Understanding the Target Search by Multiple Transcription Factors on Nucleosomal DNA.

The association of multiple Transcription Factors (TFs) in the cis-regulatory region is imperative for developmental changes in eukaryotes. The underlying process is exceedingly complex, and it is not at all clear what orchestrates the overall search process by multiple TFs. In this study, by developing a theoretical model based on a discrete-state stochastic approach, we investigated the target search mechanism of multiple TFs on nucleosomal DNA. Experimental kinetic rate constants of different TFs are taken as input to estimate the Mean-First-Passage time to recognize the binding motifs by two TFs on a dynamic nucleosome model. The theory systematically analyzes when the TFs search their binding motifs hierarchically and when simultaneously by proceeding via the formation of a protein-protein complex. Our results, validated by extensive Monte Carlo simulations, elucidate the molecular basis of the complex target search phenomenon of multiple TFs on nucleosomal DNA.

Nucleosome breathing facilitates cooperative binding of pluripotency factors Sox2 and Oct4 to DNA.

Critical lineage commitment events are staged by multiple transcription factors (TFs) binding to their cognate motifs, often positioned at nucleosome-enriched regions of chromatin. The underlying mechanism remains elusive due to difficulty in disentangling the heterogeneity in chromatin states. Using a novel coarse-grained model and molecular dynamics simulations, here we probe the association of Sox2 and Oct4 proteins that show clustered binding at the entry-exit region of a nucleosome. The model captures the conformational heterogeneity of nucleosome breathing dynamics that features repeated wrap-unwrap transitions of a DNA segment from one end of the nucleosome. During the dynamics, DNA forms bulges that diffuse stochastically and may regulate the target search dynamics of a protein by nonspecifically interacting with it. The overall search kinetics of the TF pair follows a "dissociation-compensated-association" mechanism, where Oct4 binding is facilitated by the association of Sox2. The cooperativity stems from a change in entropy caused by an alteration in the nucleosome dynamics upon TF binding. The binding pattern is consistent with a live-cell single-particle tracking experiment, suggesting the mechanism observed for clustered binding of a TF pair, which is a hallmark of cis-regulatory elements, has broader implications in understanding gene regulation in a complex chromatin environment.

Kinetic origin of nucleosome invasion by pioneer transcription factors.

Recently, a cryo-electron microscopy study has captured different stages of nucleosome breathing dynamics that show partial unwrapping of DNA from histone core to permit transient access to the DNA sites by transcription factors. In practice, however, only a subset of transcription factors named pioneer factors can invade nucleosomes and bind to specific DNA sites to trigger essential DNA metabolic processes. We propose a discrete-state stochastic model that considers the interplay of nucleosome breathing and protein dynamics explicitly and estimate the mean time to search the target DNA sites. It is found that the molecular principle governing the search process on nucleosome is very different compared to that on naked DNA. The pioneer factors minimize their search times on nucleosomal DNA by compensating their nucleosome association rates by dissociation rates. A fine balance between the two presents a tradeoff between their nuclear mobility and error associated with the search process.

Molecular dynamics simulations and biochemical characterization of Pf14-3-3 and PfCDPK1 interaction towards its role in growth of human malaria parasite.

