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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., Competing Interests: The authors declare no conflict of interest.

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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/∼CRPBSFinder., (© The Author(s) 2023. Published by Oxford University Press.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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.

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 (A 6 -tract), each with their arginine 265 guanidinium group inserted into the minor groove. The A 6 -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 A 6 -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.

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., (© The Author(s) 2020. Published by Oxford University Press on behalf of FEMS.)

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 (CRP Ec ), TTHB099 does not have a simple regulatory mechanism. However, we hypothesize that TTHB099 can be a dual-regulator similar to CRP Ec .

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., Competing Interests: The authors have declared that no competing interests exist.

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.

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.

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 Δ crp 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., (Copyright © 2019 Ritzert et al.)

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 ∼30 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., (© 2019 Evangelista et al.)

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.

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.

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 β-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 β-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 β-lactamases to date. Discovery of a similar pocket that was previously overlooked in the well-studied TEM-1 β-lactamase demonstrates the utility of exposons., (Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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., Competing Interests: The authors have declared that no competing interests exist.

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 σ 70 -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., (Copyright © 2017, American Association for the Advancement of Science.)

Cyclic adenosine monophosphate (cAMP) plays a regulatory role as second messenger in many species. In the industrial model organism Corynebacterium glutamicum, cAMP acts as effector of the global transcriptional regulator GlxR, a homolog of enterobacterial Crp. The cAMP-GlxR complex activates or represses the expression of about 200 target genes. CyaB, a membrane-bound class III adenylate cyclase, synthesizes cAMP from ATP, but another yet unknown cAMP-forming enzyme is likely present in C. glutamicum. Recently, we identified the cAMP phosphodiesterase CpdA, which catalyzes the conversion of cAMP to AMP. As a tool to search for additional cAMP-forming and degrading enzymes, we constructed a plasmid-based cAMP biosensor by fusing the promoter of cg3195, a gene strongly repressed by GlxR, to the eyfp reporter gene. In control experiments, the biosensor showed the predicted responses to increased levels of cAMP or GlxR. The biosensor was able to distinguish between C. glutamicum wild type and mutants with defects in cAMP biosynthesis or degradation. Most importantly, the sensor allowed successful sorting of mixtures of wild type and mutant strains by fluorescence activated cell sorting (FACS), thus meeting the requirements for high-throughput screening of libraries for single mutant cells with an altered cAMP level., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Many allosteric proteins form homo-oligomeric complexes to regulate a biological function. In homo-oligomers, subunits establish communication pathways that are modulated by external stimuli like ligand binding. A challenge for dissecting the communication mechanisms in homo-oligomers is identifying intermediate liganded states, which are typically transiently populated. However, their identities provide the most mechanistic information on how ligand-induced signals propagate from bound to empty subunits. Here, we dissected the directionality and magnitude of subunit communication in a reengineered single-chain version of the homodimeric transcription factor cAMP receptor protein. By combining wild-type and mutant subunits in various asymmetric configurations, we revealed a linear relationship between the magnitude of cooperative effects and the number of mutant subunits. We found that a single mutation is sufficient to change the global allosteric behavior of the dimer even when one subunit was wild type. Dimers harboring two mutations with opposite cooperative effects had different allosteric properties depending on the arrangement of the mutations. When the two mutations were placed in the same subunit, the resulting cooperativity was neutral. In contrast, when placed in different subunits, the observed cooperativity was dominated by the mutation with strongest effects over cAMP affinity relative to wild type. These results highlight the distinct roles of intrasubunit interactions and intersubunit communication in allostery. Finally, dimers bound to either one or two cAMP molecules had similar DNA affinities, indicating that both asymmetric and symmetric liganded states activate DNA interactions. These studies have revealed the multiple communication pathways that homo-oligomers employ to transduce signals., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Although cyclic AMP receptor protein (CRP) has long served as a typical example of effector-mediated protein allostery, mechanistic details into its regulation have been controversial due to discrepancy between the known crystal structure and NMR structure of apo-CRP. Here, we report that the recombinant protein corresponding to its C-terminal DNA-binding domain (CDD) forms a dimer. This result, together with structural information obtained in the present NMR study, is consistent with the previous crystal structure and validates its relevance also in solution. Therefore, our findings suggest that dissociation of the CDD may be critically involved in cAMP-induced allosteric activation of CRP., (© 2017 Federation of European Biochemical Societies.)

