Chapter 5: Molecular Dynamics Structure Prediction of a Novel Protein–DNA Complex: Two HU Proteins with a DNA Four-way Junction

The DNA four-way junction (4WJ) is a non-canonical structure of DNA that is involved in homologous replication and recombination. There are essentially two forms of 4WJ in solution, an open form (OPN) characterized in a square plane configuration by a mobile center and four duplex arms, and a stacked-X form (STX), with two pseudo-duplex strands stacked on top of one another and intersecting at the positions of strand exchange. The prokaryotic architectural protein HU is known to bind damaged or nicked duplex DNA as well as the STX form of the 4WJ with nanomolar affinity. Recent spectroscopic studies demonstrated that the stoichiometric ratio of HU:4WJ in the complex is 2:1, but the molecular structure of the complex has not been determined. A set of distances within the complex have been obtained using Förster resonance energy transfer (FRET) but the results are insufficient to provide details of the location and orientation of the proteins with respect to the DNA. In this project, we have constructed a series of possible models for the 2HU-4WJ complex using computer graphics, docking, energy minimization, and all-atom molecular dynamics (MD) simulation including explicit solvent. Three models with the proteins situated in various orientations were investigated in detail. A structure with the two HU proteins docked in a face-to-face orientation across the junction provided the closest overall agreement between calculated and observed FRET results. This structure was dynamically stable over the course of 30ns of MD simulation.