RESUMEN
The cAMP receptor proteins (CRPs) play a critical role in bacterial environmental adaptation by regulating global gene expression levels via cAMP binding. Here, we report the structure of DdrI, a CRP family protein from Deinococcus radiodurans. Combined with biochemical, kinetic, and molecular dynamics simulations analyses, our results indicate that DdrI adopts a DNA-binding conformation in the absence of cAMP and can form stable complexes with the target DNA sequence of classical CRPs. Further analysis revealed that the high-affinity cAMP binding pocket of DdrI is partially filled with Tyr113-Arg55-Glu65 sidechains, mimicking the anti-cAMP-mediated allosteric transition. Moreover, the second syn-cAMP binding site of DdrI at the protein-DNA interface is more negatively charged compared to that of classical CRPs, and manganese ions can enhance its DNA binding affinity. DdrI can also bind to a target sequence that mimics another transcription factor, DdrO, suggesting potential cross-talk between these two transcription factors. These findings reveal a class of CRPs that are independent of cAMP activation and provide valuable insights into the environmental adaptation mechanisms of D. radiodurans.IMPORTANCEBacteria need to respond to environmental changes at the gene transcriptional level, which is critical for their evolution, virulence, and industrial applications. The cAMP receptor protein (CRP) of Escherichia coli (ecCRP) senses changes in intracellular cAMP levels and is a classic example of allosteric effects in textbooks. However, the structures and biochemical activities of CRPs are not generally conserved and there exist different mechanisms. In this study, we found that the proposed CRP from Deinococcus radiodurans, DdrI, exhibited DNA binding ability independent of cAMP binding and adopted an apo structure resembling the activated CRP. Manganese can enhance the DNA binding of DdrI while allowing some degree of freedom for its target sequence. These results suggest that CRPs can evolve to become a class of cAMP-independent global regulators, enabling bacteria to adapt to different environments according to their characteristics. The first-discovered CRP family member, ecCRP (or CAP) may well not be typical of the family and be very different to the ancestral CRP-family transcription factor.
Asunto(s)
Proteínas Bacterianas , Proteína Receptora de AMP Cíclico , AMP Cíclico , Deinococcus , Unión Proteica , Deinococcus/genética , Deinococcus/metabolismo , Proteína Receptora de AMP Cíclico/metabolismo , Proteína Receptora de AMP Cíclico/genética , Proteína Receptora de AMP Cíclico/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , AMP Cíclico/metabolismo , Sitios de Unión , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Simulación de Dinámica Molecular , Conformación Proteica , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/química , Regulación Bacteriana de la Expresión GénicaRESUMEN
In this study, we utilize Protein Residue Networks (PRNs), constructed using Local Spatial Pattern (LSP) alignment, to explore the dynamic behavior of Catabolite Activator Protein (CAP) upon the sequential binding of cAMP. We employed the Degree Centrality of these PRNs to investigate protein dynamics on a sub-nanosecond time scale, hypothesizing that it would reflect changes in CAP's entropy related to its thermal motions. We show that the binding of the first cAMP led to an increase in stability in the Cyclic-Nucleotide Binding Domain A (CNBD-A) and destabilization in CNBD-B, agreeing with previous reports explaining the negative cooperativity of cAMP binding in terms of an entropy-driven allostery. LSP-based PRNs also allow for the study of Betweenness Centrality, another graph-theoretical characteristic of PRNs, providing insights into global residue connectivity within CAP. Using this approach, we were able to correctly identify amino acids that were shown to be critical in mediating allosteric interactions in CAP. The agreement between our studies and previous experimental reports validates our method, particularly with respect to the reliability of Degree Centrality as a proxy for entropy related to protein thermal dynamics. Because LSP-based PRNs can be easily extended to include dynamics of small organic molecules, polynucleotides, or other allosteric proteins, the methods presented here mark a significant advancement in the field, positioning them as vital tools for a fast, cost-effective, and accurate analysis of entropy-driven allostery and identification of allosteric hotspots.
