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.

Binding Dynamics of a Stapled Peptide Targeting the Transcription Factor NF-Y.

Transcription factors (TFs) play a central role in gene regulation, and their malfunction can result in a plethora of severe diseases. TFs are therefore interesting therapeutic targets, but their involvement in protein-protein interaction networks and the frequent lack of well-defined binding pockets render them challenging targets for classical small molecules. As an alternative, peptide-based scaffolds have proven useful, in particular with an α-helical active conformation. Peptide-based strategies often require extensive structural optimization efforts, which could benefit from a more detailed understanding of the dynamics in inhibitor/protein interactions. In this study, we investigate how truncated stapled α-helical peptides interact with the transcription factor Nuclear Factor-Y (NF-Y). We identified a 13-mer minimal binding core region, for which two crystal structures with an altered C-terminal peptide conformation when bound to NF-Y were obtained. Subsequent molecular dynamics simulations confirmed that the C-terminal part of the stapled peptide is indeed relatively flexible while still showing defined interactions with NF-Y. Our findings highlight the importance of flexibility in the bound state of peptides, which can contribute to overall binding affinity.

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.

Rigid Base Biasing in Molecular Dynamics Enables Enhanced Sampling of DNA Conformations.

All-atom simulations have become increasingly popular to study conformational and dynamical properties of nucleic acids as they are accurate and provide high spatial and time resolutions. This high resolution, however, comes at a heavy computational cost, and, within the time scales of simulations, nucleic acids weakly fluctuate around their ideal structure exploring a limited set of conformations. We introduce the RBB-NA algorithm (available as a package in the Open Source Library PLUMED), which is capable of controlling rigid base parameters in all-atom simulations of nucleic acids. With suitable biasing potentials, this algorithm can "force" a DNA or RNA molecule to assume specific values of the six rotational (tilt, roll, twist, buckle, propeller, opening) and/or the six translational parameters (shift, slide, rise, shear, stretch, stagger). The algorithm enables the use of advanced sampling techniques to probe the structure and dynamics of locally strongly deformed nucleic acids. We illustrate its performance showing some examples in which DNA is strongly twisted, bent, or locally buckled. In these examples, RBB-NA reproduces well the unconstrained simulations data and other known features of DNA mechanics, but it also allows one to explore the anharmonic behavior characterizing the mechanics of nucleic acids in the high deformation regime.

Path Sampling Simulations Reveal How the Q61L Mutation Alters the Dynamics of KRas.

Flexibility is essential for many proteins to function, but can be difficult to characterize. Experiments lack resolution in space and time, while the time scales involved are prohibitively long for straightforward molecular dynamics simulations. In this work, we present a multiple state transition path sampling simulation study of a protein that has been notoriously difficult to characterize in its active state. The GTPase enzyme KRas is a signal transduction protein in pathways for cell differentiation, growth, and division. When active, KRas tightly binds guanosine triphosphate (GTP) in a rigid state. The protein-GTP complex can also visit more flexible states, in which it is not active. KRas mutations can affect the conversion between these rigid and flexible states, thus prolonging the activation of signal transduction pathways, which may result in tumor formation. In this work, we apply path sampling simulations to investigate the dynamic behavior of KRas-4B (wild type, WT) and the oncogenic mutant Q61L (Q61L). Our results show that KRas visits several conformational states, which are the same for WT and Q61L. The multiple state transition path sampling (MSTPS) method samples transitions between the different states in a single calculation. Tracking which transitions occur shows large differences between WT and Q61L. The MSTPS results further reveal that for Q61L, a route to a more flexible state is inaccessible, thus shifting the equilibrium to more rigid states. The methodology presented here enables a detailed characterization of protein flexibility on time scales not accessible with brute-force molecular dynamics simulations.

Sequence dependence of transient Hoogsteen base pairing in DNA.

Hoogsteen (HG) base pairing is characterized by a 180° rotation of the purine base with respect to the Watson-Crick-Franklin (WCF) motif. Recently, it has been found that both conformations coexist in a dynamical equilibrium and that several biological functions require HG pairs. This relevance has motivated experimental and computational investigations of the base-pairing transition. However, a systematic simulation of sequence variations has remained out of reach. Here, we employ advanced path-based methods to perform unprecedented free-energy calculations. Our methodology enables us to study the different mechanisms of purine rotation, either remaining inside or after flipping outside of the double helix. We study seven different sequences, which are neighbor variations of a well-studied A⋅T pair in A6-DNA. We observe the known effect of A⋅T steps favoring HG stability, and find evidence of triple-hydrogen-bonded neighbors hindering the inside transition. More importantly, we identify a dominant factor: the direction of the A rotation, with the 6-ring pointing either towards the longer or shorter segment of the chain, respectively relating to a lower or higher barrier. This highlights the role of DNA's relative flexibility as a modulator of the WCF/HG dynamic equilibrium. Additionally, we provide a robust methodology for future HG proclivity studies.

