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1.
Nat Chem Biol ; 19(6): 767-777, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-36894721

RESUMO

Bacterial transcription factors (TFs) with helix-turn-helix (HTH) DNA-binding domains have been widely explored to build orthogonal transcriptional regulation systems in mammalian cells. Here we capitalize on the modular structure of these proteins to build a framework for multi-input logic gates relying on serial combinations of inducible protein-protein interactions. We found that for some TFs, their HTH domain alone is sufficient for DNA binding. By fusing the HTH domain to TFs, we established dimerization dependent rather than DNA-binding-dependent activation. This enabled us to convert gene switches from OFF-type into more widely applicable ON-type systems and to create mammalian gene switches responsive to new inducers. By combining both OFF and ON modes of action, we built a compact, high-performance bandpass filter. Furthermore, we were able to show cytosolic and extracellular dimerization. Cascading up to five pairwise fusion proteins yielded robust multi-input AND logic gates. Combinations of different pairwise fusion proteins afforded a variety of 4-input 1-output AND and OR logic gate configurations.


Assuntos
Regulação da Expressão Gênica , Fatores de Transcrição , Animais , Multimerização Proteica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Sequências Hélice-Volta-Hélice , DNA/química , Mamíferos
2.
J Cell Biochem ; 124(3): 337-358, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36715571

RESUMO

Specific interactions between transcription factors (TFs) and substrate DNA constitute the fundamental basis of gene expression. Unlike in TFs like basic helix-loop-helix or basic leucine zippers, prediction of substrate DNA is extremely challenging for helix-turn-helix (HTH). Experimental techniques like chromatin immunoprecipitation combined with massively parallel DNA sequencing remains a viable option. We characterize the molecular basis of heterogeneity in HTH-DNA interaction using in silico tools and thence validate them experimentally. Given the profound functional diversity in HTH, we focus primarily on winged-HTH (wHTH). We consider 180 wHTH TFs, whose experimental three-dimensional structures are available in DNA bound/unbound conformations. Starting with PDB-wide scanning and curation of data, we construct a phylogenetic tree, which distributes 180 wHTH sequences under multiple sub-groups. Structure-sequence alignment followed by detailed intra/intergroup analysis, covariation studies and extensive network theory analysis help us to gain deep insight into heterogeneous wHTH-substrate DNA interactions. A central aim of this study is to find a consensus to predict the substrate DNA sequence for wHTH, amidst heterogeneity. The strength of our exhaustive theoretical investigations including molecular docking are successfully tested through experimental characterization of wHTH TF from Sulfurimonas denitrificans.


Assuntos
Proteínas de Ligação a DNA , DNA , Proteínas de Ligação a DNA/metabolismo , Simulação de Acoplamento Molecular , Filogenia , Sequências Hélice-Volta-Hélice , DNA/química
3.
Nucleic Acids Res ; 50(20): 11938-11947, 2022 11 11.
Artigo em Inglês | MEDLINE | ID: mdl-36370103

RESUMO

Some transcription factors bind DNA motifs containing direct or inverted sequence repeats. Preference for each of these DNA topologies is dictated by structural constraints. Most prokaryotic regulators form symmetric oligomers, which require operators with a dyad structure. Binding to direct repeats requires breaking the internal symmetry, a property restricted to a few regulators, most of them from the AraC family. The KorA family of transcriptional repressors, involved in plasmid propagation and stability, includes members that form symmetric dimers and recognize inverted repeats. Our structural analyses show that ArdK, a member of this family, can form a symmetric dimer similar to that observed for KorA, yet it binds direct sequence repeats as a non-symmetric dimer. This is possible by the 180° rotation of one of the helix-turn-helix domains. We then probed and confirmed that ArdK shows affinity for an inverted repeat, which, surprisingly, is also recognized by a non-symmetrical dimer. Our results indicate that structural flexibility at different positions in the dimerization interface constrains transcription factors to bind DNA sequences with one of these two alternative DNA topologies.