Asunto(s)
Regulación Alostérica , Proteína Receptora de AMP Cíclico , Alineación de Secuencia , Proteína Receptora de AMP Cíclico/química , Entropía , Simulación de Dinámica Molecular , Unión Proteica , Reproducibilidad de los Resultados , Alineación de Secuencia/métodosRESUMEN
Cyclic AMP receptor proteins (CRPs) are important transcription regulators in many species. The prediction of CRP-binding sites was mainly based on position-weighted matrixes (PWMs). Traditional prediction methods only considered known binding motifs, and their ability to discover inflexible binding patterns was limited. Thus, a novel CRP-binding site prediction model called CRPBSFinder was developed in this research, which combined the hidden Markov model, knowledge-based PWMs and structure-based binding affinity matrixes. We trained this model using validated CRP-binding data from Escherichia coli and evaluated it with computational and experimental methods. The result shows that the model not only can provide higher prediction performance than a classic method but also quantitatively indicates the binding affinity of transcription factor binding sites by prediction scores. The prediction result included not only the most knowns regulated genes but also 1089 novel CRP-regulated genes. The major regulatory roles of CRPs were divided into four classes: carbohydrate metabolism, organic acid metabolism, nitrogen compound metabolism and cellular transport. Several novel functions were also discovered, including heterocycle metabolic and response to stimulus. Based on the functional similarity of homologous CRPs, we applied the model to 35 other species. The prediction tool and the prediction results are online and are available at: https://awi.cuhk.edu.cn/â¼CRPBSFinder.
Asunto(s)
Proteína Receptora de AMP Cíclico , Proteínas de Escherichia coli , Proteína Receptora de AMP Cíclico/genética , Proteína Receptora de AMP Cíclico/química , Proteína Receptora de AMP Cíclico/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Sitios de Unión/genética , Unión Proteica/genéticaRESUMEN
The family of coarse-grained models for protein dynamics known as Elastic Network Models (ENMs) require careful choice of parameters to represent well experimental measurements or fully-atomistic simulations. The most basic ENM that represents each protein residue by a node at the position of its C-alpha atom, all connected by springs of equal stiffness, up to a cut-off in distance. Even at this level a choice is required of the optimum cut-off distance and the upper limit of elastic normal modes taken in any sum for physical properties, such as dynamic correlation or allosteric effects on binding. Additionally, backbone-enhanced ENM (BENM) may improve the model by allocating a higher stiffness to springs that connect along the protein backbone. This work reports on the effect of varying these three parameters (distance and mode cutoffs, backbone stiffness) on the dynamical structure of three proteins, Catabolite Activator Protein (CAP), Glutathione S-transferase (GST), and the SARS-CoV-2 Main Protease (M pro ). Our main results are: (1) balancing B-factor and dispersion-relation predictions, a near-universal optimal value of 8.5 Å is advisable for ENMs; (2) inhomogeneity in elasticity brings the first mode containing spatial structure not well-resolved by the ENM typically within the first 20; (3) the BENM only affects modes in the upper third of the distribution, and, additionally to the ENM, is only able to model the dispersion curve better in this vicinity; (4) BENM does not typically affect fluctuation-allostery, which also requires careful treatment of the effector binding to the host protein to capture.