Length-scale-dependent elasticity in DNA from coarse-grained and all-atom models.

We investigate the influence of nonlocal couplings on the torsional and bending elasticities of DNA. Such couplings have been observed in the past by several simulation studies. Here, we use a description of DNA conformations based on the variables tilt, roll, and twist. Our analysis of both coarse-grained (oxDNA) and all-atom models indicates that these share strikingly similar features: there are strong off-site couplings for tilt-tilt and twist-twist, while they are much weaker in the roll-roll case. By developing an analytical framework to estimate bending and torsional persistence lengths in nonlocal DNA models, we show how off-site interactions generate a length-scale-dependent elasticity. Based on the simulation-generated elasticity data, the theory predicts a significant length-scale-dependent effect on torsional fluctuations but only a modest effect on bending fluctuations. These results are in agreement with experiments probing DNA mechanics from single base pair to kilobase pair scales.

Predicting the Structure and Dynamics of Membrane Protein GerAB from Bacillus subtilis.

Bacillus subtilis forms dormant spores upon nutrient depletion. Germinant receptors (GRs) in spore's inner membrane respond to ligands such as L-alanine, and trigger spore germination. In B. subtilis spores, GerA is the major GR, and has three subunits, GerAA, GerAB, and GerAC. L-Alanine activation of GerA requires all three subunits, but which binds L-alanine is unknown. To date, how GRs trigger germination is unknown, in particular due to lack of detailed structural information about B subunits. Using homology modelling with molecular dynamics (MD) simulations, we present structural predictions for the integral membrane protein GerAB. These predictions indicate that GerAB is an α-helical transmembrane protein containing a water channel. The MD simulations with free L-alanine show that alanine binds transiently to specific sites on GerAB. These results provide a starting point for unraveling the mechanism of L-alanine mediated signaling by GerAB, which may facilitate early events in spore germination.

Structural basis for osmotic regulation of the DNA binding properties of H-NS proteins.

H-NS proteins act as osmotic sensors translating changes in osmolarity into altered DNA binding properties, thus, regulating enterobacterial genome organization and genes transcription. The molecular mechanism underlying the switching process and its conservation among H-NS family members remains elusive. Here, we focus on the H-NS family protein MvaT from Pseudomonas aeruginosa and demonstrate experimentally that its protomer exists in two different conformations, corresponding to two different functional states. In the half-opened state (dominant at low salt) the protein forms filaments along DNA, in the fully opened state (dominant at high salt) the protein bridges DNA. This switching is a direct effect of ionic strength on electrostatic interactions between the oppositely charged DNA binding and N-terminal domains of MvaT. The asymmetric charge distribution and intramolecular interactions are conserved among the H-NS family of proteins. Therefore, our study establishes a general paradigm for the molecular mechanistic basis of the osmosensitivity of H-NS proteins.

Atomistic insight into the kinetic pathways for Watson-Crick to Hoogsteen transitions in DNA.

DNA predominantly contains Watson-Crick (WC) base pairs, but a non-negligible fraction of base pairs are in the Hoogsteen (HG) hydrogen bonding motif at any time. In HG, the purine is rotated ∼180° relative to the WC motif. The transitions between WC and HG may play a role in recognition and replication, but are difficult to investigate experimentally because they occur quickly, but only rarely. To gain insight into the mechanisms for this process, we performed transition path sampling simulations on a model nucleotide sequence in which an AT pair changes from WC to HG. This transition can occur in two ways, both starting with loss of hydrogen bonds in the base pair, followed by rotation around the glycosidic bond. In one route the adenine base converts from WC to HG geometry while remaining entirely within the double helix. The other route involves the adenine leaving the confines of the double helix and interacting with water. Our results indicate that this outside route is more probable. We used transition interface sampling to compute rate constants and relative free energies for the transitions between WC and HG. Our results agree with experiments, and provide highly detailed insights into the mechanisms of this important process.

The Adaptive Path Collective Variable: A Versatile Biasing Approach to Compute the Average Transition Path and Free Energy of Molecular Transitions.