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).

Class II transcription activators function by binding to a DNA site overlapping a core promoter and stimulating isomerization of an initial RNA polymerase (RNAP)-promoter closed complex into a catalytically competent RNAP-promoter open complex. Here, we report a 4.4 angstrom crystal structure of an intact bacterial class II transcription activation complex. The structure comprises Thermus thermophilus transcription activator protein TTHB099 (TAP) [homolog of Escherichia coli catabolite activator protein (CAP)], T. thermophilus RNAP σ(A) holoenzyme, a class II TAP-dependent promoter, and a ribotetranucleotide primer. The structure reveals the interactions between RNAP holoenzyme and DNA responsible for transcription initiation and reveals the interactions between TAP and RNAP holoenzyme responsible for transcription activation. The structure indicates that TAP stimulates isomerization through simple, adhesive, stabilizing protein-protein interactions with RNAP holoenzyme., (Copyright © 2016, American Association for the Advancement of Science.)

The cyclic AMP receptor protein family of transcription factors regulates various metabolic pathways in bacteria, and also play roles in response to environmental changes. Here, we identify four homologs of the CRP family in Deinococcus radiodurans, one of which tolerates extremely high levels of oxidative stress and DNA-damaging reagents. Transcriptional levels of CRP were increased under hydrogen peroxide (H2O2) treatment during the stationary growth phase, indicating that CRPs function in response to oxidative stress. By constructing all CRP single knockout mutants, we found that the dr0997 mutant showed the lowest tolerance toward H2O2, ultraviolet radiation, ionizing radiation, and mitomycin C, while the phenotypes of the dr2362, dr0834, and dr1646 mutants showed slight or no significant differences from those of the wild-type strain. Taking advantage of the conservation of the CRP-binding site in many bacteria, we found that transcription of 18 genes, including genes encoding chromosome-partitioning protein (dr0998), Lon proteases (dr0349 and dr1974), NADH-quinone oxidoreductase (dr1506), thiosulfate sulfurtransferase (dr2531), the DNA repair protein UvsE (dr1819), PprA (dra0346), and RecN (dr1447), are directly regulated by DR0997. Quantitative real-time polymerase chain reaction (qRT-PCR) analyses showed that certain genes involved in anti-oxidative responses, DNA repair, and various cellular pathways are transcriptionally attenuated in the dr0997 mutant. Interestingly, DR0997 also regulate the transcriptional levels of all CRP genes in this bacterium. These data suggest that DR0997 contributes to the extreme stress resistance of D. radiodurans via its regulatory role in multiple cellular pathways, such as anti-oxidation and DNA repair pathways.

The Escherichia coli cAMP receptor protein (CRP) requires cAMP binding to undergo a conformational change for DNA binding and transcriptional regulation. Two CRP residues, Thr(127) and Ser(128), are known to play important roles in cAMP binding through hydrogen bonding and in the cAMP-induced conformational change, but the connection between the two is not completely clear. Here, we simultaneously randomized the codons for these two residues and selected CRP mutants displaying high CRP activity in a cAMP-producing E. coli. Many different CRP mutants satisfied the screening condition for high CRP activity, including those that cannot form any hydrogen bonds with the incoming cAMP at the two positions. In vitro DNA-binding analysis confirmed that these selected CRP mutants indeed display high CRP activity in response to cAMP. These results indicate that the hydrogen bonding ability of the Thr(127) and Ser(128) residues is not critical for the cAMP-induced CRP activation. However, the hydrogen bonding ability of Thr(127) and Ser(128) was found to be important in attaining high cAMP affinity. Computational analysis revealed that most natural cAMP-sensing CRP homologs have Thr/Ser, Thr/Thr, or Thr/Asn at positions 127 and 128. All of these pairs are excellent hydrogen bonding partners and they do not elevate CRP activity in the absence of cAMP. Taken together, our analyses suggest that CRP evolved to have hydrogen bonding residues at the cAMP pocket residues 127 and 128 for performing dual functions: preserving high cAMP affinity and keeping CRP inactive in the absence of cAMP., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Allostery is a fundamental process by which ligand binding to a protein alters its activity at a distant site. Both experimental and theoretical evidence demonstrate that allostery can be communicated through altered slow relaxation protein dynamics without conformational change. The catabolite activator protein (CAP) of Escherichia coli is an exemplar for the analysis of such entropically driven allostery. Negative allostery in CAP occurs between identical cAMP binding sites. Changes to the cAMP-binding pocket can therefore impact the allosteric properties of CAP. Here we demonstrate, through a combination of coarse-grained modeling, isothermal calorimetry, and structural analysis, that decreasing the affinity of CAP for cAMP enhances negative cooperativity through an entropic penalty for ligand binding. The use of variant cAMP ligands indicates the data are not explained by structural heterogeneity between protein mutants. We observe computationally that altered interaction strength between CAP and cAMP variously modifies the change in allosteric cooperativity due to second site CAP mutations. As the degree of correlated motion between the cAMP-contacting site and a second site on CAP increases, there is a tendency for computed double mutations at these sites to drive CAP toward noncooperativity. Naturally occurring pairs of covarying residues in CAP do not display this tendency, suggesting a selection pressure to fine tune allostery on changes to the CAP ligand-binding pocket without a drive to a noncooperative state. In general, we hypothesize an evolutionary selection pressure to retain slow relaxation dynamics-induced allostery in proteins in which evolution of the ligand-binding site is occurring., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

