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The N-terminal 16-kDa domain of Escherichia coli Ada protein (N-Ada16k) repairs DNA methyl phosphotriester lesions by an irreversible methyl transfer to its cysteine residue. Upon the methylation, the sequence-specific DNA binding affinity for the promoter region of the alkylation resistance genes is enhanced by 103-fold. Then, it acts as a transcriptional regulator for the methylation damage. In this paper, we identified the methyl acceptor residue of N-Ada16k and determined the solution structure of the methylated form of N-Ada16k by using NMR and mass spectrometry. The results of a 13C-filtered 1H-13C HMBC experiment and MALDI-TOF MS and MS/MS experiments clearly showed that the methyl acceptor residue is Cys38. The solution structure revealed that it has two distinct subdomains connected by a flexible linker loop: the methyltransferase (MTase) subdomain with the zinc-thiolate center, and the helical subdomain with a helix-turn-helix motif. Interestingly, there is no potential hydrogen bond donor around Cys38, whereas the other three cysteine residues coordinated to a zinc ion have potential donors. Hence, Cys38 could retain its inherent nucleophilicity and react with a methyl phosphotriester. Furthermore, the structure comparison shows that there is no indication of a remarkable conformational change occurring upon the methylation. This implies that the electrostatic repulsion between the negatively charged DNA and the zinc-thiolate center may avoid the contact between the MTase subdomain and the DNA in the nonmethylated form. Thus, after the Cys38 methylation, the MTase subdomain can bind the cognate DNA because the negative charge of the zinc-thiolate center is reduced. [ABSTRACT FROM AUTHOR]

Abstract: Amphotericin B is a medically important antifungal antibiotic that is produced by Streptomyces nodosus. Genetic manipulation of this organism has led to production of the first amphotericin analogues by engineered biosynthesis. Here, these studies were extended by sequencing the chromosomal regions flanking the amphotericin polyketide synthase genes, and by refining the phage KC515 transduction method for disruption and replacement of S. nodosus genes. A hybrid vector was constructed from KC515 DNA and the Escherichia coli plasmid pACYC177. This vector replicated as a plasmid in E. coli and the purified DNA yielded phage plaques on Streptomyces lividans after polyethylene glycol (PEG)-mediated transfection of protoplasts. The left flank of the amphotericin gene cluster was found to include amphRI, RII, RIII and RIV genes that are similar to regulatory genes in other polyene biosynthetic gene clusters. One of these regulatory genes, amphRI, was found to have a homologue, amphRVI, located in the right flank at a distance of 127 kbp along the chromosome. However, disruption of amphRVI using the hybrid vector had no effect on the yield of amphotericin obtained from cultures grown on production medium. The hybrid vector was also used for precise deletion of the DNA coding for two modules of the AmphC polyketide synthase protein. Analysis by UV spectrophotometry revealed that the deletion mutant produced a novel pentaene, with reduced antifungal activity but apparently greater water-solubility than amphotericin B. This shows the potential for use of the new vector in engineering of this and other biosynthetic pathways in Streptomyces. [Copyright &y& Elsevier]

Regulatory complexes formed by the CytR repressor protein and the cAMP receptor protein (CRP) prevent transcription initiation from several promoters in Escherichia coli. The formation of the complexes is mediated by protein–DNA interactions and protein–protein interactions between the two regulators. Interestingly, co-binding with CRP has a profound effect on the configuration of the DNA-binding targets preferred by CytR. When binding to DNA by itself, CytR binds preferentially to two octamer repeats in direct or inverted orientation, and separated by 2 bp. However, in the presence of CRP, CytR recognizes inverted repeats separated by 10–13 bp, or direct repeats separated by 1 bp. A fixed orientation of at least one CytR octamer repeat in close proximity to a CRP-binding target is a common architectural feature at promoters optimised for repression complex formation. These observations suggest that CRP alters the DNA-binding mode of CytR. Here, we have investigated the CRP-induced changes in CytR by protein footprinting and alanine-scanning mutagenesis. Our data suggest that a flexible interdomain linker region in CytR, connecting the DNA-binding domain to the dimerization domain allows the repressor protein to interact with DNA-binding sites in a highly relaxed manner, as shown previously, and plays an active role in transcription regulation. Thus, the interactions between CRP, CytR and DNA within the repression complex appear to be more extensive than anticipated. The results support and extend the view that the high degree of adaptability observed in the CytR/CRP regulatory system is obtained though multiple adjustable interactions between the implicated factors. [Copyright &y& Elsevier]

