ABSTRACT
Accurate transcription of genetic information is crucial, involving precise recognition of the binding motifs by DNA-binding proteins. While some proteins rely on short-range hydrophobic and hydrogen bonding interactions at binding sites, others employ a DNA shape readout mechanism for specific recognition. In this mechanism, variations in DNA shape at the binding motif resulted from either inherent flexibility or binding of proteins at adjacent sites are sensed and capitalized by the searching proteins to locate them specifically. Through extensive computer simulations, we investigate both scenarios to uncover the underlying mechanism and origin of specificity in the DNA shape readout mechanism. Our findings reveal that deformation in shape at the binding motif creates an entropy funnel, allowing information about altered shapes to manifest as fluctuations in minor groove widths. This signal enhances the efficiency of nonspecific search of nearby proteins by directing their movement toward the binding site, primarily driven by a gain in entropy. We propose this as a generic mechanism for DNA shape readout, where specificity arises from the alignment between the molecular frustration of the searching protein and the ruggedness of the entropic funnel governed by molecular features of the protein and arrangement of the DNA bases at the binding site, respectively.
ABSTRACT
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.
Subject(s)
DNA Damage , DNA Repair , DNA , Molecular Dynamics Simulation , DNA/metabolism , DNA-Binding Proteins/metabolism , Nucleotides/metabolism , Protein Binding , HumansABSTRACT
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.
ABSTRACT
Nearly three-fourths of all eukaryotic DNA is occupied by nucleosomes, protein-DNA complexes comprising octameric histone core proteins and â¼150 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.
Subject(s)
Histones , Nucleosomes , Histones/metabolism , DNA/metabolism , Transcription Factors/geneticsABSTRACT
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.
Subject(s)
DNA, Single-Stranded , Replication Protein A , Humans , Replication Protein A/metabolism , Protein Binding/genetics , Proteins/metabolism , Thermodynamics , KineticsABSTRACT
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.
Subject(s)
Nucleosomes , Transcription Factors , Transcription Factors/genetics , Transcription Factors/metabolism , DNA/chemistry , Protein Binding , Kinetics , Binding SitesABSTRACT
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 â¼ 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.
ABSTRACT
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.
Subject(s)
DNA , NucleosomesABSTRACT
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.
ABSTRACT
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.
Subject(s)
Nucleosomes , Transcription Factors , Cryoelectron Microscopy , DNA/genetics , Histones , Transcription Factors/metabolismABSTRACT
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.
Subject(s)
DNA, Single-Stranded , Replication Protein A , DNA Repair , DNA Replication , Protein Binding , Replication Protein A/genetics , Replication Protein A/metabolismABSTRACT
Coronavirus disease 2019 (COVID-19) is a severe acute respiratory syndrome caused by Coronavirus 2 (SARS-CoV-2). In the light of its rapid global spreading, on 11 March 2020, the World Health Organization has declared it a pandemic. Interestingly, the global spreading of the disease is not uniform, but has so far left some countries relatively less affected. The reason(s) for this anomalous behavior are not fully understood, but distinct hypotheses have been proposed. Here we discuss the plausibility of two of them: the universal vaccination with Bacillus Calmette-Guerin (BCG) and the widespread use of the antimalarial drug chloroquine (CQ). Both have been amply discussed in the recent literature with positive and negative conclusions: we felt that a comprehensive presentation of the data available on them would be useful. The analysis of data for countries with over 1000 reported COVID-19 cases has shown that the incidence and mortality were higher in countries in which BCG vaccination is either absent or has been discontinued, as compared with the countries with universal vaccination. We have performed a similar analysis of the data available for CQ, a widely used drug in the African continent and in other countries in which malaria is endemic; we discuss it here because CQ has been used as the drug to treat COVID-19 patients. Several African countries no longer recommend it officially for the fight against malaria, due to the development of resistance to Plasmodium, but its use across the continent is still diffuse. Taken together, the data in the literature have led to the suggestion of a possible inverse correlation between BCG immunization and COVID-19 disease incidence and severity.
Subject(s)
Antiviral Agents/therapeutic use , BCG Vaccine/therapeutic use , Betacoronavirus/drug effects , Chloroquine/therapeutic use , Coronavirus Infections/prevention & control , Pandemics/prevention & control , Pneumonia, Viral/prevention & control , Africa/epidemiology , COVID-19 , Coronavirus Infections/drug therapy , Coronavirus Infections/epidemiology , Humans , Incidence , Pneumonia, Viral/drug therapy , Pneumonia, Viral/epidemiology , SARS-CoV-2 , Vaccination , COVID-19 Drug TreatmentABSTRACT
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 ± 3.6â 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.