The Catabolite Activator Protein (CAP) is a showcase example for entropic allostery. For full activation and DNA binding, the homodimeric protein requires the binding of two cyclic AMP (cAMP) molecules in an anti-cooperative manner, the source of which appears to be largely of entropic nature according to previous experimental studies. We here study at atomic detail the allosteric regulation of CAP with Molecular dynamics (MD) simulations. We recover the experimentally observed entropic penalty for the second cAMP binding event with our recently developed force covariance entropy estimator and reveal allosteric communication pathways with Force Distribution Analyses (FDA). Our observations show that CAP binding results in characteristic changes in the interaction pathways connecting the two cAMP allosteric binding sites with each other, as well as with the DNA binding domains. We identified crucial relays in the mostly symmetric allosteric activation network, and suggest point mutants to test this mechanism. Our study suggests inter-residue forces, as opposed to coordinates, as a highly sensitive measure for structural adaptations that, even though minute, can very effectively propagate allosteric signals.

Mycobacterium smegmatis is a fast-growing, saprophytic, mycobacterial species that contains two cAMP-receptor protein (CRP) homologues designated herein as Crp1 and Crp2. Phylogenetic analysis suggests that Crp1 (Msmeg_0539) is uniquely present in fast-growing environmental mycobacteria, whereas Crp2 (Msmeg_6189) occurs in both fast- and slow-growing species. A crp1 mutant of M. smegmatis was readily obtained, but crp2 could not be deleted, suggesting it was essential for growth. A total of 239 genes were differentially regulated in response to crp1 deletion (loss of function), including genes coding for mycobacterial energy generation, solute transport and catabolism of carbon sources. To assess the role of Crp2 in M. smegmatis, the crp2 gene was overexpressed (gain of function) and transcriptional profiling studies revealed that 58 genes were differentially regulated. Identification of the CRP promoter consensus in M. smegmatis showed that both Crp1 and Crp2 recognized the same consensus sequence (TGTGN8CACA). Comparison of the Crp1- and Crp2-regulated genes revealed distinct but overlapping regulons with 11 genes in common, including those of the succinate dehydrogenase operon (MSMEG_0417-0420, sdh1). Expression of the sdh1 operon was negatively regulated by Crp1 and positively regulated by Crp2. Electrophoretic mobility shift assays with purified Crp1 and Crp2 demonstrated that Crp1 binding to the sdh1 promoter was cAMP-independent whereas Crp2 binding was cAMP-dependent. These data suggest that Crp1 and Crp2 respond to distinct signalling pathways in M. smegmatis to coordinate gene expression in response to carbon and energy supply., (© 2015 The Authors.)