Bacillus subtilis GabR is a member of a poorly characterized but widespread family of chimeric bacterial proteins that have apparent DNA binding and aminotransferase domains. GabR positively regulates expression of the gabTD operon responsible for utilization of γ-aminobutyric acid (GABA) and represses the divergently transcribed gabR gene. Purified GabR bound specifically to the DNA region overlapping the -35 region of the gabT promoter and the -10 and +1 regions of the gabR promoter. Two 6 bp direct repeats located at the ends of this region appeared to be essential for GabR binding. In transcription reactions in vitro, GabR alone repressed expression from the gabR promoter but activated expression from the gabT promoter only in the presence of GABA and pyridoxal 5′-phosphate, an essential cofactor of aminotransferases. A similar requirement for pyridoxal 5′-phosphate and GABA for GabR-mediated transcription activation was shown in vivo. In vitro this requirement could be partially satisfied with pyridoxamine 5′-phosphate and succinic semialdehyde, the products of a GABA-dependent aminotransferase half-reaction. We hypothesize that the GabR-catalyzed aminotransferase-like reaction between GABA and pyridoxal 5′-phosphate is essential for GabR action as a transcriptional activator. [Copyright &y& Elsevier]

The transcriptional regulator DntR, a member of the LysR family, is a central element in a prototype bacterial cell-based biosensor for the detection of hazardous contamination of soil and groundwater by dinitrotoluenes. To optimise the sensitivity of the biosensor for such compounds we have chosen a rational design of the inducer-binding cavity based on knowledge of the three-dimensional structure of DntR. We report two crystal structures of DntR with acetate (resolution 2.6 Å) and thiocyanate (resolution 2.3 Å), respectively, occupying the inducer-binding cavity. These structures allow for the construction of models of DntR in complex with salicylate (Kd4 μM) and 2,4-dinitrotoluene that provide a basis for the design of mutant DntR with enhanced specificity for dinitrotoluenes. In both crystal structures DntR crystallises as a homodimer with a “head-to-tail” arrangement of monomers in the asymmetric unit. Analysis of the crystal structure has allowed the building of a full-length model of DntR in its biologically active homotetrameric form consisting of two “head-to-head” dimers. The implications of this model for the mechanism of transcription regulation by LysR proteins are discussed. [Copyright &y& Elsevier]

The excisionase (Xis) protein from bacteriophage λ is the best characterized member of a large family of recombination directionality factors that control integrase-mediated DNA rearrangements. It triggers phage excision by cooperatively binding to sites X1 and X2 within the phage, bending DNA significantly and recruiting the phage-encoded integrase (Int) protein to site P2. We have determined the co-crystal structure of Xis with its X2 DNA-binding site at 1.7 A˚ resolution. Xis forms a unique winged-helix motif that interacts with the major and minor grooves of its binding site using an α-helix and an ordered β-hairpin (wing), respectively. Recognition is achieved through an elaborate water-mediated hydrogen-bonding network at the major groove interface, while the preformed hairpin forms largely non-specific interactions with the minor groove. The structure of the complex provides insights into how Xis recruits Int cooperatively, and suggests a plausible mechanism by which it may distort longer DNA fragments significantly. It reveals a surface on the protein that is likely to mediate Xis–Xis interactions required for its cooperative binding to DNA. [Copyright &y& Elsevier]

