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DNA-binding proteins play a central role in all aspects of genetic activity within an organism. MD simulations have been used to probe the nature of DNA-protein complexes thereby giving valuable insights into the genetic processes that they control. The majority of DNA-protein simulations employ either the CHARMM or AMBER force-fields (see Chapter 2). Currently, simulations of DNA-protein complexes are performed either by solvating the complex within a water sphere and use a switch/shift potential for electrostatic interactions or implement periodic boundary conditions with PME treatment of electrostatics. 171

Roxtrom et al. reported the first MD simulation of a fully charged protein-DNA complex in the

nanosecond timescale.172 They performed 1ns simulation of a zinc finger-DNA complex. Zinc fingers are important DNA-binding domains found in many transcription factors. They bind in the major groove of DNA where the larger size of the groove allows access to a greater number of base pairs, thereby increasing the scope for sequence specific binding. Zif268-DNA was modelled with the GROMOS87 force-field. Explicit water molecules were included using the SPC water model with the modifications of Daura et. al.173 Results showed the DNA to be quite

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conformation was observed for the Zif268 after 850ps. Although these results agreed well with experiment, a 1ns trajectory may well be too short to pick up significant dynamical changes in the protein-DNA complex.

As the computer power available has increased, it has become possible to perform longer simulations of these complex systems. Tsui et. al. studied the hydration of a zinc finger-DNA

complex using both NMR and MD simulations.174 They performed two 2 ns simulations of the same system in explicit water. This was the first time multiple simulations of nanosecond timescales had been performed to compare the accuracy of MD for predicting hydration

patterns. The standard AMBER force field parameters and the TIP3P model of water were used for all simulations 175176. Patterns of hydration and the trajectories of water molecules

intimately associated with the zinc finger-DNA complex were analysed. It was not possible to calculate accurate residence times from these nanosecond timescale simulations as long residence water molecules can remain bound for up to 103 ps. Nevertheless the results gave excellent agreement with experimental intermolecular NOEs. Furthermore the MD results were instrumental in interpreting the otherwise ambiguous protein-water NOEs in terms of water residence times.

These simulations provide an excellent example of a system in which MD simulations with explicit water have provided atomic level detail of protein-water-DNA interactions which could not otherwise have been identified from experimental data alone.

MD simulations have also been used to study several other DNA-protein complexes. One of the best studied DNA-protein complexes is the estrogen receptor DNA-binding domain (ERDBD). Eriksson and Nilsson reported an MD study on the comparative DNA-binding of ERDBD both as a dimer and a monomer.177 They used the CHARMM22 force-field modified for the zinc ions

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and the side chains of the coordinating cysteins. The protein monomer-DNA simulation was run for 1.3 ns whereas the protein dimer-DNA was run for 0.5 ns. Results showed a considerable difference in the structure and dynamics of ERDBD complexed as a monomer to DNA compared with the dimer in complex with DNA. The monomer-DNA complex showed

significant rmsd from the starting structure whereas the dimer was very well behaved. The MD simulations suggested that dimerisation facilitates the DNA binding of ERDBD by ordering the ZnII region of the protein.

Few simulation studies of protein-DNA complexes have considered both the bound and free DNA. One such study was reported by Tang and Nilsson.178 They conducted an MD simulation study of the human sex-determining region Y (hSRY) protein interacting with DNA. Analysis of their results demonstrated the hydrophobic nature of the DNA-protein interaction. They were also able to show that both hSRY and DNA undergo significant conformational changes during binding which enable an almost perfect fit in the dimer.

Crystal structures of protein-DNA complexes often reveal ordered water molecules at the protein-DNA interface. For example the structure of the trp repressor-DNA complex has three ordered water molecules at the protein-DNA interface that can hydrogen bond with the base pairs and the protein side chains. The presence of these ordered water molecules begs a molecular explanation of their presence and role. Reddy etal. reported a study in which they

address this issue.179 They analysed X-ray/NMR structures of 109 protein-DNA complexes that contained interfacial water molecules. Hydrogen atoms were added to the protein-DNA

complex and water oxygen atoms and the systems were energy–minimised using the Amber 6 suite of programs. To test the validity of this method, MD simulations were performed on 35 of these complexes with explicit solvent and counterions. The simulations were run for 100 ps

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with 5 of them extended to 200 ps as a check of convergence. The results of the MD simulations were essentially identical to the minimisations and the conclusions unchanged.

The water molecules were split into four broad classes; class I contained water molecules that made hydrogen bonds with the protein as well as the DNA, class II contained water molecules that made contact with either the protein or the DNA, but not both, water molecules proximal to hydrophobic atoms of either protein or DNA were placed in group III and water molecules hydrogen-bonding to other water molecules as in bulk solvent made up group IV. About 6% of the water molecules belonged to class I and were responsible for water mediated protein-DNA interactions. They concluded that majority of the water molecules belonged to class II. The role of these waters was to buffer electrostatic repulsions between the protein and DNA. There were very few water molecules in class III.

The simulations of protein-DNA complexes reported in this section illustrate the quality of current force-fields. The importance of accurate representation of the solvent in MD simulations of nucleic acids is also highlighted.