Assuntos
DNA , Fatores de Transcrição , Fatores de Transcrição/metabolismo , Sequência de Bases , Sequência de Aminoácidos , Sequências Hélice-Volta-Hélice , DNA/química , Inversão de Sequência , Sítios de Ligação
4.
Nucleic Acids Res ; 50(15): 8615-8625, 2022 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-35947749

RESUMO

Many bacteria use CRISPR-Cas systems to defend against invasive mobile genetic elements (MGEs). In response, MGEs have developed strategies to resist CRISPR-Cas, including the use of anti-CRISPR (Acr) proteins. Known acr genes may be followed in an operon by a putative regulatory Acr-associated gene (aca), suggesting the importance of regulation. Although ten families of helix-turn-helix (HTH) motif containing Aca proteins have been identified (Aca1-10), only three have been tested and shown to be transcriptional repressors of acr-aca expression. The AcrIIA1 protein (a Cas9 inhibitor) also contains a functionally similar HTH containing repressor domain. Here, we identified and analysed Aca and AcrIIA1 homologs across all bacterial genomes. Using HMM models we found aca-like genes are widely distributed in bacteria, both with and without known acr genes. The putative promoter regions of acr-aca operons were analysed and members of each family of bacterial Aca tested for regulatory function. For each Aca family, we predicted a conserved inverted repeat binding site within a core promoter. Promoters containing these sites directed reporter expression in E. coli and were repressed by the cognate Aca protein. These data demonstrate that acr repression by Aca proteins is widely conserved in nature.


Assuntos
Proteínas Associadas a CRISPR , Proteínas Associadas a CRISPR/genética , Escherichia coli/genética , Sistemas CRISPR-Cas , Óperon/genética , Sequências Hélice-Volta-Hélice , Bactérias/genética , Proteínas de Bactérias/genética
5.
J Biol Inorg Chem ; 27(4-5): 485-495, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35796835

RESUMO

Most pathogenic bacteria require ferrous iron (Fe2+) in order to sustain infection within hosts. The ferrous iron transport (Feo) system is the most highly conserved prokaryotic transporter of Fe2+, but its mechanism remains to be fully characterized. Most Feo systems are composed of two proteins: FeoA, a soluble SH3-like accessory protein, and FeoB, a membrane protein that translocates Fe2+ across a lipid bilayer. Some bacterial feo operons encode FeoC, a third soluble, winged-helix protein that remains enigmatic in function. We previously demonstrated that selected FeoC proteins bind O2-sensitive [4Fe-4S] clusters via Cys residues, leading to the proposal that some FeoCs could sense O2 to regulate Fe2+ transport. However, not all FeoCs conserve these Cys residues, and FeoC from the causative agent of cholera (Vibrio cholerae) notably lacks any Cys residues, precluding cluster binding. In this work, we determined the NMR structure of VcFeoC, which is monomeric and conserves the helix-turn-helix domain seen in other FeoCs. In contrast, however, the structure of VcFeoC reveals a truncated winged ß-sheet in which the cluster-binding domain is notably absent. Using homology modeling, we predicted the structure of VcNFeoB and used docking to identify an interaction site with VcFeoC, which is confirmed by NMR spectroscopy. These findings provide the first atomic-level structure of VcFeoC and contribute to a better understanding of its role vis-à-vis FeoB.


Assuntos
Vibrio cholerae , Proteínas de Bactérias/química , Regulação Bacteriana da Expressão Gênica , Sequências Hélice-Volta-Hélice , Ferro/metabolismo , Óperon , Vibrio cholerae/genética , Vibrio cholerae/metabolismo
6.
Nat Commun ; 13(1): 2883, 2022 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-35610211

RESUMO

RNA-binding proteins play key roles in controlling gene expression in many organisms, but relatively few have been identified and characterised in detail in Gram-positive bacteria. Here, we globally analyse RNA-binding proteins in methicillin-resistant Staphylococcus aureus (MRSA) using two complementary biochemical approaches. We identify hundreds of putative RNA-binding proteins, many containing unconventional RNA-binding domains such as Rossmann-fold domains. Remarkably, more than half of the proteins containing helix-turn-helix (HTH) domains, which are frequently found in prokaryotic transcription factors, bind RNA in vivo. In particular, the CcpA transcription factor, a master regulator of carbon metabolism, uses its HTH domain to bind hundreds of RNAs near intrinsic transcription terminators in vivo. We propose that CcpA, besides acting as a transcription factor, post-transcriptionally regulates the stability of many RNAs.