The origin of metal ion selectivity by members of the SmtB/ArsR family of bacterial metal-sensing transcriptional repressors and the mechanism of negative allosteric regulation of DNA binding is poorly understood. Here, we report that two homologous zinc sensors, Staphylococcus aureus CzrA and cyanobacterial SmtB, are “winged” helix homodimeric DNA-binding proteins that bind Zn(II) to a pair of tetrahedral, interhelical binding sites, with two ligands derived from the α5 helix of one subunit, Asp84 Oδ1 (Asp104 in SmtB), His86 Nδ1 (His106), and two derived from the α5 helix of the other, His97′ Nδ1 (His117′) and His100′ Nϵ2 (Glu120′). Formation of the metal chelate drives a quaternary structural switch mediated by an intersubunit hydrogen-binding network that originates with the non-liganding Nϵ2 face of His97 in CzrA (His117 in SmtB) that stabilizes a low-affinity, DNA-binding conformation. The structure of the Zn1 SmtB homodimer shows that both metal-binding sites of the dimer must be occupied for the quaternary structural switch to occur. Thus, a critical zinc-ligating histidine residue obligatorily couples formation of the metal-sensing coordination chelate to changes in the conformation and dynamics of the putative DNA-binding helices. [Copyright &y& Elsevier]

A method for discerning protein structures containing the DNA-binding helix-turn-helix (HTH) motif has been developed. The method uses statistical models based on geometrical measurements of the motif. With a decision tree model, key structural features required for DNA binding were identified. These include a high average solvent-accessibility of residues within the recognition helix and a conserved hydrophobic interaction between the recognition helix and the second alpha helix preceding it. The Protein Data Bank was searched using a more accurate model of the motif created using the Adaboost algorithm to identify structures that have a high probability of containing the motif, including those that had not been reported previously. [Copyright &y& Elsevier]

Active partition of the F plasmid to dividing daughter cells is assured by interactions between proteins SopA and SopB, and a centromere, sopC. A close homologue of the sop operon is present in the linear prophage N15 and, together with sopC-like sequences, it ensures stability of this replicon. We have exploited this sequence similarity to construct hybrid sop operons with the aim of locating specific interaction determinants within the SopA and SopB proteins that are needed for partition function and for autoregulation of sopAB expression. Centromere binding was found to be specified entirely by a central 25 residue region of SopB strongly predicted to form a helix-turn-helix structure. SopB protein also carries a species-specific SopA-interaction determinant within its N-terminal 45 amino acids, and, as shown by Escherichia coli two-hybrid analysis, a dimerization domain within its C-terminal 75 (F) or 97 (N15) residues. Promoter-operator binding specificity was located within an N-terminal 66 residue region of SopA, which is predicted to contain a helix-turn-helix motif. Two other regions of SopA protein, one next to the ATPase Walker A-box, the other C-terminal, specify interaction with SopB. Yeast two-hybrid analysis indicated that these regions contact SopB directly. Evidence for the involvement of the SopA N terminus in autoinhibition of SopA function was obtained, revealing a possible new aspect of the role of SopB in SopA activation. [Copyright &y& Elsevier]

Many of the targets of structural genomics will be proteins with little or no structural similarity to those currently in the database. Therefore, novel function prediction methods that do not rely on sequence or fold similarity to other known proteins are needed. We present an automated approach to predict nucleic-acid-binding (NA-binding) proteins, specifically DNA-binding proteins. The method is based on characterizing the structural and sequence properties of large, positively charged electrostatic patches on DNA-binding protein surfaces, which typically coincide with the DNA-binding-sites. Using an ensemble of features extracted from these electrostatic patches, we predict DNA-binding proteins with high accuracy. We show that our method does not rely on sequence or structure homology and is capable of predicting proteins of novel-binding motifs and protein structures solved in an unbound state. Our method can also distinguish NA-binding proteins from other proteins that have similar, large positive electrostatic patches on their surfaces, but that do not bind nucleic acids. [Copyright &y& Elsevier]