Scaffold proteins play pivotal role as modulators of cellular processes by operating as multipurpose conformation clamps. 14-3-3 proteins are gold-standard scaffold modules that recognize phosphoSer/Thr (pS/pT) containing conserved motifs, and confer conformational changes leading to modulation of functional parameters of their target proteins. Modulation in functional activity of kinases has been attributed to their interaction with 14-3-3 proteins. Herein, we have annotated and characterized PF3D7_0818200 as 14-3-3 isoform I in Plasmodium falciparum 3D7, and its interaction with one of the key kinases of the parasite, Calcium-Dependent Protein Kinase 1 (CDPK1) by performing various analytical biochemistry and biophysical assays. Molecular dynamics simulation studies indicated that CDPK1 polypeptide sequence (61KLGpS64) behaves as canonical Mode I-type (RXXpS/pT) consensus 14-3-3 binding motif, mediating the interaction. The 14-3-3I/CDPK1 interaction was validated in vitro with ELISA and SPR, which confirmed that the interaction is phosphorylation dependent, with binding affinity constant of 670 ± 3.6 nM. The interaction of 14-3-3I with CDPK1 was validated with well characterized optimal 14-3-3 recognition motifs: Mode I-type ARSHpSYPA and Mode II-type RLYHpSLPA, by simulation studies and ITC. This interaction was found to marginally enhance CDPK1 functional activity. Furthermore, interaction antagonizing peptidomimetics showed growth inhibitory impact on the parasite indicating crucial physiological role of 14-3-3/CDPK1 interaction. Overall, this study characterizes 14-3-3I as a scaffold protein in the malaria parasite and unveils CDPK1 as its previously unidentified target. This sets a precedent for the rational design of 14-3-3 based PPI inhibitors by utilizing 14-3-3 recognition motif peptides, as a potential antimalarial strategy.

Structural Basis of Enhanced Facilitated Diffusion of DNA-Binding Protein in Crowded Cellular Milieu.

Although the fast association between DNA-binding proteins (DBPs) and DNA is explained by a facilitated diffusion mechanism, in which DBPs adopt a weighted combination of three-dimensional diffusion and one-dimensional (1D) sliding and hopping modes of transportation, the role of cellular environment that contains many nonspecifically interacting proteins and other biomolecules is mostly overlooked. By performing large-scale computational simulations with an appropriately tuned model of protein and DNA in the presence of nonspecifically interacting bulk and DNA-bound crowders (genomic crowders), we demonstrate the structural basis of the enhanced facilitated diffusion of DBPs inside a crowded cellular milieu through, to our knowledge, novel 1D scanning mechanisms. In this one-dimensional scanning mode, the protein can float along the DNA under the influence of nonspecific interactions of bulk crowder molecules. The search mode is distinctly different compared to usual 1D sliding and hopping dynamics in which protein diffusion is regulated by the DNA electrostatics. In contrast, the presence of genomic crowders expedites the target search process by transporting the protein over DNA segments through the formation of a transient protein-crowder bridged complex. By analyzing the ruggedness of the associated potential energy landscape, we underpin the molecular origin of the kinetic advantages of these search modes and show that they successfully explain the experimentally observed acceleration of facilitated diffusion of DBPs by molecular crowding agents and crowder-concentration-dependent enzymatic activity of transcription factors. Our findings provide crucial insights into gene regulation kinetics inside the crowded cellular milieu.

Mechanism of Dynamic Binding of Replication Protein A to ssDNA.

Replication protein A (RPA) serves as a hub protein inside eukaryotic cells, where it coordinates crucial DNA metabolic processes and activates the DNA-damage response system. A characteristic feature of its action is to associate with single-stranded DNA (ssDNA) intermediates before handing them over to downstream proteins. The length of ssDNA intermediates differs for different pathways. This means that RPA must have mechanisms for selective processing of ssDNA intermediates based on their length, the knowledge of which is fundamental to elucidate when and how DNA repair and replication processes are symphonized. By employing extensive molecular dynamics simulations, we investigated the mechanism of binding of RPA to ssDNA of different lengths. We show that the binding involves dynamic equilibrium with a stable intermediate, the population of which increases with the length of ssDNA. The vital underlying factors are decoded through collective variable principal component analysis. It suggests a differently orchestrated set of interactions that define the action of RPA based on the length of ssDNA intermediates. We further estimated the association kinetics that matches excellently well with previous experimental studies and probed the diffusion mechanism of RPA to ssDNA. RPA diffuses on short ssDNA through progressive "bulge" formation. With long ssDNA, we observed a conformational change in ssDNA coupled with its binding to RPA in a cooperative fashion. This unanticipated binding mechanism successfully explains how the "short-lived", long ssDNA intermediates are processed quickly in vivo. This study thus reveals the molecular basis of several recent experimental observations related to RPA binding to ssDNA and provides novel insights into the RPA functioning in DNA repair and replication.

