90 ( 90°, and β′ for (-) adduct ) 270 ( 90°; region 2, χ ) 230 ( 130°, R′ ) 0-100° and 285-355°, β′ for (+) adduct ) 90 ( 90°, and β′ for (-) adduct ) 270 ( 90°; region 3, χ ) 0 ( 180°, R′ ) 180 ( 90°, β′ for (+) adduct ) 90 ( 90°, and β′ for (-) adduct ) 270 ( 90°; and region 4, χ ) 0 ( 180°, R′ ) 0 ( 180°, β′ for (+) adduct ) 270 ( 90°, and β′ for (-) adduct ) 90 ( 90°. On the basis of this grouping, we computed the combined fractional statistical weight, Wj, for each region (containing one low-energy well) by summing the individual statistical weights,
Figure 2. Three-dimensional χ, R′, and β′ energy topographies to 5 kcal/mol. II and III denote the two low-energy domains. Torsion angles cycle over 360°, so 359° is actually contiguous with 0°. (A) (+)-cis-anti-[BP]-N2-dG adduct and (B) (-)-cis-anti[BP]-N2-dG adduct. Pi, of each point in the region: nj
Wj )
∑P
i
i)1
where nj is the number of conformers in each one of the four regions (j ) 1, 2, 3, or 4). In practice, only conformers with energies ∆Ei of