ModE is a bacterial transcriptional regulator that orchestrates many aspects of molybdenum metabolism by binding to specific DNA sequences in a molybdate-dependent fashion. We present the crystal structure of Escherichia coli ModE in complex with molybdate, which was determined at 2.75 A˚ from a merohedrally twinned crystal (twin fraction≈0.30) with space group P43. We now have structures of ModE in both its “switched on” (ligand-bound) and “switched off” (apo) states. Comparison with the apo structure shows that ligand binding leads to extensive conformational changes not only in the molybdate-binding domain, but also in the DNA-binding domain. The most obvious difference is the loss of the pronounced asymmetry between the two chains of the ModE dimer, which had been a characteristic property of the apo structure. Another major change concerns the relative orientation of the two DNA-interacting winged helix-turn-helix motifs. Manual docking of an idealized DNA structure suggests that this conformational change should improve DNA binding of the activated molybdate-bound ModE. [Copyright &y& Elsevier]

The Ets family of transcription factors characterized by an evolutionarily-conserved DNA-binding domain regulates expression of a variety of viral and cellular genes by binding to a purine-rich GGAA/T core sequence in cooperation with other transcriptional factors and co-factors. Most Ets family proteins are nuclear targets for activation of Ras-MAP kinase signaling pathway and some of them affect proliferation of cells by regulating the immediate early response genes and other growth-related genes. Some of them also regulate apoptosis-related genes. Several Ets family proteins are preferentially expressed in specific cell lineages and are involved in their development and differentiation by increasing the enhancer or promoter activities of the genes encoding growth factor receptors and integrin families specific for the cell lineages. Many Ets family proteins also modulate gene expression through protein-protein interactions with other cellular partners. Deregulated expression or formation of chimeric fusion proteins of Ets family due to proviral insertion or chromosome translocation is associated with leukemias and specific types of solid tumors. Several Ets family proteins also participate in malignancy of tumor cells including invasion and metastasis by activating the transcription of several protease genes and angiogenesis-related genes. [Copyright &y& Elsevier]

The cI protein of bacteriophage λ (λcI) activates transcription from promoter PRM through an acidic patch on the surface of its DNA-binding domain. Genetic evidence suggests that this acidic patch stimulates transcription from PRM through contact with the C-terminal domain (region 4) of the σ70 subunit of Escherichia coli RNA polymerase. Here, we identify two basic residues in region 4 of σ70 that are critical for λcI-mediated activation of transcription from PRM. On the basis of structural modeling, we propose that one of these σ70 residues, K593, facilitates the interaction between λcI and region 4 of σ70 by inducing a bend in the DNA upstream of the −35 element, whereas the other, R588, interacts directly with a critical acidic residue within the activating patch of λcI. Residue R588 of σ70 has been shown to play an important role in promoter recognition; our findings suggest that the R588 side-chain has a dual function at PRM, facilitating the interaction of region 4 with the promoter −35 element and participating directly in the protein–protein interaction with λcI. [Copyright &y& Elsevier]

The deformed (Dfd) and ultrabithorax (Ubx) homeoproteins regulate developmental gene expression in Drosophila melanogaster by binding to specific DNA sequences within its genome. DNA binding is largely accomplished via a highly conserved helix-turn-helix DNA-binding domain that is known as a homeodomain (HD). Despite nearly identical DNA recognition helices and similar target DNA sequence preferences, the in vivo functions of the two proteins are quite different. We have previously revealed differences between the two HDs in their interactions with DNA. In an effort to define the individual roles of the HD N-terminal arm and recognition helix in sequence-specific binding, we have characterized the structural details of two Dfd/Ubx chimeric HDs in complex with both the Dfd and Ubx-optimal-binding site sequences. We utilized hydroxyl radical cleavage of DNA to assess the positioning of the proteins on the binding sites. The effects of missing nucleosides and purine methylation on HD binding were also analyzed. Our results show that both the Dfd and Ubx HDs have similar DNA-binding modes when in complex with the Ubx-optimal site. There are subtle but reproducible differences in these modes that are completely interchanged when the Dfd N-terminal arm is replaced with the corresponding region of the Ubx HD. In contrast, we showed previously that the Dfd-optimal site sequence elicits a very different binding mode for the Ubx HD, while the Dfd HD maintains a mode similar to that elicited by the Ubx-optimal site. Our current methylation interference studies suggest that this alternate binding mode involves interaction of the Ubx N-terminal arm with the minor groove on the opposite face of DNA relative to the major groove that is occupied by the recognition helix. As judged by hydroxyl radical footprinting and the missing nucleoside experiment, it appears that interaction of the Ubx recognition helix with the DNA major groove is reduced. Replacing the Dfd N-terminal arm with that of Ubx does not elicit a complete interchange of the DNA-binding mode. Although the position of the chimera relative to DNA, as judged by hydroxyl radical footprinting, is similar to that of the Dfd HD, the missing nucleoside and methylation interference patterns resemble those of the Ubx HD. Repositioning of amino acid side-chains without wholesale structural alteration in the polypeptide appears to occur as a function of N-terminal arm identity and DNA-binding site sequence. Complete interchange of binding modes was achieved only by replacement of the Dfd N-terminal arm and the recognition helix plus 13 carboxyl-terminal residues with the corresponding residues of Ubx. The position of the N-terminal arm in the DNA minor groove appears to differ in a manner that depends on the two base-pair differences between the Dfd and Ubx-optimal-binding sites. Thus, N-terminal arm position dictates the binding mode and the interaction of the recognition helix with nucleosides in the major groove. [Copyright &y& Elsevier]

