Multiple conformations of an intercalated (-)-(7S,8R,9S,10R)-N-6-[10-(7,8,9,10-tetrahydrobenzo[a]pyrenyl)]-2 '-deoxyadenosyl adduct in the N-ras codon 61 sequence

The structure of the (-)-(7S,8R,9S,10R)-N-6-[10-(7,8,9,10-tetrahydrobenzo[a ]pyrenyl)]-2'-deoxyadenosyl adduct at A(7) of 5'-d(CGGA<(CAA)under bar>GAAG )-3'.5'-d(CTTCTTGTCCG)-3', derived from trans addition of the exocyclic N-6 -amino group of dA to (-)-(7S,8R,9R,10S)-7,8-dihydroxy-9,10-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene [(-)-DE2], was detennined using molecular dynamics simulations restrained by 532 NOEs from H-1 NMR. This was named the SRSR(6 1,3) adduct, derived from the N-ras protooncogene at and adjacent to the nu cleotides encoding amino acid 61 (underlined) of the p21 gene product. The solution structure of this adduct was best described as a mixture of two co nformations in rapid equilibrium on the NMR time scale. The two populations differed in the pseudorotation angle of the sugar ring for the 5'-neighbor ing base A(6), as detennined from scalar coupling data. One population, est imated to be present at 53%, had the A(6) deoxyribose in the C2'-endo confo rmation, while in the second conformation the A(6) deoxyribose was in the C 3'-endo conformation. NOEs between C-5, A(6), and (SRSR)A(7) were either di srupted or weakened, as were those in the complementary strand between C-15 , T-16, and T-17. Major groove NOEs were observed between the benzo[a]pyren e aromatic protons, H1, H2, H3, H4, H5, and H6. and T-16 CH3. Minor groove NOEs were observed between H1, H2, and H3 of benzo[a]pyrene and T-16 H1' an d H2' and T-17 H1' and H2'. The benzo[a]pyrene protons H10, H11, and H12 sh owed NOEs to A(6) H1', H2', and H2 ". The chemical shifts of the pyrenyl mo iety were dispersed over a 1.9 ppm range. Upfield chemical shifts of 2.4 pp m for T-16 N3H, 1.1 ppm for T-17 N3H, 1.3 and 1.0 ppm for T-16 H6 and CH3, 0.85 ppm for T-16 H1', and 0.80 and 0.90 ppm for C-15 H2' and H2 " were obs erved. These observations were consistent with intercalation of the pyrenyl moiety toward the 5' direction of (SRSR)A(7). The results were compared to the isomeric SRSR(61,2) adduct [I. S. Zegar, S. J. Kim, T. N. Johansen, P. J. Horton, C. M. Harris, T. M. Harris, and M. P. Stone (1996) Biochemistry 35, 6212-6224] and revealed the role of DNA sequence in modulating the con formation of this benzo[a]pyrene adduct.