Pet is a cytotoxic autotransporter protein secreted by the pathogenic enteroaggregative Escherichia coli strain 042. Expression of Pet is co-dependent on two global transcription regulators: CRP (cyclic AMP receptor protein) and Fis (factor for inversion stimulation). At the pet promoter CRP binds to a single site centred at position -40.5 upstream of the start site for transcription. Due to the suboptimal positioning of this site, CRP alone activates transcription poorly and requires Fis to bind upstream to promote full activation. Here, we show that CRP and Fis control the expression of other important autotransporter toxins, namely Sat from uropathogenic E. coli (UPEC) and SigA from Shigella sonnei, and that this regulation has been conserved in different pathogens. Furthermore, we investigate the mechanism of Fis-mediated co-activation, exploiting a series of semi-synthetic promoters, with similar architecture to the pet promoter. We show that, when bound at position -40.5, CRP recruits RNA polymerase inefficiently and that Fis compensates by aiding polymerase recruitment through a direct protein-protein interaction. We demonstrate that other suitably positioned upstream transcription factors, which directly recruit RNA polymerase, can also compensate for the inappropriate positioning of CRP. We propose that this is a simple 'shared-recruitment' mechanism, by which co-dependence of promoters on two transcription factors could evolve.

The cyclic AMP receptor protein (CRP) family of transcription factors consists of global regulators of bacterial gene expression. Here, we identify two paralogous CRPs in the genome of Mycobacterium smegmatis that have 78% identical sequences and characterize them biochemically and functionally. The two proteins (MSMEG_0539 and MSMEG_6189) show differences in cAMP binding affinity, trypsin sensitivity, and binding to a CRP site that we have identified upstream of the msmeg_3781 gene. MSMEG_6189 binds to the CRP site readily in the absence of cAMP, while MSMEG_0539 binds in the presence of cAMP, albeit weakly. msmeg_6189 appears to be an essential gene, while the Δmsmeg_0539 strain was readily obtained. Using promoter-reporter constructs, we show that msmeg_3781 is regulated by CRP binding, and its transcription is repressed by MSMEG_6189. Our results are the first to characterize two paralogous and functional CRPs in a single bacterial genome. This gene duplication event has subsequently led to the evolution of two proteins whose biochemical differences translate to differential gene regulation, thus catering to the specific needs of the organism.

The understanding of how the architecture of cis-regulatory elements at bacterial promoters determines their final output is of central interest in modern biology. In this work, we attempt to gain insight into this process by analyzing complex promoter architectures in the model organism Escherichia coli. By focusing on the relationship between different TFs at the genomic scale in terms of their binding site arrangement and their effect on the target promoters, we found no strong constraint limiting the combinatorial assembly of TF pairs in E. coli. More strikingly, overlapping binding sites were found equally associated with both equivalent (both TFs have the same effect on the promoter) and opposite (one TF activates while the other repress the promoter) effects on gene expression. With this information on hand, we set an in silico approach to design overlapping sites for three global regulators (GRs) of E. coli, specifically CRP, Fis, and IHF. Using random sequence assembly and an evolutionary algorithm, we were able to identify potential overlapping operators for all TF pairs. In order to validate our prediction, we constructed two lac promoter variants containing overlapping sites for CRP and IHF designed in silico. By assaying the synthetic promoters using a GFP reporter system, we demonstrated that these variants were functional and activated by CRP and IHF in vivo. Taken together, presented results add new information on the mechanisms of signal integration in bacterial promoters and provide new strategies for the engineering of synthetic regulatory circuits in bacteria.

Surface plasmon resonance monitoring of the binding of transcription factors cAMP receptor protein (CRP) and nitrogen control factor of cyanobacteria (NtcA) from Synechocystis sp. PCC6803 to promoter fragments of glnA, glnN (NtcA regulon) and cccS (CRP regulon), revealed exclusive CRP binding to cccS, whereas NtcA was bound to all three promoters with different affinities, which were strongly increased by the NtcA activator 2-oxoglutarate. Effective NtcA affinity for 2-oxoglutarate varied with the promoter. High-affinity promoters and the NtcA-coactivating protein PII-interacting protein X (PipX) increased NtcA affinity towards 2-oxoglutarate, suggesting PipX-stabilization of the 2-oxoglutarate-bound NtcA conformation. PipX binding to NtcA required 2-oxoglutarate and was much tighter (Kd≈85 nM) than to the PipX-sequestering PII protein. NtcA appears to require more strongly PipX and 2-oxoglutarate (2OG) for estimulating gene expression at promoters having "imperfect" NtcA binding sites., (Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

The crystal structure of cyclic AMP-catabolite activator protein (CAP) from Escherichia coli containing cobalt(II) chloride and ammonium sulfate is reported at 1.97 Å resolution. Each of the two CAP subunits in the asymmetric unit binds one cobalt(II) ion, in each case coordinated by N-terminal domain residues His19, His21 and Glu96 plus an additional acidic residue contributed via a crystal contact. The three identified N-terminal domain cobalt-binding residues are part of a region of CAP that is important for transcription activation at class II CAP-dependent promoters. Sulfate anions mediate additional crystal lattice contacts and occupy sites corresponding to DNA backbone phosphate positions in CAP-DNA complex structures.

