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1.
Nat Commun ; 12(1): 2949, 2021 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-34011959

RESUMO

The antibiotic trimethoprim (TMP) is used to treat a variety of Escherichia coli infections, but its efficacy is limited by the rapid emergence of TMP-resistant bacteria. Previous laboratory evolution experiments have identified resistance-conferring mutations in the gene encoding the TMP target, bacterial dihydrofolate reductase (DHFR), in particular mutation L28R. Here, we show that 4'-desmethyltrimethoprim (4'-DTMP) inhibits both DHFR and its L28R variant, and selects against the emergence of TMP-resistant bacteria that carry the L28R mutation in laboratory experiments. Furthermore, antibiotic-sensitive E. coli populations acquire antibiotic resistance at a substantially slower rate when grown in the presence of 4'-DTMP than in the presence of TMP. We find that 4'-DTMP impedes evolution of resistance by selecting against resistant genotypes with the L28R mutation and diverting genetic trajectories to other resistance-conferring DHFR mutations with catalytic deficiencies. Our results demonstrate how a detailed characterization of resistance-conferring mutations in a target enzyme can help identify potential drugs against antibiotic-resistant bacteria, which may ultimately increase long-term efficacy of antimicrobial therapies by modulating evolutionary trajectories that lead to resistance.


Assuntos
Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/microbiologia , Resistência a Trimetoprima/genética , Trimetoprima/análogos & derivados , Substituição de Aminoácidos , Antibacterianos/química , Antibacterianos/farmacologia , Cristalografia por Raios X , Evolução Molecular Direcionada , Desenho de Fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Antagonistas do Ácido Fólico/química , Antagonistas do Ácido Fólico/farmacologia , Genes Bacterianos , Genótipo , Humanos , Modelos Moleculares , Mutação , Tetra-Hidrofolato Desidrogenase/química , Tetra-Hidrofolato Desidrogenase/genética , Trimetoprima/química , Trimetoprima/farmacologia
2.
Methods Mol Biol ; 2274: 295-304, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34050481

RESUMO

The application of smartphones as detectors is essential to achieve ubiquitous measurement targeting biomolecules. Because bioluminescence (BL), as a tag for a target sample, does not require an excitation light source, it can be combined with a smartphone to constitute a compact and mobile measurement system. A method was recently established to detect the spectral change of ratiometric indicators based on bioluminescence resonance energy transfer with a smartphone camera. For example, it was possible to detect changes in the BL color of the Ca2+ indicator quantitatively and easily calculate the concentration of free Ca2+ by setting appropriate image acquisition conditions in a smartphone application. In this paper, we describe techniques to obtain scientifically relevant and reliable BL data with such a convenient instrument. This protocol expands the potential of the smartphone as a personal imaging device with high mobility that can be used anywhere.


Assuntos
Técnicas Biossensoriais/métodos , Cálcio/metabolismo , Proteínas de Escherichia coli/química , Medições Luminescentes/métodos , Proteínas Luminescentes/química , Smartphone/instrumentação , Transferência Ressonante de Energia de Fluorescência/métodos , Humanos
3.
Nat Commun ; 12(1): 3237, 2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-34050149

RESUMO

Crosslinking mass spectrometry has developed into a robust technique that is increasingly used to investigate the interactomes of organelles and cells. However, the incomplete and noisy information in the mass spectra of crosslinked peptides limits the numbers of protein-protein interactions that can be confidently identified. Here, we leverage chromatographic retention time information to aid the identification of crosslinked peptides from mass spectra. Our Siamese machine learning model xiRT achieves highly accurate retention time predictions of crosslinked peptides in a multi-dimensional separation of crosslinked E. coli lysate. Importantly, supplementing the search engine score with retention time features leads to a substantial increase in protein-protein interactions without affecting confidence. This approach is not limited to cell lysates and multi-dimensional separation but also improves considerably the analysis of crosslinked multiprotein complexes with a single chromatographic dimension. Retention times are a powerful complement to mass spectrometric information to increase the sensitivity of crosslinking mass spectrometry analyses.


Assuntos
Redes Neurais de Computação , Mapeamento de Interação de Proteínas/métodos , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia de Fase Reversa/métodos , Reagentes para Ligações Cruzadas , Escherichia coli , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Fatores de Tempo
4.
Int J Mol Sci ; 22(9)2021 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-33946272