In the past decade, great progress has been made in the development of enhanced sampling methods, aimed at overcoming the time-scale limitations of molecular dynamics (MD) simulations. Many sampling schemes rely on adding an external bias to favor the sampling of transitions and to estimate the underlying free energy landscape. Nevertheless, sampling molecular processes described by many order parameters, or collective variables (CVs), such as complex biomolecular transitions, remains often very challenging. The computational cost has a prohibitive scaling with the dimensionality of the CV-space. Inspiration can be taken from methods that focus on localizing transition pathways: the CV-space can be projected onto a path-CV that connects two stable states, and a bias can be exerted onto a one-dimensional parameter that captures the progress of the transition along the path-CV. In principle, such a sampling scheme can handle an arbitrarily large number of CVs. A standard enhanced sampling technique combined with an adaptive path-CV can then locate the mean transition pathway and obtain the free energy profile along the path. In this chapter, we discuss the adaptive path-CV formalism and its numerical implementation. We apply the path-CV with several enhanced sampling methods-steered MD, metadynamics, and umbrella sampling-to a biologically relevant process: the Watson-Crick to Hoogsteen base-pairing transition in double-stranded DNA. A practical guide is provided on how to recognize and circumvent possible pitfalls during the calculation of a free energy landscape that contains multiple pathways. Examples are presented on how to perform enhanced sampling simulations using PLUMED, a versatile plugin that can work with many popular MD engines.

Predicting the mechanism and rate of H-NS binding to AT-rich DNA.

Bacteria contain several nucleoid-associated proteins that organize their genomic DNA into the nucleoid by bending, wrapping or bridging DNA. The Histone-like Nucleoid Structuring protein H-NS found in many Gram-negative bacteria is a DNA bridging protein and can structure DNA by binding to two separate DNA duplexes or to adjacent sites on the same duplex, depending on external conditions. Several nucleotide sequences have been identified to which H-NS binds with high affinity, indicating H-NS prefers AT-rich DNA. To date, highly detailed structural information of the H-NS DNA complex remains elusive. Molecular simulation can complement experiments by modelling structures and their time evolution in atomistic detail. In this paper we report an exploration of the different binding modes of H-NS to a high affinity nucleotide sequence and an estimate of the associated rate constant. By means of molecular dynamics simulations, we identified three types of binding for H-NS to AT-rich DNA. To further sample the transitions between these binding modes, we performed Replica Exchange Transition Interface Sampling, providing predictions of the mechanism and rate constant of H-NS binding to DNA. H-NS interacts with the DNA through a conserved QGR motif, aided by a conserved arginine at position 93. The QGR motif interacts first with phosphate groups, followed by the formation of hydrogen bonds between acceptors in the DNA minor groove and the sidechains of either Q112 or R114. After R114 inserts into the minor groove, the rest of the QGR motif follows. Full insertion of the QGR motif in the minor groove is stable over several tens of nanoseconds, and involves hydrogen bonds between the bases and both backbone and sidechains of the QGR motif. The rate constant for the process of H-NS binding to AT-rich DNA resulting in full insertion of the QGR motif is in the order of 10(6) M-1s-1, which is rate limiting compared to the non-specific association of H-NS to the DNA backbone at a rate of 10(8) M-1s-1.

Allostery in Its Many Disguises: From Theory to Applications.

Allosteric regulation plays an important role in many biological processes, such as signal transduction, transcriptional regulation, and metabolism. Allostery is rooted in the fundamental physical properties of macromolecular systems, but its underlying mechanisms are still poorly understood. A collection of contributions to a recent interdisciplinary CECAM (Center Européen de Calcul Atomique et Moléculaire) workshop is used here to provide an overview of the progress and remaining limitations in the understanding of the mechanistic foundations of allostery gained from computational and experimental analyses of real protein systems and model systems. The main conceptual frameworks instrumental in driving the field are discussed. We illustrate the role of these frameworks in illuminating molecular mechanisms and explaining cellular processes, and describe some of their promising practical applications in engineering molecular sensors and informing drug design efforts.

Cis-to- Trans Isomerization of Azobenzene Derivatives Studied with Transition Path Sampling and Quantum Mechanical/Molecular Mechanical Molecular Dynamics.