Disparity in anomalous diffusion of proteins searching for their target DNA sites in a crowded medium is controlled by the size, shape and mobility of macromolecular crowders.

Using extensive computer simulations, we analyzed the role of physical properties of molecular crowding agents such as size, shape and mobility in the target search dynamics of DNA binding proteins. Our main result is that the sub-diffusive dynamics of a protein inside a crowded medium strongly depends on the crowder properties and also on the protein's mode of diffusion. For instance, while scanning the DNA one-dimensionally, the protein dynamics does not vary with the change in crowder properties. Conversely, the diffusion exponent varies non-monotonically during 3D diffusion and is maximally affected when the crowders match the protein physically. The investigation shows that the effect stems from the ruggedness of the associated potential energy landscape, which is regulated by the protein-crowder and DNA-crowder interactions. Our findings have broad significance in understanding the target search dynamics of proteins on DNA in crowded cellular milieu and selecting appropriate crowding agents when designing in vitro experiments.

Searching target sites on DNA by proteins: Role of DNA dynamics under confinement.

DNA-binding proteins (DBPs) rapidly search and specifically bind to their target sites on genomic DNA in order to trigger many cellular regulatory processes. It has been suggested that the facilitation of search dynamics is achieved by combining 3D diffusion with one-dimensional sliding and hopping dynamics of interacting proteins. Although, recent studies have advanced the knowledge of molecular determinants that affect one-dimensional search efficiency, the role of DNA molecule is poorly understood. In this study, by using coarse-grained simulations, we propose that dynamics of DNA molecule and its degree of confinement due to cellular crowding concertedly regulate its groove geometry and modulate the inter-communication with DBPs. Under weak confinement, DNA dynamics promotes many short, rotation-decoupled sliding events interspersed by hopping dynamics. While this results in faster 1D diffusion, associated probability of missing targets by jumping over them increases. In contrast, strong confinement favours rotation-coupled sliding to locate targets but lacks structural flexibility to achieve desired specificity. By testing under physiological crowding, our study provides a plausible mechanism on how DNA molecule may help in maintaining an optimal balance between fast hopping and rotation-coupled sliding dynamics, to locate target sites rapidly and form specific complexes precisely.

Search by proteins for their DNA target site: 1. The effect of DNA conformation on protein sliding.

The recognition of DNA-binding proteins (DBPs) to their specific site often precedes by a search technique in which proteins slide, hop along the DNA contour or perform inter-segment transfer and 3D diffusion to dissociate and re-associate to distant DNA sites. In this study, we demonstrated that the strength and nature of the non-specific electrostatic interactions, which govern the search dynamics of DBPs, are strongly correlated with the conformation of the DNA. We tuned two structural parameters, namely curvature and the extent of helical twisting in circular DNA. These two factors are mutually independent of each other and can modulate the electrostatic potential through changing the geometry of the circular DNA conformation. The search dynamics for DBPs on circular DNA is therefore markedly different compared with linear B-DNA. Our results suggest that, for a given DBP, the rotation-coupled sliding dynamics is precluded in highly curved DNA (as well as for over-twisted DNA) because of the large electrostatic energy barrier between the inside and outside of the DNA molecule. Under such circumstances, proteins prefer to hop in order to explore interior DNA sites. The change in the balance between sliding and hopping propensities as a function of DNA curvature or twisting may result in different search efficiency and speed.

Search by proteins for their DNA target site: 2. The effect of DNA conformation on the dynamics of multidomain proteins.