The transcription factors Dorsal and Twist regulate dorsoventral axis formation during Drosophila embryogenesis. Dorsal and Twist bind to closely linked DNA elements in a number of promoters and synergistically activate transcription. We have identified a novel protein named Dorsal-interacting protein 3 (Dip3) that may play a role in this synergy. Dip3 functions as a coactivator to stimulate synergistic activation by Dorsal and Twist, but does not stimulate simple activation of promoters containing only Dorsal or only Twist binding sites. In addition, Dip3 is able to bind DNA in a sequence specific manner and activate transcription directly. Dip3 possesses an N-terminal MADF domain and a C-terminal BESS domain, an architecture that is conserved in at least 14 Drosophila proteins, including Adf-1 and Stonewall. The MADF domain directs sequence specific DNA binding to a site consisting of multiple trinucleotide repeats, while the BESS domain directs a variety of protein–protein interactions, including interactions with itself, with Dorsal, and with a TBP-associated factor. We assess the possibility that the MADF and BESS domains are related to the SANT domain, a well-characterized motif found in many transcriptional regulators and coregulators. [Copyright &y& Elsevier]

In the mammalian hippocampus, there is a trisynaptic loop that has been often referred to in studies on learning and memory mechanisms and their physiological correlate, the long-term potentiation (LTP). The three sets of synapses are formed by the fibers of perforant pathway terminating on granule cells and by the mossy fibers and Schaeffer collaterals making connections with the pyramidal cells. Each of the three types of synapses can develop LTP. LTP is accompanied by changes in gene expression and it is the nuclear transcription, involving specific transcription factors, that is the starting point for the series of biological amplifications and consolidations both necessary for such sustained changes. The transcription factors are proteins that control gene expression, development and functional formation in every eukaryotic cell. Two categories of transcription factors have been defined to date: general factors that comprise at least 20 proteins to form multiple preinitiation complex at the TATA box (TATA rich sequence) or regulatory factors that bind to promoter or enhancer regions at specific sites on the DNA close to, or distant from, the TATA box. Transcription factors have been divided into five different major classes according to unique protein motifs. These include basic domain, zinc-finger, helix–turn–helix, β-Scaffold factors with minor groove contacts and other transcription factors not specifically classified. Much evidence has been accumulating in favor of the participation of several transcription factors in the consolidation of memory in the mammalian hippocampus following a spatial memory task. It is, therefore, of great importance that the involvement of transcription factors in de novo protein synthesis relevant to the synaptic mechanisms that mediate the formation of long-term memory should be summarized and discussed. No specific correlation between transduction of extracellular signals and expression of nuclear transcription factors, however, has been demonstrated to date. [Copyright &y& Elsevier]