Azobenzene-based molecular photoswitches are becoming increasingly important for the development of photoresponsive, functional soft-matter material systems. Upon illumination with light, fast interconversion between a more stable trans and a metastable cis configuration can be established resulting in pronounced changes in conformation, dipole moment or hydrophobicity. A rational design of functional photosensitive molecules with embedded azo moieties requires a thorough understanding of isomerization mechanisms and rates, especially the thermally activated relaxation. For small azo derivatives considered in the gas phase or simple solvents, Eyring's classical transition state theory (TST) approach yields useful predictions for trends in activation energies or corresponding half-life times of the cis isomer. However, TST or improved theories cannot easily be applied when the azo moiety is part of a larger molecular complex or embedded into a heterogeneous environment, where a multitude of possible reaction pathways may exist. In these cases, only the sampling of an ensemble of dynamic reactive trajectories (transition path sampling, TPS) with explicit models of the environment may reveal the nature of the processes involved. In the present work we show how a TPS approach can conveniently be implemented for the phenomenon of relaxation-isomerization of azobenzenes starting with the simple examples of pure azobenzene and a push-pull derivative immersed in a polar (DMSO) and apolar (toluene) solvent. The latter are represented explicitly at a molecular mechanical (MM) and the azo moiety at a quantum mechanical (QM) level. We demonstrate for the push-pull azobenzene that path sampling in combination with the chosen QM/MM scheme produces the expected change in isomerization pathway from inversion to rotation in going from a low to a high permittivity (explicit) solvent model. We discuss the potential of the simulation procedure presented for comparative calculation of reaction rates and an improved understanding of activated states.

Mechanism of environmentally driven conformational changes that modulate H-NS DNA-bridging activity.

Bacteria frequently need to adapt to altered environmental conditions. Adaptation requires changes in gene expression, often mediated by global regulators of transcription. The nucleoid-associated protein H-NS is a key global regulator in Gram-negative bacteria and is believed to be a crucial player in bacterial chromatin organization via its DNA-bridging activity. H-NS activity in vivo is modulated by physico-chemical factors (osmolarity, pH, temperature) and interaction partners. Mechanistically, it is unclear how functional modulation of H-NS by such factors is achieved. Here, we show that a diverse spectrum of H-NS modulators alter the DNA-bridging activity of H-NS. Changes in monovalent and divalent ion concentrations drive an abrupt switch between a bridging and non-bridging DNA-binding mode. Similarly, synergistic and antagonistic co-regulators modulate the DNA-bridging efficiency. Structural studies suggest a conserved mechanism: H-NS switches between a 'closed' and an 'open', bridging competent, conformation driven by environmental cues and interaction partners.

Influence of Glu/Arg, Asp/Arg, and Glu/Lys Salt Bridges on α-Helical Stability and Folding Kinetics.

Using a combination of ultraviolet circular dichroism, temperature-jump transient-infrared spectroscopy, and molecular dynamics simulations, we investigate the effect of salt bridges between different types of charged amino-acid residue pairs on α-helix folding. We determine the stability and the folding and unfolding rates of 12 alanine-based α-helical peptides, each of which has a nearly identical composition containing three pairs of positively and negatively charged residues (either Glu(-)/Arg(+), Asp(-)/Arg(+), or Glu(-)/Lys(+)). Within each set of peptides, the distance and order of the oppositely charged residues in the peptide sequence differ, such that they have different capabilities of forming salt bridges. Our results indicate that stabilizing salt bridges (in which the interacting residues are spaced and ordered such that they favor helix formation) speed up α-helix formation by up to 50% and slow down the unfolding of the α-helix, whereas salt bridges with an unfavorable geometry have the opposite effect. Comparing the peptides with different types of charge pairs, we observe that salt bridges between side chains of Glu(-) and Arg(+) are most favorable for the speed of folding, probably because of the larger conformational space of the salt-bridging Glu(-)/Arg(+) rotamer pairs compared to Asp(-)/Arg(+) and Glu(-)/Lys(+). We speculate that the observed impact of salt bridges on the folding kinetics might explain why some proteins contain salt bridges that do not stabilize the final, folded conformation.

The Trigger Factor Chaperone Encapsulates and Stabilizes Partial Folds of Substrate Proteins.