Asunto(s)
Proteasas 3C de Coronavirus , Proteína Receptora de AMP Cíclico , Glutatión Transferasa , Regulación Alostérica , Proteasas 3C de Coronavirus/química , Proteína Receptora de AMP Cíclico/química , Elasticidad , Glutatión Transferasa/química , Humanos , Simulación de Dinámica Molecular , Conformación ProteicaRESUMEN
The cyclic AMP receptor protein (CRP) is one of the best-known transcription factors, regulating about 400 genes. The histone-like nucleoid structuring protein (H-NS) is one of the nucleoid-forming proteins and is responsible for DNA packaging and gene repression in prokaryotes. In this study, the binding of ppGpp to CRP and H-NS was determined by fluorescence spectroscopy. CRP from Escherichia coli exhibited intrinsic fluorescence at 341 nm when excited at 280 nm. The fluorescence intensity decreased in the presence of ppGpp. The dissociation constant of 35 ± 3 µM suggests that ppGpp binds to CRP with a similar affinity to cAMP. H-NS also shows intrinsic fluorescence at 329 nm. The fluorescence intensity was decreased by various ligands and the calculated dissociation constant for ppGpp was 80 ± 11 µM, which suggests that the binding site was occupied fully by ppGpp under starvation conditions. This study suggests the modulatory effects of ppGpp in gene expression regulated by CRP and H-NS. The method described here may be applicable to many other proteins.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteína Receptora de AMP Cíclico/metabolismo , Proteínas de Unión al ADN/metabolismo , Guanosina Tetrafosfato/metabolismo , Proteínas Bacterianas/química , Sitios de Unión , AMP Cíclico/metabolismo , Proteína Receptora de AMP Cíclico/química , Proteínas de Unión al ADN/química , Escherichia coli , Espectrometría de FluorescenciaRESUMEN
We demonstrate here that the α subunit C-terminal domain of Escherichia coli RNA polymerase (αCTD) recognizes the upstream promoter (UP) DNA element via its characteristic minor groove shape and electrostatic potential. In two compositionally distinct crystallized assemblies, a pair of αCTD subunits bind in tandem to the UP element consensus A-tract that is 6 bp in length (A6-tract), each with their arginine 265 guanidinium group inserted into the minor groove. The A6-tract minor groove is significantly narrowed in these crystal structures, as well as in computationally predicted structures of free and bound DNA duplexes derived by Monte Carlo and molecular dynamics simulations, respectively. The negative electrostatic potential of free A6-tract DNA is substantially enhanced compared to that of generic DNA. Shortening the A-tract by 1 bp is shown to "knock out" binding of the second αCTD through widening of the minor groove. Furthermore, in computationally derived structures with arginine 265 mutated to alanine in either αCTD, either with or without the "knockout" DNA mutation, contact with the DNA is perturbed, highlighting the importance of arginine 265 in achieving αCTD-DNA binding. These results demonstrate that the importance of the DNA shape in sequence-dependent recognition of DNA by RNA polymerase is comparable to that of certain transcription factors.
Asunto(s)
ADN Bacteriano/química , ADN Bacteriano/metabolismo , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Proteína Receptora de AMP Cíclico/química , Proteína Receptora de AMP Cíclico/genética , Proteína Receptora de AMP Cíclico/metabolismo , ADN Bacteriano/genética , ARN Polimerasas Dirigidas por ADN/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Técnicas de Inactivación de Genes , Genes Bacterianos , Modelos Moleculares , Mutación , Conformación de Ácido Nucleico , Regiones Promotoras Genéticas , Dominios Proteicos , Electricidad EstáticaRESUMEN
Transcription activation by the Escherichia coli CRP at Class II promoters is dependent on direct interactions between RNA polymerase and CRP, therefore the spatial proximity between both proteins plays a significant role in the ability of CRP to activate transcription. Using both in vivo and in vitro techniques, here we demonstrate that the CRP K100 positive charge, adjacent to AR2, is required for full promoter activity when CRP is optimally positioned. Accordingly, K100 mediated activation is very position-dependent and our data confirm that the largest impact of the K100 status on transcription activation occurs when the spacing between the CRP binding site and the A2 of the -10 element is 22 bp. From the results of this study and the progress in the understanding about open complex DNA scrunching, we propose that CRP-dependent promoters should now be numbered by the distance from the center of the DNA site for CRP and the most highly conserved base at position 2 of the -10 hexamer in bacterial promoters.