Assuntos
Staphylococcus aureus Resistente à Meticilina , Proteínas de Bactérias/metabolismo , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Sequências Hélice-Volta-Hélice/genética , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/metabolismo , Ligação Proteica , Proteoma/metabolismo , RNA/metabolismo , Fatores de Transcrição/metabolismo
7.
J Physiol ; 599(21): 4831-4844, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34605028

RESUMO

Pain and thermosensation rely on temperature-sensitive ion channels at peripheral nerve endings for transducing thermal cues into electrical signals. Members of the transient receptor potential (TRP) family are prominent candidates for temperature transducers in mammals. These thermal TRP channels possess an unprecedentedly steep temperature dependence, allowing them to discriminate small temperature variations. Thermodynamically, it is understood that the strong temperature sensitivity of the channel arises because opening of the channel undergoes reactions involving large enthalpy and entropy changes. However, the underlying molecular mechanisms have remained elusive. Here we investigated the molecular basis for heat activation of TRPV2, a thermal TRP channel in the vanilloid subfamily with the strongest temperature dependence among TRP channels. We unravel a minimum molecular region in the proximal N-terminus which dictates the slope temperature sensitivity of the channel. Structurally, the region comprises a helix-turn-helix motif and is positioned among the TRP helix from the C-terminus, the S2-S3 linker from the transmembrane domain and the ankyrin repeats from the distal N-terminus. Chimeric exchanges of the subregion alone sufficed to diminish the high temperature dependence in the wild-type TRPV2. Our results support a pivotal role for the structural assembly around the TRP domain in the gating of thermal TRP channels by temperature. The findings also shed insight into how the proximal N-terminal domain plays its role in the heat activation of vanilloid receptors. KEY POINTS: The vanilloid receptor subtype 2 (TRPV2) is a heat-sensitive transient receptor potential (TRP) channel with the strongest temperature dependence among thermal TRP channels. The channel also has a high temperature activation threshold above 50°C which has rendered it difficult to study by conventional patch-clamp methods. Here we utilize fast laser temperature jumps to address the challenges of technical accessibility and explore the molecular basis underlying the high temperature dependence of the channel. We unravel a short helix-turn-helix motif in the proximal N-terminus, which controls the heat activation profile of the channel. Chimeric exchanges of the subregion alone sufficed to diminish the high temperature dependence in the wild-type TRPV2. Our results provide insights on how the proximal N-terminal domain plays its role in the heat activation of vanilloid receptors.


Assuntos
Canais de Cátion TRPV , Canais de Potencial de Receptor Transitório , Animais , Sequências Hélice-Volta-Hélice , Temperatura Alta , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo , Temperatura , Canais de Potencial de Receptor Transitório/genética , Canais de Potencial de Receptor Transitório/metabolismo
8.
J Biol Chem ; 297(4): 101167, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34487759

RESUMO

ToxR represents an essential transcription factor of Vibrio cholerae, which is involved in the regulation of multiple, mainly virulence associated genes. Its versatile functionality as activator, repressor or coactivator suggests a complex regulatory mechanism, whose clarification is essential for a better understanding of the virulence expression system of V. cholerae. Here, we provide structural information elucidating the organization and binding behavior of the cytoplasmic DNA-binding domain of ToxR (cToxR), containing a winged helix-turn-helix (wHTH) motif. Our analysis reveals unexpected structural features of this domain expanding our knowledge of a poorly defined subfamily of wHTH proteins. cToxR forms an extraordinary long α-loop and furthermore has an additional C-terminal beta strand, contacting the N-terminus and thus leading to a compact fold. The identification of the exact interactions between ToxR and DNA contributes to a deeper understanding of this regulatory process. Our findings not only show general binding of the soluble cytoplasmic domain of ToxR to DNA, but also indicate a higher affinity for the toxT motif. These results support the current theory of ToxR being a "DNA-catcher" to enable binding of the transcription factor TcpP and thus activation of virulence-associated toxT transcription. Although, TcpP and ToxR interaction is assumed to be crucial in the activation of the toxT genes, we could not detect an interaction event of their isolated cytoplasmic domains. We therefore conclude that other factors are needed to establish this protein-protein interaction, e.g., membrane attachment, the presence of their full-length proteins and/or other intermediary proteins that may facilitate binding.