How chaperones interact with protein chains to assist in their folding is a central open question in biology. Obtaining atomistic insight is challenging in particular, given the transient nature of the chaperone-substrate complexes and the large system sizes. Recent single-molecule experiments have shown that the chaperone Trigger Factor (TF) not only binds unfolded protein chains, but can also guide protein chains to their native state by interacting with partially folded structures. Here, we used all-atom MD simulations to provide atomistic insights into how Trigger Factor achieves this chaperone function. Our results indicate a crucial role for the tips of the finger-like appendages of TF in the early interactions with both unfolded chains and partially folded structures. Unfolded chains are kinetically trapped when bound to TF, which suppresses the formation of transient, non-native end-to-end contacts. Mechanical flexibility allows TF to hold partially folded structures with two tips (in a pinching configuration), and to stabilize them by wrapping around its appendages. This encapsulation mechanism is distinct from that of chaperones such as GroEL, and allows folded structures of diverse size and composition to be protected from aggregation and misfolding interactions. The results suggest that an ATP cycle is not required to enable both encapsulation and liberation.

Short hydrogen bonds and negative charge in photoactive yellow protein promote fast isomerization but not high quantum yield.

Biological signal transduction by photoactive yellow protein (PYP) in halophilic purple sulfur bacteria is initiated by trans-to-cis isomerization of the p-coumaric acid chromophore (pCa) of PYP. pCa is engaged in two short hydrogen bonds with protein residues E46 and Y42, and it is negatively charged at the phenolate oxygen. We investigated the role in the isomerization process of the E46 short hydrogen bond and that of the negative charge on the anionic phenolate moiety of the chromophore. We used wild-type PYP and the mutant E46A, in protonated and deprotonated states (referred to as pE46A and dpE46A, respectively), to reduce the number of hydrogen bond interactions between the pCa phenolate oxygen and the protein and to vary the negative charge density in the chromophore-binding pocket. Their effects on the yield and rate of chromophore isomerization were determined by ultrafast spectroscopy. Molecular dynamics simulations were used to relate these results to structural changes in the mutant protein. We found that deprotonated pCa in E46A has a slower isomerization rate as the main part of this reaction was associated with time constants of 1 and 6 ps, significantly slower than the 0.6 ps time constant in wild-type PYP. The quantum yield of isomerization in dpE46A was estimated to be 30 ± 2%, and that of pE46A was 32 ± 3%, very close to the value determined for wtPYP of 32 ± 2%. Relaxation of the isomerized product state I0 to I1 was faster in dpE46A. We conclude that the negative charge on pCa stabilized by the short hydrogen bonds with E46 and Y42 affects the rate of isomerization but not the quantum yield of isomerization. With this information, we propose a scheme for the potential energy surfaces involved in the isomerization and suggest protein motions near the pCa backbone as key events in successful isomerization.

Multiscale approach to the determination of the photoactive yellow protein signaling state ensemble.

The nature of the optical cycle of photoactive yellow protein (PYP) makes its elucidation challenging for both experiment and theory. The long transition times render conventional simulation methods ineffective, and yet the short signaling-state lifetime makes experimental data difficult to obtain and interpret. Here, through an innovative combination of computational methods, a prediction and analysis of the biological signaling state of PYP is presented. Coarse-grained modeling and locally scaled diffusion map are first used to obtain a rough bird's-eye view of the free energy landscape of photo-activated PYP. Then all-atom reconstruction, followed by an enhanced sampling scheme; diffusion map-directed-molecular dynamics are used to focus in on the signaling-state region of configuration space and obtain an ensemble of signaling state structures. To the best of our knowledge, this is the first time an all-atom reconstruction from a coarse grained model has been performed in a relatively unexplored region of molecular configuration space. We compare our signaling state prediction with previous computational and more recent experimental results, and the comparison is favorable, which validates the method presented. This approach provides additional insight to understand the PYP photo cycle, and can be applied to other systems for which more direct methods are impractical.

Solvent-Exposed Salt Bridges Influence the Kinetics of α-Helix Folding and Unfolding.

Salt bridges are known to play an essential role in the thermodynamic stability of the folded conformation of many proteins, but their influence on the kinetics of folding remains largely unknown. Here, we investigate the effect of Glu-Arg salt bridges on the kinetics of α-helix folding using temperature-jump transient-infrared spectroscopy and steady-state UV circular dichroism. We find that geometrically optimized salt bridges (Glu- and Arg+ are spaced four peptide units apart, and the Glu/Arg order is such that the side-chain rotameric preferences favor salt-bridge formation) significantly speed up folding and slow down unfolding, whereas salt bridges with unfavorable geometry slow down folding and slightly speed up unfolding. Our observations suggest a possible explanation for the surprising fact that many biologically active proteins contain salt bridges that do not stabilize the native conformation: these salt bridges might have a kinetic rather than a thermodynamic function.