Asunto(s)
Proteína Receptora de AMP Cíclico/genética , Escherichia coli/genética , Regiones Promotoras Genéticas/genética , Activación Transcripcional/genética , Proteína Receptora de AMP Cíclico/química , ARN Polimerasas Dirigidas por ADN/metabolismoRESUMEN
Transcription factors (TFs) have been extensively researched in certain well-studied organisms, but far less so in others. Following the whole-genome sequencing of a new organism, TFs are typically identified through their homology with related proteins in other organisms. However, recent findings demonstrate that structurally similar TFs from distantly related bacteria are not usually evolutionary orthologs. Here we explore TTHB099, a cAMP receptor protein (CRP)-family TF from the extremophile Thermus thermophilus HB8. Using the in vitro iterative selection method Restriction Endonuclease Protection, Selection and Amplification (REPSA), we identified the preferred DNA-binding motif for TTHB099, 5'-TGT(A/g)NBSYRSVN(T/c)ACA-3', and mapped potential binding sites and regulated genes within the T. thermophilus HB8 genome. Comparisons with expression profile data in TTHB099-deficient and wild type strains suggested that, unlike E. coli CRP (CRPEc), TTHB099 does not have a simple regulatory mechanism. However, we hypothesize that TTHB099 can be a dual-regulator similar to CRPEc.
Asunto(s)
Proteína Receptora de AMP Cíclico/química , Proteína Receptora de AMP Cíclico/metabolismo , ADN/metabolismo , Thermus thermophilus/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Proteína Receptora de AMP Cíclico/genética , ADN/química , Enzimas de Restricción del ADN/metabolismo , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica , Regiones Promotoras Genéticas , Unión Proteica , Homología de Secuencia de Aminoácido , Thermus thermophilus/genéticaRESUMEN
Transcription activation by cyclic AMP (cAMP) receptor protein (CAP) is the classic paradigm of transcription regulation in bacteria. CAP was suggested to activate transcription on class-II promoters via a recruitment and isomerization mechanism. However, whether and how it modifies RNA polymerase (RNAP) to initiate transcription remains unclear. Here, we report cryo-electron microscopy (cryo-EM) structures of an intact Escherichia coli class-II CAP-dependent transcription activation complex (CAP-TAC) with and without de novo RNA transcript. The structures reveal two distinct architectures of TAC and raise the possibility that CAP binding may induce substantial conformational changes in all the subunits of RNAP and transiently widen the main cleft of RNAP to facilitate DNA promoter entering and formation of the initiation open complex. These structural changes vanish during further RNA transcript synthesis. The observations in this study may reveal a possible on-pathway intermediate and suggest a possibility that CAP activates transcription by inducing intermediate state, in addition to the previously proposed stabilization mechanism.
Asunto(s)
Proteína Receptora de AMP Cíclico/química , Proteínas de Escherichia coli/química , Microscopía por Crioelectrón , Proteína Receptora de AMP Cíclico/metabolismo , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/metabolismo , Visualización de Datos , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Complejos Multiproteicos/química , Conformación Proteica , ARN/química , ARN/metabolismo , Factor sigma/química , Factor sigma/metabolismo , Transcripción GenéticaRESUMEN
The abundance of protein dimers and multidomain proteins is a testimony to their importance in various cellular functions. Several mechanisms exist, explaining how they assemble. The energy landscape theory has shown that, irrespective of the mechanism followed, folding and binding of dimers and multidomain proteins are funneled processes. Using a structure based model, we have characterized the folding landscape and dimerization mechanism of the DNA binding domain (DBD) in a complex multidomain, homodimeric transcription factor, catabolite activator protein (CAP). The DBD is tethered to the nucleotide binding domain (NBD) of CAP. Our investigation revealed that, as the tethered DBD of CAP transitions from an unfolded to the folded state, complementary folding and backtracking occur between the individual subunits within the DBD. This redistributes the entropies of the DBDs in both the subunits and might play a role in consequently modulating the free energy surface to reduce the entropic folding barrier. This redistribution of entropies forms the basis of an unusual intersubunit assisted folding mechanism whereby each subunit acts as a chaperone for the other. We have also investigated the effect of tethering on the folding landscape of DBD and found that the folding landscape can change depending on the tethering conditions.