Assuntos
Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , Fatores de Transcrição/química , Vibrio cholerae/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Sequências Hélice-Volta-Hélice , Domínios Proteicos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Vibrio cholerae/genética , Vibrio cholerae/metabolismo
9.
Nucleic Acids Res ; 49(17): 9938-9952, 2021 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-34428286

RESUMO

Casposase, a homolog of Cas1 integrase, is encoded by a superfamily of mobile genetic elements known as casposons. While family 2 casposase has been well documented in both function and structure, little is known about the other three casposase families. Here, we studied the family 1 casposase lacking the helix-turn-helix (HTH) domain from Candidatus Nitrosopumilus koreensis AR1 (Ca. N. koreensis). The determinants for integration by Ca. N. koreensis casposase were extensively investigated, and it was found that a 13-bp target site duplication (TSD) sequence, a minimal 3-bp leader and three different nucleotides of the TSD sequences are indispensable for target specific integration. Significantly, the casposase can site-specifically integrate a broad range of terminal inverted repeat (TIR)-derived oligonucleotides ranging from 7-nt to ∼4000-bp, and various oligonucleotides lacking the 5'-TTCTA-3' motif at the 3' end of TIR sequence can be integrated efficiently. Furthermore, similar to some Cas1 homologs, the casposase utilizes a 5'-ATAA-3' motif in the TSD as a molecular ruler to dictate nucleophilic attack at 9-bp downstream of the end of the ruler during the spacer-side integration. By characterizing the family 1 Ca. N. koreensis casposase, we have extended our understanding on mechanistic similarities and evolutionary connections between casposons and the adaptation elements of CRISPR-Cas immunity.


Assuntos
Proteínas Associadas a CRISPR/genética , Integrases/genética , Integrases/metabolismo , Sequências Repetidas Terminais/genética , Archaea/genética , Sistemas CRISPR-Cas/genética , Elementos de DNA Transponíveis/genética , Sequências Hélice-Volta-Hélice/genética , Sequenciamento de Nucleotídeos em Larga Escala , Oligonucleotídeos/genética
10.
Biochem J ; 478(17): 3185-3204, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34405853

RESUMO

p97 protein is a highly conserved, abundant, functionally diverse, structurally dynamic homohexameric AAA enzyme-containing N, D1, and D2 domains. A truncated p97 protein containing the N and D1 domains and the D1-D2 linker (ND1L) exhibits 79% of wild-type (WT) ATPase activity whereas the ND1 domain alone without the linker only has 2% of WT activity. To investigate the relationship between the D1-D2 linker and the D1 domain, we produced p97 ND1L mutants and demonstrated that this 22-residue linker region is essential for D1 ATPase activity. The conserved amino acid leucine 464 (L464) is critical for regulating D1 and D2 ATPase activity by p97 cofactors p37, p47, and Npl4-Ufd1 (NU). Changing leucine to alanine, proline, or glutamate increased the maximum rate of ATP turnover (kcat) of p47-regulated ATPase activities for these mutants, but not for WT. p37 and p47 increased the kcat of the proline substituted linker, suggesting that they induced linker conformations facilitating ATP hydrolysis. NU inhibited D1 ATPase activities of WT and mutant ND1L proteins, but activated D2 ATPase activity of full-length p97. To further understand the mutant mechanism, we used single-particle cryo-EM to visualize the full-length p97L464P and revealed the conformational change of the D1-D2 linker, resulting in a movement of the helix-turn-helix motif (543-569). Taken together with the biochemical and structural results we conclude that the linker helps maintain D1 in a competent conformation and relays the communication to/from the N-domain to the D1 and D2 ATPase domains, which are ∼50 Šaway.


