Molecular contacts in the Cren7-DNA complex: A quantitative investigation for electrostatic interaction.

The molecular association of proteins with nucleic acids leading to the formation of macromolecular complexes is a crucial step in several biological processes. Stabilization of these complexes involves electrostatic interactions between ion pairs (salt bridges) of nucleic acid phosphates and protein side chains. The crenarchaeal DNA binding protein, Cren7 plays a key role in the regulation of chromosomal structure and gene expression in eukaryotic extremophiles. However, the molecular contacts that occur at the interface of protein-DNA complexes and their contribution to the electrostatic interaction have not been fully elucidated. This work presents a quantitative description of the mechanism of the electrostatic interaction between the protein and DNA. We have identified a few residues located at the Cren7-DNA interface that could potentially be responsible for the interaction. Structural studies using circular dichroism indicate mutation of these surface residues minimally affect their structure and stability. The binding affinity of these mutants for the DNA duplexes was examined from reverse titration, biolayer interferometry, and fluorescence anisotropy measurements with calf thymus DNA, polynucleotides, and small DNA oligonucleotides. The resulting kinetic parameters highlight a difference in electrostatic interactions potentials exhibited by residues positioned at different locations of the protein-DNA interface. Computational studies attribute this difference to their surrounding atmosphere and energetic stabilization parameters. The biophysical approach described here can be extended for other proteins that play a crucial role in DNA bending and compaction, to properly evaluate the role of specific residues on the mechanisms of DNA binding.