Asunto(s)
Proteína Receptora de AMP Cíclico/química , ADN/química , Sitios de Unión , Simulación de Dinámica Molecular , Dominios Proteicos , Pliegue de ProteínaRESUMEN
The theory for allostery has evolved to a modern energy landscape ensemble theory, the major feature of which is the existence of multiple microstates in equilibrium. The properties of microstates are not well defined due to their transient nature. Characterization of apo protein microstates is important because the specific complex of the ligand-bound microstate defines the biological function. The information needed to link biological function and structure is a quantitative correlation of the energy landscapes between the apo and holo protein states. We employed the Escherichia coli cAMP receptor protein (CRP) system to test the features embedded in the ensemble theory because multiple crystalline apo and holo structures are available. Small angle X-ray scattering data eliminated one of the three apo states but not the other two. We defined the underlying energy landscape differences among the apo microstates by employing the computation algorithm COREX/BEST. The same connectivity patterns among residues in apo CRP are retained upon binding of cAMP. The microstates of apo CRP differ from one another by minor structural perturbations, resulting in changes in the energy landscapes of the various domains of CRP. Using the differences in energy landscapes among these apo states, we computed the cAMP binding energetics that were compared with solution biophysical results. Only one of the three apo microstates yielded data consistent with the solution data. The relative magnitude of changes in energy landscapes embedded in various apo microstates apparently defines the ultimate outcome of the cooperativity of binding.
Asunto(s)
Regulación Alostérica/fisiología , Proteína Receptora de AMP Cíclico/química , Biología Computacional/métodos , AMP Cíclico/metabolismo , Proteína Receptora de AMP Cíclico/metabolismo , Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Modelos Moleculares , Conformación Proteica , TermodinámicaRESUMEN
Cyclic AMP (cAMP) receptor protein (Crp) is an important transcriptional regulator of Yersinia pestis Expression of crp increases during pneumonic plague as the pathogen depletes glucose and forms large biofilms within lungs. To better understand control of Y. pestis Crp, we determined a 1.8-Å crystal structure of the protein-cAMP complex. We found that compared to Escherichia coli Crp, C helix amino acid substitutions in Y. pestis Crp did not impact the cAMP dependency of Crp to bind DNA promoters. To investigate Y. pestis Crp-regulated genes during plague pneumonia, we performed RNA sequencing on both wild-type and Δcrp mutant bacteria growing in planktonic and biofilm states in minimal media with glucose or glycerol. Y. pestis Crp was found to dramatically alter expression of hundreds of genes in a manner dependent upon carbon source and growth state. Gel shift assays confirmed direct regulation of the malT and ptsG promoters, and Crp was then linked to Y. pestis growth on maltose as a sole carbon source. Iron regulation genes ybtA and fyuA were found to be indirectly regulated by Crp. A new connection between carbon source and quorum sensing was revealed as Crp was found to regulate production of acyl-homoserine lactones (AHLs) through direct and indirect regulation of genes for AHL synthetases and receptors. AHLs were subsequently identified in the lungs of Y. pestis-infected mice when crp expression was highest in Y. pestis biofilms. Thus, in addition to the well-studied pla gene, other Crp-regulated genes likely have important functions during plague infection.IMPORTANCE Bacterial pathogens have evolved extensive signaling pathways to translate environmental signals into changes in gene expression. While Crp has long been appreciated for its role in regulating metabolism of carbon sources in many bacterial species, transcriptional profiling has revealed that this protein regulates many other aspects of bacterial physiology. The plague pathogen Y. pestis requires this global regulator to survive in blood, skin, and lungs. During disease progression, this organism adapts to changes within these niches. In addition to regulating genes for metabolism of nonglucose sugars, we found that Crp regulates genes for virulence, metal acquisition, and quorum sensing by direct or indirect mechanisms. Thus, this single transcriptional regulator, which responds to changes in available carbon sources, can regulate multiple critical behaviors for causing disease.