Assuntos
Leucina/metabolismo , Domínios Proteicos/genética , Transdução de Sinais/genética , Proteína com Valosina/química , Proteína com Valosina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação/genética , Ativação Enzimática/genética , Células HeLa , Sequências Hélice-Volta-Hélice/genética , Humanos , Hidrólise , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação , Ligação Proteica/genética , Transfecção , Proteína com Valosina/genética
11.
PLoS Genet ; 17(7): e1009550, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34228715

RESUMO

Quorum sensing is a chemical communication process that bacteria use to coordinate group behaviors. In the global pathogen Vibrio cholerae, one quorum-sensing receptor and transcription factor, called VqmA (VqmAVc), activates expression of the vqmR gene encoding the small regulatory RNA VqmR, which represses genes involved in virulence and biofilm formation. Vibriophage VP882 encodes a VqmA homolog called VqmAPhage that activates transcription of the phage gene qtip, and Qtip launches the phage lytic program. Curiously, VqmAPhage can activate vqmR expression but VqmAVc cannot activate expression of qtip. Here, we investigate the mechanism underlying this asymmetry. We find that promoter selectivity is driven by each VqmA DNA-binding domain and key DNA sequences in the vqmR and qtip promoters are required to maintain specificity. A protein sequence-guided mutagenesis approach revealed that the residue E194 of VqmAPhage and A192, the equivalent residue in VqmAVc, in the helix-turn-helix motifs contribute to promoter-binding specificity. A genetic screen to identify VqmAPhage mutants that are incapable of binding the qtip promoter but maintain binding to the vqmR promoter delivered additional VqmAPhage residues located immediately C-terminal to the helix-turn-helix motif as required for binding the qtip promoter. Surprisingly, these residues are conserved between VqmAPhage and VqmAVc. A second, targeted genetic screen revealed a region located in the VqmAVc DNA-binding domain that is necessary to prevent VqmAVc from binding the qtip promoter, thus restricting DNA binding to the vqmR promoter. We propose that the VqmAVc helix-turn-helix motif and the C-terminal flanking residues function together to prohibit VqmAVc from binding the qtip promoter.


Assuntos
Bacteriófagos/fisiologia , Vibrio cholerae/fisiologia , Proteínas Virais/genética , Proteínas Virais/metabolismo , Sítios de Ligação , DNA Bacteriano/metabolismo , Regulação Bacteriana da Expressão Gênica , Sequências Hélice-Volta-Hélice , Mutagênese , Regiões Promotoras Genéticas , Percepção de Quorum/genética , Vibrio cholerae/virologia , Proteínas Virais/química
12.
J Membr Biol ; 254(4): 397-407, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34189599

RESUMO

During the final step of the bacteriophage infection cycle, the cytoplasmic membrane of host cells is disrupted by small membrane proteins called holins. The function of holins in cell lysis is carried out by forming a highly ordered structure called lethal lesion, in which the accumulation of holins in the cytoplasmic membrane leads to the sudden opening of a hole in the middle of this oligomer. Previous studies showed that dimerization of holins is a necessary step to induce their higher order assembly. However, the molecular mechanism underlying the holin-mediated lesion formation is not well understood. In order to elucidate the functions of holin, we first computationally constructed a structural model for our testing system: the holin S105 from bacteriophage lambda. All atom molecular dynamic simulations were further applied to refine its structure and study its dynamics as well as interaction in lipid bilayer. Additional simulations on association between two holins provide supportive evidence to the argument that the C-terminal region of holin plays a critical role in regulating the dimerization. In detail, we found that the adhesion of specific nonpolar residues in transmembrane domain 3 (TMD3) in a polar environment serves as the driven force of dimerization. Our study therefore brings insights to the design of binding interfaces between holins, which can be potentially used to modulate the dynamics of lesion formation.


Assuntos
Bacteriófago lambda , Proteínas Virais , Sequência de Aminoácidos , Bacteriófago lambda/química , Bacteriófago lambda/metabolismo , Dimerização , Sequências Hélice-Volta-Hélice , Proteínas Virais/química , Proteínas Virais/metabolismo
13.
J Phys Chem B ; 125(25): 6791-6806, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34137249

