Deciphering Sequence-Specific DNA Binding by H-NS Using Molecular Simulation.

H-NS is a DNA organizing protein that occurs in Gram-negative bacteria. It can form long filaments between two DNA duplexes by first binding to a high-affinity AT-rich nucleotide sequence and extending from there. Using molecular dynamics simulations and steered molecular dynamics, we are able to determine the free energy of formation and dissociation of a protein-DNA complex comprising an H-NS DNA-binding domain and a specific nucleotide sequence. The molecular dynamics simulations allow detailed characterization of the interactions between the protein and a specific nucleotide sequence. To quantify the strength of the interaction, we employ an additional potential based on protein-DNA contacts to speed up dissociation of the protein-DNA complex. The work required for the dissociation results in an estimate of the free energy of dissociation/complex formation. Our protocol can provide quantitative prediction of protein-DNA complex stability, while also providing high-resolution insights into recognition mechanisms. In this chapter, we have used this approach to quantify the sequence specificity of H-NS DNA-binding domains to various nucleotide sequences, thus elucidating the mechanism with which H-NS can specifically bind to AT-rich DNA.