Two-component regulatory systems mediate most of the bacterial cells responses to a variety of signals. In Sinorhizobium meliloti, the FixL–FixJ couple controls the expression of the nitrogen fixation genes through the binding of the two-domains response regulator FixJ to the fixK and nifA promoters. Phosphorylation of the N-terminal regulatory domain activates the protein and releases the inhibition of the C-terminal DNA-binding domain that occurs in the unphosphorylated protein. Insights into the transition from the inactive to the active form are provided by the architecture of the unphosphorylated response regulator reported in this study. The relative position and orientation of the N and C-terminal domains were defined from the molecular envelope restored from small-angle X-ray scattering (SAXS) data. The involvement of the α4–β5–α5 surface of the regulatory domain, the linker region and the C-terminal helix of the DNA-binding domain in the interdomain interface of unphosphorylated FixJ was supported by biochemical investigations. These results, together with the previously reported studies on the phosphorylated regulatory domain of FixJ, emphasize the role of the α4–β5–α5 surface in mediating a flow of information in this response regulator. This first study by SAXS of proteins from two-component systems suggests that the method could be successfully applied to other members of this family and could be suitable for the study of multidomain proteins and protein–protein complexes regulated through molecular interfaces in the low micromolar range. [Copyright &y& Elsevier]

A gene regulatory protein with helix-turn-helix (HTH) DNA-binding motif, GalS contains a functional operator within the DNA sequences encoding the HTH region (Nature 369 (1994) 314). We searched for operator-like sequences within the DNA sequences encoding the DNA binding motifs of other regulatory proteins. Five such proteins, DeoR, CytR, LRP, LuxR and PurR, were found to have actual operator or operator-like sequences in the DNA sequences encoding the DNA-binding motif. Except DeoR, all of them including GalS, are known to be auto-regulated. Auto-regulation in case of DeoR has not been investigated. Seven other proteins containing a HTH motif, do not have operator-like sequences in the DNA sequences encoding the HTH motif; none of them, except MerR, are known to be auto-regulated. The DNA binding proteins may have evolved from a common ancestor containing a DNA binding site within its gene segment that encodes the DNA-binding motif to facilitate auto-regulation. We have discussed current evidence for monophyletic or polyphyletic origin of such sequences. [Copyright &y& Elsevier]

Proteins have highly conserved domains that determine their functionality. Out of the thousands of domains discovered so far across all living forms, some of the predominant clinically-relevant domains include IENR1, HNHc, HELICc, Pro-kuma_activ, Tryp_SPc, Lactamase_B, PbH1, ChtBD3, CBM49, acidPPc, G3P_acyltransf, RPOL8c, KbaA, HAMP, HisKA, Hr1, Dak2, APC2, Citrate_ly_lig, DALR, VKc, YARHG, WR1, PWI, ZnF_BED, TUDOR, MHC_II_beta, Integrin_B_tail, Excalibur, DISIN, Cadherin, ACTIN, PROF, Robl_LC7, MIT, Kelch, GAS2, B41, Cyclin_C, Connexin_CCC, OmpH, Bac_rhodopsin, AAA, Knot1, NH, Galanin, IB, Elicitin, ACTH, Cache_2, CHASE, AgrB, PRP, IGR, and Antimicrobial21. These domains are distributed in nucleases/helicases, proteases, esterases, lipases, glycosylase, GTPases, phosphatases, methyltransferases, acyltransferase, acetyltransferase, polymerase, kinase, ligase, synthetase, oxidoreductase, protease inhibitors, nucleic acid binding proteins, adhesion and immunity-related proteins, cytoskeletal component-manipulating proteins, lipid biosynthesis and metabolism proteins, membrane-associated proteins, hormone-like and signaling proteins, etc. These domains are ubiquitous stretches or folds of the proteins in pathogens and allergens. Pathogenesis alleviation efforts can benefit enormously if the characteristics of these domains are known. Hence, this review catalogs and discusses the role of such pivotal domains, suggesting hypotheses for better understanding of pathogenesis at molecular level., (© 2017 Elsevier Inc. All rights reserved.)