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.