Subject(s)
14-3-3 Proteins/metabolism , Malaria, Falciparum/parasitology , Molecular Dynamics Simulation , Plasmodium falciparum/growth & development , Protein Kinases/metabolism , Protozoan Proteins/metabolism , 14-3-3 Proteins/chemistry , Amino Acid Sequence , Animals , Phosphorylation , Phylogeny , Plasmodium falciparum/isolation & purification , Plasmodium falciparum/metabolism , Protein Conformation , Protein Kinases/chemistry , Protozoan Proteins/chemistry , Sequence HomologyABSTRACT
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.
Subject(s)
DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Diffusion , Models, Molecular , Protein ConformationABSTRACT
Preserving the genomic integrity is a fundamental requirement, primarily achieved by the DNA repair proteins through their continuous patrolling on the DNA in search of lesions. Human uracil DNA glycosylase (hUNG) is one such DNA repair protein that recognizes uracil in the duplex DNA and excises it using the extrahelical base recognition mechanism. Recent site transfer assay experiments based on full-length hUNG suggest that a crowded environment facilitates its search efficiency, which is enhanced further in the presence of a 93 residue disordered tail associated with its N-terminal. In this study, by performing extensive molecular dynamics simulations with an appropriately tuned model of protein and DNA in the presence of inert crowding agents, we probe the role of cellular crowding and the disordered region in the target search efficiency of the enzyme. Our analysis highlights a complex interplay among the shape of the enzyme, the presence of a disordered tail, and the macromolecular crowding agents that work in harmony to enhance the facilitated diffusion of hUNG protein in a crowded environment. The findings provide novel insights into the in vivo target search mechanism of DNA repair proteins.
Subject(s)
Molecular Dynamics Simulation , Uracil-DNA Glycosidase/chemistry , DNA Repair , Diffusion , Humans , Uracil-DNA Glycosidase/metabolismABSTRACT
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.
Subject(s)
DNA/metabolism , Models, Molecular , Proteins/metabolism , Binding Sites , DNA/chemistry , Diffusion , Protein Binding , Proteins/chemistry , Surface Properties , ThermodynamicsABSTRACT
Recent experiments suggest that cellular crowding facilitates the target search dynamics of proteins on DNA, the mechanism of which is not yet known. By using large scale computer simulations, we show that two competing factors, namely the width of the depletion layer that separates the crowder cloud from the DNA molecule and the degree of protein-crowder crosstalk, act in harmony to affect the target search dynamics of proteins. The impacts vary from nonspecific to specific target search regime. During a nonspecific search, dynamics of a protein is only minimally affected, whereas, a significantly different behaviour is observed when the protein starts forming a specific protein-DNA complex. We also find that the severity of impacts largely depends upon physiological crowder concentration and deviation from it leads to attenuation in the binding kinetics. Based on extensive kinetic study and binding energy landscape analysis, we further present a comprehensive molecular description of the search process that allows us to interpret the experimental findings.
Subject(s)
DNA-Binding Proteins/metabolism , DNA/metabolism , Binding Sites , DNA/chemistry , DNA-Binding Proteins/chemistry , Diffusion , Kinetics , Molecular Dynamics Simulation , Nucleic Acid Conformation , Protein Binding , Protein Structure, TertiaryABSTRACT
A wide range of cellular processes initiates upon recognizing and binding of proteins to specific DNA sites. Typically, the recognition process is incredibly fast owing to a complex mechanism that combines different 3D and 1D modes of translocation of the protein. While few studies performed on selected DNA topologies suggested that the DNA topology might alter the balance between these two modes and therefore the target search kinetics, its detailed role in the target search mechanism remains unclear. Here, we present a discrete-state stochastic approach that allows us to incorporate the topological information of DNA molecule explicitly and predict its role during the process when proteins search for their specific binding sites on DNA. Applying the theory to the closed loop and different supercoiled DNA topologies, we find that the target search efficiency of the protein is strongly influenced by the DNA topology. Furthermore, if the topology is such that it promotes juxtaposition of distant DNA sites, the number, position and relative distances between such juxtaposition sites play a crucial role in facilitating the search process by providing additional routes to approach the target site. Our predictions are validated through extensive Monte Carlo simulations.
Subject(s)
DNA-Binding Proteins/metabolism , DNA/metabolism , Binding Sites , Computer Simulation , DNA/chemistry , DNA-Binding Proteins/chemistry , Kinetics , Monte Carlo Method , Protein Binding , Stochastic Processes , Surface PropertiesABSTRACT
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.
Subject(s)
DNA-Binding Proteins/chemistry , DNA/chemistry , DNA/metabolism , DNA-Binding Proteins/metabolism , Facilitated Diffusion , Kinetics , Molecular Dynamics Simulation , Nucleic Acid Conformation , Protein BindingABSTRACT
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.