RESUMO

DNA-binding proteins play an important role in gene regulation and cellular function. The transcription factors MarA and Rob are two homologous members of the AraC/XylS family that regulate multidrug resistance. They share a common DNA-binding domain, and Rob possesses an additional C-terminal domain that permits binding of low-molecular weight effectors. Both proteins possess two helix-turn-helix (HTH) motifs capable of binding DNA; however, while MarA interacts with its promoter through both HTH-motifs, prior studies indicate that Rob binding to DNA via a single HTH-motif is sufficient for tight binding. In the present work, we perform microsecond time scale all-atom simulations of the binding of both transcription factors to different DNA sequences to understand the determinants of DNA recognition and binding. Our simulations characterize sequence-dependent changes in dynamical behavior upon DNA binding, showcasing the role of Arg40 of the N-terminal HTH-motif in allowing for specific tight binding. Finally, our simulations demonstrate that an acidic C-terminal loop of Rob can control the DNA binding mode, facilitating interconversion between the distinct DNA binding modes observed in MarA and Rob. In doing so, we provide detailed molecular insight into DNA binding and recognition by these proteins, which in turn is an important step toward the efficient design of antivirulence agents that target these proteins.


Assuntos
Proteínas de Ligação a DNA , Proteínas de Escherichia coli , Fatores de Transcrição , Proteínas de Bactérias , Sítios de Ligação , DNA/genética , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Sequências Hélice-Volta-Hélice , Transativadores/metabolismo , Fatores de Transcrição/genética
14.
Chempluschem ; 86(4): 646-649, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33856118

RESUMO

Numerous beta-amino acid containing peptides forming secondary structures have been already described, however the design of higher-order structures remains poorly explored. The methodology allowing construction of sequence patterns containing few rigid secondary element was proposed and experimentally validated. On the basis of 9/10/9/12-helix containing cis-2-aminocyclopentanecarboxylic acid (cis-ACPC) residues arranged in an ααßß sequence pattern, a conformationally stable helix-turn-helix structure was designed. The connection between two helices was also constructed using cis-ACPC residues. Five examples of designed peptides were obtained and analyzed using circular dichroism and nuclear magnetic resonance spectroscopy, which confirmed the assumed way of folding. The NMR structure was calculated for the peptide with the highest number of non-sequential contacts.


Assuntos
Peptídeos/química , Sequência de Aminoácidos , Dicroísmo Circular , Cicloleucina/química , Sequências Hélice-Volta-Hélice , Isomerismo , Ressonância Magnética Nuclear Biomolecular
15.
Biochem Biophys Res Commun ; 555: 26-31, 2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-33812055

RESUMO

Bacteria utilize two-component systems to regulate gene expression in response to changes in environmental stimuli. CssS/CssR, a two-component system in Bacillus subtilis, is responsible for overcoming envelope stresses caused by heat shock and secretion overload. During stress, the sensor component CssS is auto-phosphorylated and transfers the phosphoryl group to the response regulator CssR. Phosphorylated CssR then directly regulates the transcription of genes required to counteract the stress. Here, the crystal structure of the DNA-binding domain of CssR, determined at 1.07 Å resolution, is reported. The structure shows that the DNA-binding domain of CssR harbors a winged helix-turn-helix motif that is conserved in the OmpR/PhoB subfamily of response regulators. Based on the crystal structure, the dimeric architecture of the full-length CssR and its DNA-binding mode were suggested.


Assuntos
Bacillus subtilis/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Sítios de Ligação , Cristalografia por Raios X , DNA/química , DNA/metabolismo , Sequências Hélice-Volta-Hélice , Modelos Moleculares , Domínios Proteicos , Multimerização Proteica
16.
mBio ; 12(2)2021 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-33727356