Asunto(s)
Biopelículas/crecimiento & desarrollo , Proteína Receptora de AMP Cíclico/genética , Regulación Bacteriana de la Expresión Génica , Plancton/crecimiento & desarrollo , Percepción de Quorum , Yersinia pestis/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biología Computacional/métodos , Proteína Receptora de AMP Cíclico/química , Proteína Receptora de AMP Cíclico/metabolismo , Ontología de Genes , Humanos , Modelos Biológicos , Modelos Moleculares , Peste/metabolismo , Peste/microbiología , Regiones Promotoras Genéticas , Conformación ProteicaRESUMEN
The Escherichia coli cAMP receptor protein, CRP, is a homodimeric global transcription activator that employs multiple mechanisms to modulate the expression of hundreds of genes. These mechanisms require different interfacial interactions among CRP, RNA, and DNA of varying sequences. The involvement of such a multiplicity of interfaces requires a tight control to ensure the desired phenotype. CRP-dependent promoters can be grouped into three classes. For decades scientists in the field have been puzzled over the differences in mechanisms between class I and II promoters. Using a new crystal structure, IR spectroscopy, and computational analysis, we defined the energy landscapes of WT and 14 mutated CRPs to determine how a homodimeric protein can distinguish nonpalindromic DNA sequences and facilitate communication between residues located in three different activation regions (AR) in CRP that are â¼30 Å apart. We showed that each mutation imparts differential effects on stability among the subunits and domains in CRP. Consequently, the energetic landscapes of subunits and domains are different, and CRP is asymmetric. Hence, the same mutation can exert different effects on ARs in class I or II promoters. The effect of a mutation is transmitted through a network by long-distance communication not necessarily relying on physical contacts between adjacent residues. The mechanism is simply the sum of the consequences of modulating the synchrony of dynamic motions of residues at a distance, leading to differential effects on ARs in different subunits. The computational analysis is applicable to any system and potentially with predictive capability.
Asunto(s)
Proteína Receptora de AMP Cíclico/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Regiones Promotoras Genéticas , Sitios de Unión , Proteína Receptora de AMP Cíclico/química , Proteína Receptora de AMP Cíclico/genética , Dimerización , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Dominios Proteicos , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismoRESUMEN
We present a rare comparison of structures of the same protein but generated by different potentials. We used four popular water potentials (SPC, TIP3P, TIP4P, TIP5P) in conjunction with the equally popular ff99SB. However, the ff12SB protein potential was used with TI3P only. Simulations (60 ns) were run on the catabolite activator protein (CAP), which is a textbook case of allosteric interaction. Overall, all potentials generated largely similar structures but failed to reproduce a crucial structural feature determined by NMR experiment. This example shows the need to develop next-generation potentials. Graphical abstract Catabolite activator protein.
Asunto(s)
Proteína Receptora de AMP Cíclico/química , Simulación de Dinámica Molecular , Agua/química , ADN/metabolismo , Difusión , Ligandos , Estructura Secundaria de Proteína , Subunidades de Proteína/química , Homología Estructural de Proteína , TermodinámicaRESUMEN
The transcription mechanism of genetic information from DNA to RNA is efficiently controlled by regulatory proteins, such as catabolite activator protein (CAP), and their ligands. When cyclic AMP (cAMP) binds to CAP, the complex forms a dimer and binds specifically to DNA to activate the transcription mechanism. On the other hand, when cyclic GMP (cGMP) binds to CAP, the complex has no marked effect on the mechanism. In our previous study, based on molecular dynamics (MD) and ab initio fragment molecular orbital (FMO) methods, we elucidated which residues of CAP are important for the specific interactions between CAP and DNA in the CAP-monomer+DNA + cAMP complex. However, this monomer model for CAP cannot describe real interactions between the CAP-dimer and DNA because CAPs form a dimer before binding to DNA. Accordingly, here, we investigated stable structures and their electronic states for the CAP-dimer+DNA complex with cAMP or cGMP ligand, to clarify the influence of ligand-binding on the interactions between CAP-dimer and DNA. The MD simulations elucidated that the DNA-binding domains of CAP-dimer behave differently depending on the ligand bound to the CAP-dimer. In addition, FMO calculations revealed that the binding energy between CAP-dimer and DNA for the CAP-dimer+DNA + cAMP complex is larger than that for the CAP-dimer+DNA + cGMP complex, being consistent with experiments. It was also highlighted that the Arg185 and Lys188 residues of CAP-dimer are important for the binding between CAP-dimer and DNA. These results provide useful information for proposing new compounds that efficiently control the transcription mechanism.
