SOLUTION STRUCTURE OF A CONSERVED DNA-SEQUENCE FROM THE HIV-1 GENOME - RESTRAINED MOLECULAR-DYNAMICS SIMULATION WITH DISTANCE AND TORSION ANGLE RESTRAINTS DERIVED FROM 2-DIMENSIONAL NMR-SPECTRA

The three-dimensional solution structure of a trisdecamer DNA duplex s equence, D(AGCTTGCCTTGAG).d(CTCAAGGCAAGCT), from a conserved region of HIV-1 genome's long terminal repeat, has been investigated using NMR spectroscopy and restrained molecular dynamics calculations. Interprot on distances derived from two-dimensional nuclear Overhauser enhanceme nt (2D NOE) experiments, using the iterative complete relaxation matri x algorithm MARDIGRAS, and torsion angles for sugar rings, estimated f rom simulated fitting of double-quantum-filtered correlation (2QF-COSY ) spectra, were obtained [Mujeeb, A., Kerwin, S. M., Egan, W. M., Keny on, G. L., & James, T. L. (1992) Biochemistry 31, 9325-38]. These stru ctural restraints have now been employed as the basis for structure re finement using restrained molecular dynamics (rMD) to search conformat ional space for structures consistent with the experimental restraints . Specifically, upper and lower bounds on the restraints were incorpor ated into the AMBER (version 4.0) total potential energy function of t he system, the bounds being used to define the width of a flat-well pe nalty term in the AMBER force field. Confidence in the time-averaged s tructure obtained is engendered by convergence to essentially the same structure (root-mean-square deviation approximately 0.9 angstrom) whe n two quite different DNA models, A-DNA and B-DNA (RMSD approximately 6.5 angstrom), were employed as starting structures and when various i nitial trajectories were used for the rMD runs. The derived structure is further supported by the total energy calculated, the restraint vio lation energy, the restraint deviations, and the fit with experimental data. For the latter, the sixth-root residual index indicated a good fit of the determined structure with experimental 2D NOE spectral inte nsities (R1x < 0.07), and the RMS difference between vicinal proton co upling constants calculated for the derived structure and experimental coupling constants were also in reasonable agreement (J(RMS) = 0.9 Hz ). While the structure of the trisdecamer is basically in the B-DNA fa mily, some structural parameters manifest interesting local variations . The helix parameters indicate that, compared with classical B-DNA, t he structure is longitudinally more compressed. Local structural varia tions at the two TG steps in particular together create bending into t he major groove of the duplex. Comparison of the two-CTTG-tetrads in t he duplex reveals that they have similar structures, with the TT moiet ies being almost identical; however, the -CTTG-pur sequence has a larg er roll and slide for the -TG- step than for the -CTTG-pyr sequence, i n accord with published X-ray crystallographic conclusions.