RESUMO

Sequence-specific DNA-binding domains (DBDs) are conserved in all domains of life. These proteins carry out a variety of cellular functions, and there are a number of distinct structural domains already described that allow for sequence-specific DNA binding, including the ubiquitous helix-turn-helix (HTH) domain. In the facultative pathogen Vibrio cholerae, the chitin sensor ChiS is a transcriptional regulator that is critical for the survival of this organism in its marine reservoir. We recently showed that ChiS contains a cryptic DBD in its C terminus. This domain is not homologous to any known DBD, but it is a conserved domain present in other bacterial proteins. Here, we present the crystal structure of the ChiS DBD at a resolution of 1.28 Å. We find that the ChiS DBD contains an HTH domain that is structurally similar to those found in other DNA-binding proteins, like the LacI repressor. However, one striking difference observed in the ChiS DBD is that the canonical tight turn of the HTH is replaced with an insertion containing a ß-sheet, a variant which we term the helix-sheet-helix. Through systematic mutagenesis of all positively charged residues within the ChiS DBD, we show that residues within and proximal to the ChiS helix-sheet-helix are critical for DNA binding. Finally, through phylogenetic analyses we show that the ChiS DBD is found in diverse proteobacterial proteins that exhibit distinct domain architectures. Together, these results suggest that the structure described here represents the prototypical member of the ChiS-family of DBDs.IMPORTANCE Regulating gene expression is essential in all domains of life. This process is commonly facilitated by the activity of DNA-binding transcription factors. There are diverse structural domains that allow proteins to bind to specific DNA sequences. The structural basis underlying how some proteins bind to DNA, however, remains unclear. Previously, we showed that in the major human pathogen Vibrio cholerae, the transcription factor ChiS directly regulates gene expression through a cryptic DNA-binding domain. This domain lacked homology to any known DNA-binding protein. In the current study, we determined the structure of the ChiS DNA-binding domain (DBD) and found that the ChiS-family DBD is a cryptic variant of the ubiquitous helix-turn-helix (HTH) domain. We further demonstrate that this domain is conserved in diverse proteins that may represent a novel group of transcriptional regulators.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Sequências Hélice-Volta-Hélice/genética , Domínios Proteicos , Vibrio cholerae/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/classificação , Proteínas de Ligação a DNA/química , Mutagênese , Ligação Proteica , Vibrio cholerae/metabolismo
17.
Mol Microbiol ; 116(1): 140-153, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33561892

RESUMO

The essential transcription factor PoxCxrA is required for cellulase and xylanase gene expression in the filamentous fungus Penicillium oxalicum that is potentially applied in biotechnological industry as a result of the existence of the integrated cellulolytic and xylolytic system. However, the regulatory mechanism of cellulase and xylanase gene expression specifically associated with PoxCxrA regulation in fungi is poorly understood. In this study, the novel regulator PoxCbh (POX06865), containing a centromere protein B-type helix-turn-helix domain, was identified through screening for the PoxCxrA regulon under Avicel induction and genetic analysis. The mutant ∆PoxCbh showed significant reduction in cellulase and xylanase production, ranging from 28.4% to 59.8%. Furthermore, PoxCbh was found to directly regulate the expression of important cellulase and xylanase genes, as well as the known regulatory genes PoxNsdD and POX02484, and its expression was directly controlled by PoxCxrA. The PoxCbh-binding DNA sequence in the promoter region of the cellobiohydrolase 1 gene cbh1 was identified. These results expand our understanding of the diverse roles of centromere protein B-like protein, the regulatory network of cellulase and xylanase gene expression, and regulatory mechanisms in fungi.


Assuntos
Proteína B de Centrômero/genética , Proteínas Cromossômicas não Histona/biossíntese , Regulação Fúngica da Expressão Gênica/genética , Sequências Hélice-Volta-Hélice/genética , Penicillium/genética , Penicillium/metabolismo , Celulase/biossíntese , Celulase/genética , Celulose 1,4-beta-Celobiosidase/genética , Proteína B de Centrômero/biossíntese , Proteínas Cromossômicas não Histona/genética , Endo-1,4-beta-Xilanases/biossíntese , Endo-1,4-beta-Xilanases/genética , Fatores de Transcrição/genética
18.
Yi Chuan ; 43(1): 66-73, 2021 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-33509775

RESUMO

In bacteria, GntR family transcription regulators are the widespread family of transcription factors. Members of this family consist of two functional domains, a conserved N-terminal DNA-binding domain that contains a typical helix-turn-helix (HTH) motif and a C-terminal effector-binding or oligomerization domain. Usually, the amino acid sequences of N-terminal DNA-binding domains are highly conserved, but differ in the C-terminal effector-binding or oligomerization domains. In the past several decades, many GntR family transcription regulators have been characterized in a number of bacteria. These regulators control a variety of cellular processes such as cell motility, glucose metabolism, bacterial resistance, pathogenesis and virulence. In this review, we summarized the discovery, C-terminal domains, biological function and regulation mode of GntR family transcription regulators. This review will help researchers to obtain more knowledge about the functions and mechanisms of the GntR family transcriptional regulatory factors.