Multidomain transcription factors, which are especially abundant in eukaryotic genomes, are advantageous to accelerate the search kinetics for target site because they can follow the intersegment transfer via the monkey-bar mechanism in which the protein forms a bridged intermediate between two distant DNA regions. Monkey-bar dynamics highly depends on the properties of the multidomain protein (the affinity of each of the constituent domains to the DNA and the length of the linker) and the DNA molecules (their inter-distance and inter-angle). In this study, we investigate using coarse-grained molecular dynamics simulations how the local conformation of the DNA may affect the DNA search performed by a multidomain protein Pax6 in comparison to that of the isolated domains. Our results suggest that in addition to the common rotation-coupled translation along the DNA major groove, for curved DNA the tethered domains may slide in a rotation-decoupled sliding mode. Furthermore, the multidomain proteins move by longer jumps on curved DNA compared with those performed by the single domain protein. The long jumps originate from the DNA curvature bringing two sequentially distant DNA sites into close proximity with each other and they suggest that multidomain proteins may move on highly curved DNA faster than linear DNA.

Thermodynamic Protein Destabilization by GFP Tagging: A Case of Interdomain Allostery.

The Engrailed Homeodomain (EnHD) transcription factor of Drosophila melanogaster was fused to the enhanced green fluorescent protein (eGFP) either at its C- or N-terminus via three- or ten-residue flexible linkers. Here, we show that EnHD undergoes destabilization upon fusing it to eGFP regardless of the linker length used and whether the tethering is to its N- or C-terminus. The destabilization is reflected in melting points that are lower by up to 9°C. Thermodynamic analysis and coarse-grained molecular dynamic simulations indicate that this destabilization is due to eGFP-promoted entropic stabilization of the denatured state ensemble of EnHD. Our results provide, therefore, an example for destabilizing interdomain allostery. They are also important given the widespread use of eGFP tagging in cell biology, as they indicate that such tagging can cause unintended protein destabilization and concomitant effects.

Exploring Protein-Peptide Binding Specificity through Computational Peptide Screening.

The binding of short disordered peptide stretches to globular protein domains is important for a wide range of cellular processes, including signal transduction, protein transport, and immune response. The often promiscuous nature of these interactions and the conformational flexibility of the peptide chain, sometimes even when bound, make the binding specificity of this type of protein interaction a challenge to understand. Here we develop and test a Monte Carlo-based procedure for calculating protein-peptide binding thermodynamics for many sequences in a single run. The method explores both peptide sequence and conformational space simultaneously by simulating a joint probability distribution which, in particular, makes searching through peptide sequence space computationally efficient. To test our method, we apply it to 3 different peptide-binding protein domains and test its ability to capture the experimentally determined specificity profiles. Insight into the molecular underpinnings of the observed specificities is obtained by analyzing the peptide conformational ensembles of a large number of binding-competent sequences. We also explore the possibility of using our method to discover new peptide-binding pockets on protein structures.

Hybrid Monte Carlo with non-uniform step size.

The Hybrid Monte Carlo method offers a rigorous and potentially efficient approach to the simulation of dense systems, by combining numerical integration of Newton's equations of motion with a Metropolis accept-or-reject step. The Metropolis step corrects for sampling errors caused by the discretization of the equations of motion. The integration is usually performed using a uniform step size. Here, we present simulations of the Lennard-Jones system showing that the use of smaller time steps in the tails of each integration trajectory can reduce errors in energy. The acceptance rate is 10-15 percentage points higher in these runs, compared to simulations with the same trajectory length and the same number of integration steps but a uniform step size. We observe similar effects for the harmonic oscillator and a coarse-grained peptide model, indicating generality of the approach.

The role of nucleotide opening dynamics in facilitated target search by DNA-repair proteins.

Preserving the genomic integrity stands a fundamental necessity, primarily achieved by the DNA repair proteins through their continuous patrolling on the DNA in search of lesions. However, comprehending how even a single base-pair lesion can be swiftly and specifically recognized amidst millions of base-pair sites remains a formidable challenge. In this study, we employ extensive molecular dynamics simulations using an appropriately tuned model of both protein and DNA to probe the underlying molecular principles. Our findings reveal that the dynamics of a non-canonical base generate an entropic signal that guides the one-dimensional search of a repair protein, thereby facilitating the recognition of the lesion site. The width of the funnel perfectly aligns with the one-dimensional diffusion length of DNA-binding proteins. The generic mechanism provides a physical basis for rapid recognition and specificity of DNA damage sensing and recognition.