The Escherichia coli cyclic AMP receptor protein (CRP) is a prokaryotic global transcription activator protein that controls the expression of many different genes. Wild-type CRP can bind to special DNA sequences in the presence of cAMP. The substitution of Asp53 by His results in the CRP* phenotype, which does not require exogenous cAMP. In the present study, the D53H CRP mutant was overexpressed, purified and crystallized. cAMP-free D53H CRP crystals were obtained and diffracted to a resolution of 2.9 Å. Based on the systematic absences of the crystals, the space group is likely to be P2(1)2(1)2(1), with unit-cell parameters a = 76.66, b = 152.14, c = 176.11 Å. The asymmetric unit was confirmed to contain four protein dimers, with a Matthews coefficient of 2.71 Å(3) Da(-1) and a solvent content of 54.68%.

The ability to inhibit binding or enzymatic activity is key to preventing aberrant behaviors of proteins. Allosteric inhibition is desirable as it offers several advantages over competitive inhibition, but the mechanisms of action remain poorly understood in most cases. Here we show that allosteric inhibition can be effected by destabilizing a low-populated conformational state that serves as an on-pathway intermediate for ligand binding, without altering the protein's ground-state structure. As standard structural approaches are typically concerned with changes in the ground-state structure of proteins, the presence of such a mechanism can go easily undetected. Our data strongly argue for the routine use of NMR tools suited to detect and characterize transiently formed conformational states in allosteric systems. Structure information on such important intermediates can ultimately result in more efficient design of allosteric inhibitors.

The Escherichia coli cyclic AMP receptor protein (CRP) is a well known transcription activator protein. In this study, CRP was overexpressed, purified and cocrystallized with cAMP and a 38 bp full-length double-stranded DNA fragment. The full-length segment differed from the half-site fragments used in previous crystallization experiments and is more similar to the environment in vivo. CRP-cAMP-DNA crystals were obtained and diffracted to 2.9 Å resolution. The crystals belonged to space group P3121, with unit-cell parameters a = b = 76.03, c = 144.00 Å. The asymmetric unit was found to contain one protein molecule and half a 38 bp full-length double-stranded DNA fragment, with a Matthews coefficient of 2.62 Å(3) Da(-1) and a solvent content of 53.14%.

Escherichia coli cyclic AMP Receptor Protein (CRP) undergoes conformational changes with cAMP binding and allosterically promotes CRP to bind specifically to the DNA. In that, the structural and dynamic properties of apo CRP prior to cAMP binding are of interest for the comprehension of the activation mechanism. Here, the dynamics of apo CRP monomer/dimer and holo CRP dimer were studied by Molecular Dynamics (MD) simulations and Gaussian Network Model (GNM). The interplay of the inter-domain hinge with the cAMP and DNA binding domains are pre-disposed in the apo state as a conformational switch in the CRP's allosteric communication mechanism. The hinge at L134-D138 displaying intra- and inter-subunit coupled fluctuations with the cAMP and DNA binding domains leads to the emergence of stronger coupled fluctuations between the two domains and describes an on state. The flexible regions at K52-E58, P154/D155 and I175 maintain the dynamic coupling of the two domains. With a shift in the inter-domain hinge position towards the N terminus, nevertheless, the latter correlations between the domains loosen and become disordered; L134-D138 dynamically interacts only with the cAMP and DNA binding domains of its own subunit, and an off state is assumed. We present a mechanistic view on how the structural dynamic units are hierarchically built for the allosteric functional mechanism; from apo CRP monomer to apo-to-holo CRP dimers.

