Molecular dynamics of protein-nucleic acid complexes

Abstract: Nucleic acids are essential molecules when it comes to replication, transcription and translation of the genetic information and nearly all functions of nucleic acids are carried out in complex with proteins. The proteins need to be able to discriminate between different sites on the nucleic acids for correct binding. The recognition of a specific nucleotide sequence is determined by atomic interactions between amino acids and the nucleotides. In this thesis, molecular dynamics simulations have been used to gain insight into two different modes of recognition mechanisms: base flipping and indirect recognition of the DNA BII state. The ADAR2 (adenosine deaminase acting on RNA) protein deaminates adenosines into inosines in mostly double stranded RNA. The target amino group on the adenosine is not accessible to the protein when the base is in the stack of the helix and therefore it is believed that the protein might utilize a base flipping mechanism. The mechanism for discrimination of one adenosine from another is unknown and in paper I a study was performed on two different RNA molecules; one containing the R/G site which is selectively deaminated in vivo and one containing the 46-site which is not selectively deaminated. It was found that spontaneous base flipping occurred for the adenosine at the R/G site but not at the 46-site and the flipped base spent a significant time at an angle of ~50° (minor groove flipping) from the helix. The free energy required for flipping ±180° (perpendicular to the helix) was calculated using a forced base flipping method. This showed that flipping the adenosine at the R/G site required less energy than at the 46-site. In addition, a local minimum at ~50° was found which corresponds to the conformation for the spontaneously flipped base in the molecular dynamics simulation. The conclusion was that the adenosine at the R/G site is more flexible and that minor groove flipping is preferred over major groove flipping. In paper II, the ADAR2 deaminase domain was docked to three different RNA molecules containing the R/G site with the adenosine flipped to +50°, +185° and -180°. The adenosine had to be fully flipped to reach into the catalytic pocket and the most probable complex was that with the adenosine flipped to +185°. A possible role for the conserved R455 residue was investigated, which might be to guide the adenosine into the catalytic site. The transcription factor Ndt80 binds to its cognate DNA through different types of interactions, where recognition of the BII state of the phosphate backbone is of major importance. In paper III we showed that by inverting a central GC base pair, the backbone shifted from BII to BI conformation due to a rearrangement of the R177 side chain. In the wild type structure R177 makes a bidentate interaction with the guanine base and when the base was changed to a cytosine the R177 side chain instead made strong hydrogen bonds to the phosphate backbone. Free energy perturbation calculations were performed which estimated the difference in free energy between the wild type structure and the structure with the inverted GC pair to 6.5 kcal/mol. During the work on paper III we discovered that the current CHARMM27 force field used in the simulations was unable to reproduce the BII state. The BII state is linked to the epsilon and zeta torsion angles of the DNA backbone and the parameters associated with those angles were optimized in paper IV. The new parameters were tested on five different DNA sequences and the BI?BII equilibrium was clearly improved.