Interdomain dynamics in human Replication Protein A regulates kinetics and thermodynamics of its binding to ssDNA.

Human Replication Protein A (hRPA) is a multidomain protein that interacts with ssDNA intermediates to provide the latter much-needed stability during DNA metabolism and maintain genomic integrity. Although the ssDNA organization with hRPA was studied recently through experimental means, characterizing the underlying mechanism at the atomic level remains challenging because of the dynamic domain architecture of hRPA and poorly understood heterogeneity of ssDNA-protein interactions. Here, we used a computational framework, precisely tailored to capture protein-ssDNA interactions, and investigated the binding of hRPA with a 60 nt ssDNA. Two distinct binding mechanisms are realized based on the hRPA domain flexibility. For a rigid domain architecture of hRPA, ssDNA binds sequentially with hRPA domains, resulting in slow association kinetics. The binding pathway involves the formation of stable and distinct intermediate states. On contrary, for a flexible domain architecture of hRPA, ssDNA binds synergistically to the A and B domains followed by the rest of hRPA. The domain dynamics in hRPA alleviates the free energy cost of domain orientation necessary for specific binding with ssDNA, leading to fast association kinetics along a downhill binding free energy landscape. An ensemble of free energetically degenerate intermediate states is encountered that makes it arduous to characterize them structurally. An excellent match between our results with the available experimental observations provides new insights into the rich dynamics of hRPA binding to ssDNA and in general paves the way to investigate intricate details of ssDNA-protein interactions, crucial for cellular functioning.

How Do Nucleosome Dynamics Regulate Protein Search on DNA?

Nearly three-fourths of all eukaryotic DNA is occupied by nucleosomes, protein-DNA complexes comprising octameric histone core proteins and ∼150 base pairs of DNA. In addition to acting as a DNA compaction vehicle, the dynamics of nucleosomes regulate the DNA site accessibility for the nonhistone proteins, thereby controlling regulatory processes involved in determining the cell identity and cell fate. Here, we propose an analytical framework to analyze the role of nucleosome dynamics on the target search process of transcription factors through a simple discrete-state stochastic description of the search process. By considering the experimentally determined kinetic rates associated with protein and nucleosome dynamics as the only inputs, we estimate the target search time of a protein via first-passage probability calculations separately during nucleosome breathing and sliding dynamics. Although both the nucleosome dynamics permit transient access to the DNA sites that are otherwise occluded by the histone proteins, our result suggests substantial differences between the protein search mechanism on a nucleosome performing breathing and sliding dynamics. Furthermore, we identify the molecular factors that influence the search efficiency and demonstrate how these factors together portray a highly dynamic landscape of gene regulation. Our analytical results are validated using extensive Monte Carlo simulations.

Unusual weak and delayed GTPase activity of FtsZ from human pathogenic bacteria Helicobacter pylori.

Actively treadmilling FtsZ acts as the pivotal scaffold for bacterial cell divisome components providing them with a circumferential ride along the site of future division. FtsZ from slow growing Helicobacter pylori (HpFtsZ), a class I carcinogen which thrives abundantly in the acidic environment is poorly understood. We studied HpFtsZ as a function of pH, cations and time and compared it with well-studied E. coli FtsZ (EcFtsZ). HpFtsZ shows pH dependent GTPase activity which is inhibited under acidic conditions. Mg+2 ions play an indispensable role in its GTPase activity, however, higher Mg+2 levels negatively affect its activity. As compared to EcFtsZ, HpFtsZ exhibits lower and slower nucleotide hydrolyzing activity. Molecular Dynamics Simulation studies of FtsZ reveal that GTP binding induces a rewiring of the hydrogen bond network which results in reduction of the binding cleft volume leading to the spontaneous release of GTP. The GTPase activity is linked to the extent of reduction in the binding cleft volume, which is also supported by the binding free energy analysis. Evidently, HpFtsZ is a pH sensitive GTPase with low efficiency that may reflect on the overall slow growth rate of H. pylori.