Transduction of biological signals at the molecular level involves the activation and/or inhibition of allosteric proteins. In the transcription factor cAMP receptor protein (CRP) from Escherichia coli, the allosteric activation, or apo-holo transition, involves rigid body motions of domains and structural rearrangements within the hinge region connecting the cAMP- and DNA-binding domains. During this apo-holo transition, residue 138 is converted as part of the elongated D-helix to the position of the N-terminal capping residue of a shorter D-helix. The goal of the current study is to elucidate the role of residue 138 in modulating the allostery between cAMP and DNA binding. By systematically mutating residue 138, we found that mutants with higher N-terminal capping propensities lead to increased cooperativity of cAMP binding and a concomitant increase in affinity for lac-DNA. Furthermore, mutants with higher N-terminal capping propensity correlate with properties characteristic of holo-CRP, particularly, increase in protein structural dynamics. Overall, our results provide a quantitative characterization of the role of residue 138 in the isomerization equilibrium between the apo and holo forms of CRP, and in turn the thermodynamic underpin to the molecular model of allostery revealed by the high resolution structural studies.

How the interplay between protein structure and internal dynamics regulates protein function is poorly understood. Often, ligand binding, post-translational modifications and mutations modify protein activity in a manner that is not possible to rationalize solely on the basis of structural data. It is likely that changes in the internal motions of proteins have a major role in regulating protein activity, but the nature of their contributions remains elusive, especially in quantitative terms. Here we show that changes in conformational entropy can determine whether protein-ligand interactions will occur, even among protein complexes with identical binding interfaces. We have used NMR spectroscopy to determine the changes in structure and internal dynamics that are elicited by the binding of DNA to several variants of the catabolite activator protein (CAP) that differentially populate the inactive and active DNA-binding domain states. We found that the CAP variants have markedly different affinities for DNA, despite the CAP−DNA-binding interfaces being essentially identical in the various complexes. Combined with thermodynamic data, the results show that conformational entropy changes can inhibit the binding of CAP variants that are structurally poised for optimal DNA binding or can stimulate the binding activity of CAP variants that only transiently populate the DNA-binding-domain active state. Collectively, the data show how changes in fast internal dynamics (conformational entropy) and slow internal dynamics (energetically excited conformational states) can regulate binding activity in a way that cannot be predicted on the basis of the protein's ground-state structure.

Allostery is a fundamental process by which distant sites within a protein system sense each other. Allosteric regulation is such an efficient mechanism that it is used to control protein activity in most biological processes, including signal transduction, metabolism, catalysis, and gene regulation. Over recent years, our view and understanding of the fundamental principles underlying allostery have been enriched and often utterly reshaped. This has been especially so for powerful techniques such as nuclear magnetic resonance spectroscopy, which offers an atomic view of the intrinsic motions of proteins. Here, I discuss recent results on the catabolite activator protein (CAP) that have drastically revised our view about how allosteric interactions are modulated. CAP has provided the first experimentally identified system showing that (i) allostery can be mediated through changes in protein motions, in the absence of changes in the mean structure of the protein, and (ii) favorable changes in protein motions may activate allosteric proteins that are otherwise structurally inactive., (© 2012 New York Academy of Sciences.)

By binding to short and highly conserved DNA sequences in genomes, DNA-binding proteins initiate, enhance or repress biological processes. Accurately identifying such binding sites, often represented by position weight matrices (PWMs), is an important step in understanding the control mechanisms of cells. When given coordinates of a DNA-binding domain (DBD) bound with DNA, a potential function can be used to estimate the change of binding affinity after base substitutions, where the changes can be summarized as a PWM. This technique provides an effective alternative when the chromatin immunoprecipitation data are unavailable for PWM inference. To facilitate the procedure of predicting PWMs based on protein-DNA complexes or even structures of the unbound state, the web server, DBD2BS, is presented in this study. The DBD2BS uses an atom-level knowledge-based potential function to predict PWMs characterizing the sequences to which the query DBD structure can bind. For unbound queries, a list of 1066 DBD-DNA complexes (including 1813 protein chains) is compiled for use as templates for synthesizing bound structures. The DBD2BS provides users with an easy-to-use interface for visualizing the PWMs predicted based on different templates and the spatial relationships of the query protein, the DBDs and the DNAs. The DBD2BS is the first attempt to predict PWMs of DBDs from unbound structures rather than from bound ones. This approach increases the number of existing protein structures that can be exploited when analyzing protein-DNA interactions. In a recent study, the authors showed that the kernel adopted by the DBD2BS can generate PWMs consistent with those obtained from the experimental data. The use of DBD2BS to predict PWMs can be incorporated with sequence-based methods to discover binding sites in genome-wide studies. Available at: http://dbd2bs.csie.ntu.edu.tw/, http://dbd2bs.csbb.ntu.edu.tw/, and http://dbd2bs.ee.ncku.edu.tw.