Asunto(s)
Proteína Receptora de AMP Cíclico/química , ADN/química , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Aminoácidos/química , Sitios de Unión , AMP Cíclico/química , Proteína Receptora de AMP Cíclico/metabolismo , ADN/metabolismo , Ligandos , Conformación Molecular , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de ProteínaRESUMEN
Proteins are dynamic molecules that undergo conformational changes to a broad spectrum of different excited states. Unfortunately, the small populations of these states make it difficult to determine their structures or functional implications. Computer simulations are an increasingly powerful means to identify and characterize functionally relevant excited states. However, this advance has uncovered a further challenge: it can be extremely difficult to identify the most salient features of large simulation data sets. We reasoned that many functionally relevant conformational changes are likely to involve large, cooperative changes to the surfaces that are available to interact with potential binding partners. To examine this hypothesis, we introduce a method that returns a prioritized list of potentially functional conformational changes by segmenting protein structures into clusters of residues that undergo cooperative changes in their solvent exposure, along with the hierarchy of interactions between these groups. We term these groups exposons to distinguish them from other types of clusters that arise in this analysis and others. We demonstrate, using three different model systems, that this method identifies experimentally validated and functionally relevant conformational changes, including conformational switches, allosteric coupling, and cryptic pockets. Our results suggest that key functional sites are hubs in the network of exposons. As a further test of the predictive power of this approach, we apply it to discover cryptic allosteric sites in two different ß-lactamase enzymes that are widespread sources of antibiotic resistance. Experimental tests confirm our predictions for both systems. Importantly, we provide the first evidence, to our knowledge, for a cryptic allosteric site in CTX-M-9 ß-lactamase. Experimentally testing this prediction did not require any mutations and revealed that this site exerts the most potent allosteric control over activity of any pockets found in ß-lactamases to date. Discovery of a similar pocket that was previously overlooked in the well-studied TEM-1 ß-lactamase demonstrates the utility of exposons.
Asunto(s)
Sitio Alostérico , Modelos Moleculares , Proteínas/química , Solventes/química , Proteína Receptora de AMP Cíclico/química , Proteínas de Escherichia coli/química , Conformación Proteica , beta-Lactamasas/químicaRESUMEN
Allosteric transcription factors undergo binding events at inducer binding sites as well as at distinct DNA binding domains, and it is difficult to disentangle the structural and functional consequences of these two classes of interactions. We compare the ability of two statistical mechanical models-the Monod-Wyman-Changeux (MWC) and the Koshland-Némethy-Filmer (KNF) models of protein conformational change-to characterize the multi-step activation mechanism of the broadly acting cyclic-AMP receptor protein (CRP). We first consider the allosteric transition resulting from cyclic-AMP binding to CRP, then analyze how CRP binds to its operator, and finally investigate the ability of CRP to activate gene expression. We use these models to examine a beautiful recent experiment that created a single-chain version of the CRP homodimer, creating six mutants using all possible combinations of the wild type, D53H, and S62F subunits. We demonstrate that the MWC model can explain the behavior of all six mutants using a small, self-consistent set of parameters whose complexity scales with the number of subunits, providing a significant benefit over previous models. In comparison, the KNF model not only leads to a poorer characterization of the available data but also fails to generate parameter values in line with the available structural knowledge of CRP. In addition, we discuss how the conceptual framework developed here for CRP enables us to not merely analyze data retrospectively, but has the predictive power to determine how combinations of mutations will interact, how double mutants will behave, and how each construct would regulate gene expression.