Coupled folding-binding in a hydrophobic/polar protein model: impact of synergistic folding and disordered flanks.

Coupled folding-binding is central to the function of many intrinsically disordered proteins, yet not fully understood. With a continuous three-letter protein model, we explore the free-energy landscape of pairs of interacting sequences and how it is impacted by 1), variations in the binding mechanism; and 2), the addition of disordered flanks to the binding region. In particular, we focus on two sequences, one with 16 and one with 35 amino acids, which make a stable dimeric three-helix bundle at low temperatures. Three distinct binding mechanisms are realized by altering the stabilities of the individual monomers: docking, coupled folding-binding of a single α-helix, and synergistic folding and binding. Compared to docking, the free-energy barrier for binding is reduced when the single α-helix is allowed to fold upon binding, but only marginally. A greater reduction is found for synergistic folding, which in addition results in a binding transition state characterized by very few interchain contacts. Disordered flanking chain segments attached to the α-helix sequence can, despite a negligible impact on the dimer stability, lead to a downhill free-energy surface in which the barrier for binding is eliminated.

Understanding protein diffusion on force-induced stretched DNA conformation.

DNA morphology is subjected to environmental conditions and is closely coupled with its function. For example, DNA experiences stretching forces during several biological processes, including transcription and genome transactions, that significantly alter its conformation from that of B-DNA. Indeed, a well-defined 1.5 times extended conformation of dsDNA, known as Σ-DNA, has been reported in DNA complexes with proteins such as Rad51 and RecA. A striking feature in Σ-DNA is that the nucleobases are partitioned into triplets of three locally stacked bases separated by an empty rise gap of ∼ 5 Å. The functional role of such a DNA base triplet was hypothesized to be coupled with the ease of recognition of DNA bases by DNA-binding proteins (DBPs) and the physical origin of three letters (codon/anti-codon) in the genetic code. However, the underlying mechanism of base-triplet formation and the ease of DNA base-pair recognition by DBPs remain elusive. To investigate, here, we study the diffusion of a protein on a force-induced stretched DNA using coarse-grained molecular dynamics simulations. Upon pulling at the 3' end of DNA by constant forces, DNA exhibits a conformational transition from B-DNA to a ladder-like S-DNA conformation via Σ-DNA intermediate. The resulting stretched DNA conformations exhibit non-uniform base-pair clusters such as doublets, triplets, and quadruplets, of which triplets are energetically more stable than others. We find that protein favors the triplet formation compared to its unbound form while interacting non-specifically along DNA, and the relative population of it governs the ruggedness of the protein-DNA binding energy landscape and enhances the efficiency of DNA base recognition. Furthermore, we analyze the translocation mechanism of a DBP under different force regimes and underscore the significance of triplet formation in regulating the facilitated diffusion of protein on DNA. Our study, thus, provides a plausible framework for understanding the structure-function relationship between triplet formation and base recognition by a DBP and helps to understand gene regulation in complex regulatory processes.

Superstructure Detection in Nucleosome Distribution Shows Common Pattern within a Chromosome and within the Genome.

Nucleosome positioning plays an important role in crucial biological processes such as replication, transcription, and gene regulation. It has been widely used to predict the genome's function and chromatin organisation. So far, the studies of patterns in nucleosome positioning have been limited to transcription start sites, CTCFs binding sites, and some promoter and loci regions. The genome-wide organisational pattern remains unknown. We have developed a theoretical model to coarse-grain nucleosome positioning data in order to obtain patterns in their distribution. Using hierarchical clustering on the auto-correlation function of this coarse-grained nucleosome positioning data, a genome-wide clustering is obtained for Candida albicans. The clustering shows the existence beyond hetero- and eu-chromatin inside the chromosomes. These non-trivial clusterings correspond to different nucleosome distributions and gene densities governing differential gene expression patterns. Moreover, these distribution patterns inside the chromosome appeared to be conserved throughout the genome and within species. The pipeline of the coarse grain nucleosome positioning sequence to identify underlying genomic organisation used in our study is novel, and the classifications obtained are unique and consistent.