Lac operon transcription activation through CRP dimer depends on cAMP second messenger. The formation of CRP homodimers is mediated by protein-protein interactions between the monomers. Cyclic AMP ligand binding brings CRP dimer to an active state via conformational changes. Molecular modeling studies in our lab showed the importance of monomer in transcription activation through its pre-existing conformational state. Until now CRP experiments were carried out at protein concentrations higher than that of CRP dimer dissociation value making all CRP monomers dimer and ignore the importance of CRP monomer in allosteric activation. Labeling CRP monomers with fluorophores exterminate using excess protein concentration and allow monitoring CRP monomer behavior. CRP monomer exchange accelerates in the presence of non specific DNA whereas the exchange is inhibited in the presence of specific DNA and cAMP ligand. Degree of subunit exchange depends on the stability of CRP dimer. Cyclic AMP forms a single molecule from two monomers and addition of specific DNA further stabilizes CRP dimer and decreases monomer exchange. On the other hand, addition of non specific DNA increases CRP monomer exchange and may explain the mechanism of CRP monomer removal and dissociation of CRP dimer:cAMP:DNA complex. The exchange behavior of CRP in the presence of different factors implies importance of monomer in transcription complex association and dissociation.

A "bendy" protein sensor: A DNA-based sensor that uses folded DNA (through DNA kinks) and protein-induced bending to detect DNA-binding proteins is presented. Single-molecule sensing of a transcriptional activator (catabolite activator protein, CAP, which bends its DNA site by 80°) is demonstrated in solution and on surfaces, both in buffers and in cell lysates. The method should allow detection of a wide range of DNA-bending proteins., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The cAMP receptor protein (CRP) requires cAMP for an allosteric change and regulates more than 150 genes in Escherichia coli. In this study, the modular half of cAMP receptor protein was used to investigate the allosteric signal transmission pathway induced by cAMP binding. The activation of CRP upon cAMP binding is indicated to be realignment of the two subunits within the CRP dimer. The interaction of loop 3 and Phe136 do not involve in signal transmission., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Flexibility is critical for a folded protein to bind to other molecules (ligands) and achieve its functions. The conformational selection theory suggests that a folded protein deforms continuously and its ligand selects the most favorable conformations to bind to. Therefore, one of the best options to study protein-ligand binding is to sample conformations broadly distributed over the protein-folded state. This article presents a new sampler, called kino-geometric sampler (KGS). This sampler encodes dominant energy terms implicitly by simple kinematic and geometric constraints. Two key technical contributions of KGS are (1) a robotics-inspired Jacobian-based method to simultaneously deform a large number of interdependent kinematic cycles without any significant break-up of the closure constraints, and (2) a diffusive strategy to generate conformation distributions that diffuse quickly throughout the protein folded state. Experiments on four very different test proteins demonstrate that KGS can efficiently compute distributions containing conformations close to target (e.g., functional) conformations. These targets are not given to KGS, hence are not used to bias the sampling process. In particular, for a lysine-binding protein, KGS was able to sample conformations in both the intermediate and functional states without the ligand, while previous work using molecular dynamics simulation had required the ligand to be taken into account in the potential function. Overall, KGS demonstrates that kino-geometric constraints characterize the folded subset of a protein conformation space and that this subset is small enough to be approximated by a relatively small distribution of conformations., (Copyright © 2011 Wiley Periodicals, Inc.)

A recurrent theme of many structural studies of homo-oligomeric protein systems is concerned with verification that the conformation observed in a crystal represents the functionally relevant structure. An asymmetric conformation adopted by two chemically identical subunits in homo-oligomers can represent an intrinsic property of a protein or be an artifact induced by crystal packing forces. Solution NMR studies can distinguish between these two possibilities. Using methyl-based NMR spectroscopy, we provide evidence for symmetry in the absence of ligands in several homodimeric proteins that are either asymmetric functionally and/or adopt different conformations of the two subunits in available X-ray structures., (© 2011 American Chemical Society)