Assuntos
Bactérias/genética , Proteínas de Bactérias , Proteínas de Ligação a DNA , Sequências Hélice-Volta-Hélice , Fatores de Transcrição/genética , Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Regulação Bacteriana da Expressão Gênica
19.
Biochim Biophys Acta Proteins Proteom ; 1869(4): 140601, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33422669

RESUMO

Amoebiasis is the third leading cause of death among protozoon parasitic diseases in the lower-middle income countries. Understanding the molecular events that control gene expression such as transcription factors, their DNA binding mode and target sequences can help to develop new antiamoebic drugs against Entamoeba histolytica. In this paper we performed a genome and structural analysis of a specific transcription factor. The genome of E. histolytica codifies for 9 EhMybSHAQKYF proteins, which are a family within a large group of 34 Myb-DNA-binding domain (Myb-DBD) containing proteins. Here we compared Entamoeba Myb-SHAQKYF proteins with Myb-like proteins from the Reveille (RVE) family, important regulators of plant circadian networks. This comparison could lead to stablish their role in E. histolytica life cycle. We show that the ehmybshaqkyf genes are differentially expressed in trophozoites under basal cell culture conditions. An in-silico analysis predicts that members of this group harbor a highly conserved and structured Myb-DBD and a large portion of intrinsically disordered residues. As the Myb-DBD of these proteins harbors a distinctive Q[VI]R[ST]HAQK[YF]F sequence in its putative third α-helix, we consider relevant to determine the three-dimensional (3D) structure of one of them. An NMR structure of the Myb-DBD of EhMybS3 shows that this protein is composed of three α-helices stabilized by a hydrophobic core, similar to Myb proteins of different kingdoms. It is remarkable that despite not sharing similarities in their amino acid sequences, the structure of the Myb-DBD of the EhMybS3 is well conserved in this early branching eukaryote.


Assuntos
Entamoeba histolytica/genética , Regulação da Expressão Gênica , Proteínas de Protozoários/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Sequências Hélice-Volta-Hélice , Interações Hidrofóbicas e Hidrofílicas , Filogenia , Conformação Proteica , Proteínas de Protozoários/química , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/química
20.
Nucleic Acids Res ; 48(20): 11721-11736, 2020 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-33125059

RESUMO

The genome packaging motor of tailed bacteriophages and herpesviruses is a powerful nanomachine built by several copies of a large (TerL) and a small (TerS) terminase subunit. The motor assembles transiently at the portal vertex of an empty precursor capsid (or procapsid) to power genome encapsidation. Terminase subunits have been studied in-depth, especially in classical bacteriophages that infect Escherichia coli or Salmonella, yet, less is known about the packaging motor of Pseudomonas-phages that have increasing biomedical relevance. Here, we investigated the small terminase subunit from three Podoviridae phages that infect Pseudomonas aeruginosa. We found TerS is polymorphic in solution but assembles into a nonamer in its high-affinity heparin-binding conformation. The atomic structure of Pseudomonas phage PaP3 TerS, the first complete structure for a TerS from a cos phage, reveals nine helix-turn-helix (HTH) motifs asymmetrically arranged around a ß-stranded channel, too narrow to accommodate DNA. PaP3 TerS binds DNA in a sequence-specific manner in vitro. X-ray scattering and molecular modeling suggest TerS adopts an open conformation in solution, characterized by dynamic HTHs that move around an oligomerization core, generating discrete binding crevices for DNA. We propose a model for sequence-specific recognition of packaging initiation sites by lateral interdigitation of DNA.


Assuntos
DNA/metabolismo , Endodesoxirribonucleases/química , Fagos de Pseudomonas/enzimologia , Proteínas Virais/química , Sequência de Bases , DNA/química , Endodesoxirribonucleases/metabolismo , Sequências Hélice-Volta-Hélice , Modelos Moleculares , Ligação Proteica , Pseudomonas aeruginosa/virologia , Proteínas Virais/metabolismo
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