Asunto(s)
Proteína Receptora de AMP Cíclico/genética , Proteína Receptora de AMP Cíclico/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Mutación , Algoritmos , Regulación Alostérica , Sitios de Unión , Proteína Receptora de AMP Cíclico/química , Escherichia coli/genética , Proteínas de Escherichia coli/química , Regulación Bacteriana de la Expresión Génica , Modelos Estadísticos , Conformación Proteica , Dominios ProteicosRESUMEN
ZnO quantum dots (QDs) are very well known for their antimicrobial activity against several bacteria, however, we still do not know any protein targets of ZnO QDs. In order to determine possible protein target, interaction of ZnO QDs was studied with CRP (Cyclic AMP Receptor Protein), a global transcription regulator protein. Binding between ZnO QDs and E. coli CRP was mainly studied by isothermal titration calorimetry (ITC), structural changes of protein were monitored by fluorescence and circular dichroism spectroscopy, and in-vitro transcription assay was used to asses CRP activity. Result shows that both electrostatic and hydrophobic interactions are involved in CRP-ZnO binding. Different spectroscopic investigation revealed that ZnO binding to CRP leads to extensive unfolding and destabilization, which ultimately leads to protein aggregation. It was also observed that in presence of ZnO dimerization ability of CRP was sharply reduced. In-vitro transcription assay also shows that CRP activity gets severely compromised on ZnO binding. All our data suggests that ZnO QD binding to CRP and consequent structural and functional changes most probably plays a crucial role in ZnO QD induced antimicrobial action.
Asunto(s)
Antibacterianos/química , Antibacterianos/farmacología , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Puntos Cuánticos/química , Transcriptoma , Óxido de Zinc/química , Sitios de Unión , Dicroismo Circular , Proteína Receptora de AMP Cíclico/química , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Unión Proteica , Conformación Proteica , Estabilidad Proteica , Desplegamiento Proteico , Relación Estructura-Actividad , Termodinámica , Activación TranscripcionalRESUMEN
In bacteria, the activation of gene transcription at many promoters is simple and only involves a single activator. The cyclic adenosine 3',5'-monophosphate receptor protein (CAP), a classic activator, is able to activate transcription independently through two different mechanisms. Understanding the class I mechanism requires an intact transcription activation complex (TAC) structure at a high resolution. Here we report a high-resolution cryo-electron microscopy structure of an intact Escherichia coli class I TAC containing a CAP dimer, a σ70-RNA polymerase (RNAP) holoenzyme, a complete class I CAP-dependent promoter DNA, and a de novo synthesized RNA oligonucleotide. The structure shows how CAP wraps the upstream DNA and how the interactions recruit RNAP. Our study provides a structural basis for understanding how activators activate transcription through the class I recruitment mechanism.
Asunto(s)
Proteína Receptora de AMP Cíclico/química , ARN Polimerasas Dirigidas por ADN/química , Proteínas de Escherichia coli/química , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Factor sigma/química , Activación Transcripcional , Microscopía por Crioelectrón , Proteína Receptora de AMP Cíclico/ultraestructura , ADN Bacteriano/química , ADN Bacteriano/ultraestructura , ARN Polimerasas Dirigidas por ADN/ultraestructura , Proteínas de Escherichia coli/ultraestructura , Regiones Promotoras Genéticas , Factor sigma/ultraestructuraRESUMEN
The advent of Chromatin Immunoprecipitation sequencing (ChIP-Seq) has allowed the identification of genomic regions bound by a DNA binding protein in-vivo on a genome-wide scale. The impact of the DNA binding protein on gene expression can be addressed using transcriptome experiments in appropriate genetic settings. Overlaying the above two sources of data enables us to dissect the direct and indirect effects of a DNA binding protein on gene expression. Application of these techniques to Nucleoid Associated Proteins (NAPs) and Global Transcription Factors (GTFs) has underscored the complex relationship between DNA-protein interactions and gene expression change, highlighting the role of combinatorial control. Here, we demonstrate the usage of ChIP-Seq to infer binding properties and transcriptional effects of NAPs such as Fis and HNS, and the GTF CRP in the model organism Escherichia coli K12 MG1655 (E. coli).