Bacterial histone HBb from Bdellovibrio bacteriovorus compacts DNA by bending.

Histones are essential for genome compaction and transcription regulation in eukaryotes, where they assemble into octamers to form the nucleosome core. In contrast, archaeal histones assemble into dimers that form hypernucleosomes upon DNA binding. Although histone homologs have been identified in bacteria recently, their DNA-binding characteristics remain largely unexplored. Our study reveals that the bacterial histone HBb (Bd0055) is indispensable for the survival of Bdellovibrio bacteriovorus, suggesting critical roles in DNA organization and gene regulation. By determining crystal structures of free and DNA-bound HBb, we unveil its distinctive dimeric assembly, diverging from those of eukaryotic and archaeal histones, while also elucidating how it binds and bends DNA through interaction interfaces reminiscent of eukaryotic and archaeal histones. Building on this, by employing various biophysical and biochemical approaches, we further substantiated the ability of HBb to bind and compact DNA by bending in a sequence-independent manner. Finally, using DNA affinity purification and sequencing, we reveal that HBb binds along the entire genomic DNA of B. bacteriovorus without sequence specificity. These distinct DNA-binding properties of bacterial histones, showcasing remarkable similarities yet significant differences from their archaeal and eukaryotic counterparts, highlight the diverse roles histones play in DNA organization across all domains of life.

Histones, traditionally known for organizing and regulating DNA in eukaryotes and archaea, have recently been discovered in bacteria, opening up a new frontier in our understanding of genome organization across the domains of life. Our study investigates the largely unexplored DNA-binding properties of bacterial histones, focusing on HBb in Bdellovibrio bacteriovorus. We reveal that HBb is essential for bacterial survival and exhibits DNA-binding properties similar to archaeal and eukaryotic histones. However, unlike eukaryotic and archaeal histones, which wrap DNA, HBb bends DNA without sequence specificity. This work not only broadens our understanding of DNA organization across different life forms but also suggests that bacterial histones may have diverse roles in genome organization.

Decoding dissociation of sequence-specific protein-DNA complexes with non-equilibrium simulations.

Sequence-specific protein-DNA interactions are crucial in processes such as DNA organization, gene regulation and DNA replication. Obtaining detailed insights into the recognition mechanisms of protein-DNA complexes through experiments is hampered by a lack of resolution in both space and time. Here, we present a molecular simulation approach to quantify the sequence specificity of protein-DNA complexes, that yields results fast, and is generally applicable to any protein-DNA complex. The approach is based on molecular dynamics simulations in combination with a sophisticated steering potential and results in an estimate of the free energy difference of dissociation. We provide predictions of the nucleotide specific binding affinity of the minor groove binding Histone-like Nucleoid Structuring (H-NS) protein, that are in agreement with experimental data. Furthermore, our approach offers mechanistic insight into the process of dissociation. Applying our approach to the major groove binding ETS domain in complex with three different nucleotide sequences identified the high affinity consensus sequence, quantitatively in agreement with experiments. Our protocol facilitates quantitative prediction of protein-DNA complex stability, while also providing high resolution insights into recognition mechanisms. As such, our simulation approach has the potential to yield detailed and quantitative insights into biological processes involving sequence-specific protein-DNA interactions.

Tuning the Binding Strength of Even and Uneven Hydrogen-Bonded Arrays with Remote Substituents.

We investigated the tunability of hydrogen bond strength by altering the charge accumulation around the frontier atoms with remote substituents. For pyridine···H2O with NH2 and CN substituted at different positions on pyridine, we find that the electron-withdrawing CN group decreases the negative charge accumulation around the frontier atom N, resulting in weakening of the hydrogen bond, whereas the electron-donating NH2 group increases the charge accumulation around N, resulting in strengthening of the hydrogen bond. By applying these design principles on DDAA-AADD, DADA-ADAD, DAA-ADD, and ADA-DAD hydrogen-bonded dimers, we find that the effect of the substituent is delocalized over the whole molecular system. As a consequence, systems with an equal number of hydrogen bond donor (D) and acceptor (A) atoms are not tunable in a predictable way because of cancellation of counteracting strengthening and weakening effects. Furthermore, we show that the position of the substituent and long-range electrostatics can play an important role as well. Overall, the design principles presented in this work are suitable for monomers with an unequal number of donor and acceptor atoms and can be exploited to tune the binding strength of supramolecular building blocks.

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.