RESUMO

Agmatine is the product of the decarboxylation of L-arginine by the enzyme arginine decarboxylase. This amine has been attributed to neurotransmitter functions, anticonvulsant, anti-neurotoxic, and antidepressant in mammals and is a potential therapeutic agent for diseases such as Alzheimer's, Parkinson's, and cancer. Agmatinase enzyme hydrolyze agmatine into urea and putrescine, which belong to one of the pathways producing polyamines, essential for cell proliferation. Agmatinase from Escherichia coli (EcAGM) has been widely studied and kinetically characterized, described as highly specific for agmatine. In this study, we analyze the amino acids involved in the high specificity of EcAGM, performing a series of mutations in two loops critical to the active-site entrance. Two structures in different space groups were solved by X-ray crystallography, one at low resolution (3.2 Å), including a guanidine group; and other at high resolution (1.8 Å) which presents urea and agmatine in the active site. These structures made it possible to understand the interface interactions between subunits that allow the hexameric state and postulate a catalytic mechanism according to the Mn2+ and urea/guanidine binding site. Molecular dynamics simulations evaluated the conformational dynamics of EcAGM and residues participating in non-binding interactions. Simulations showed the high dynamics of loops of the active site entrance and evidenced the relevance of Trp68, located in the adjacent subunit, to stabilize the amino group of agmatine by cation-pi interaction. These results allow to have a structural view of the best-kinetic characterized agmatinase in literature up to now.


Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/química , Ureo-Hidrolases/química , Agmatina/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Conformação Proteica , Multimerização Proteica , Especificidade por Substrato , Ureo-Hidrolases/metabolismo
5.
Nat Commun ; 12(1): 1988, 2021 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-33790291

RESUMO

Bacteria respond to environmental changes by inducing transcription of some genes and repressing others. Sialic acids, which coat human cell surfaces, are a nutrient source for pathogenic and commensal bacteria. The Escherichia coli GntR-type transcriptional repressor, NanR, regulates sialic acid metabolism, but the mechanism is unclear. Here, we demonstrate that three NanR dimers bind a (GGTATA)3-repeat operator cooperatively and with high affinity. Single-particle cryo-electron microscopy structures reveal the DNA-binding domain is reorganized to engage DNA, while three dimers assemble in close proximity across the (GGTATA)3-repeat operator. Such an interaction allows cooperative protein-protein interactions between NanR dimers via their N-terminal extensions. The effector, N-acetylneuraminate, binds NanR and attenuates the NanR-DNA interaction. The crystal structure of NanR in complex with N-acetylneuraminate reveals a domain rearrangement upon N-acetylneuraminate binding to lock NanR in a conformation that weakens DNA binding. Our data provide a molecular basis for the regulation of bacterial sialic acid metabolism.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas Repressoras/metabolismo , Ácidos Siálicos/metabolismo , Regulação Alostérica , Sequência de Bases , Sítios de Ligação/genética , Cristalografia por Raios X , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Modelos Moleculares , Ácido N-Acetilneuramínico/metabolismo , Motivos de Nucleotídeos/genética , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Proteínas Repressoras/genética
6.
Nat Commun ; 12(1): 2249, 2021 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-33883550

RESUMO

The RNA chaperone Hfq, acting as a hexamer, is a known mediator of post-transcriptional regulation, expediting basepairing between small RNAs (sRNAs) and their target mRNAs. However, the intricate details associated with Hfq-RNA biogenesis are still unclear. Previously, we reported that the stringent response regulator, RelA, is a functional partner of Hfq that facilitates Hfq-mediated sRNA-mRNA regulation in vivo and induces Hfq hexamerization in vitro. Here we show that RelA-mediated Hfq hexamerization requires an initial binding of RNA, preferably sRNA to Hfq monomers. By interacting with a Shine-Dalgarno-like sequence (GGAG) in the sRNA, RelA stabilizes the initially unstable complex of RNA bound-Hfq monomer, enabling the attachment of more Hfq subunits to form a functional hexamer. Overall, our study showing that RNA binding to Hfq monomers is at the heart of RelA-mediated Hfq hexamerization, challenges the previous concept that only Hfq hexamers can bind RNA.


Assuntos
Proteínas de Escherichia coli/metabolismo , GTP Pirofosfoquinase/metabolismo , Fator Proteico 1 do Hospedeiro/metabolismo , RNA Bacteriano/metabolismo , Substituição de Aminoácidos , Sequência de Bases , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , GTP Pirofosfoquinase/química , GTP Pirofosfoquinase/genética , Fator Proteico 1 do Hospedeiro/química , Modelos Biológicos , Ligação Proteica , Multimerização Proteica , Estabilidade Proteica , Estrutura Quaternária de Proteína , Subunidades Proteicas , RNA Bacteriano/química , RNA Bacteriano/genética , Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , Deleção de Sequência
7.
Int J Mol Sci ; 22(7)2021 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-33808202

RESUMO

Active transport of sugars into bacteria occurs through symporters driven by ion gradients. LacY is the most well-studied proton sugar symporter, whereas vSGLT is the most characterized sodium sugar symporter. These are members of the major facilitator (MFS) and the amino acid-Polyamine organocation (APS) transporter superfamilies. While there is no structural homology between these transporters, they operate by a similar mechanism. They are nano-machines driven by their respective ion electrochemical potential gradients across the membrane. LacY has 12 transmembrane helices (TMs) organized in two 6-TM bundles, each containing two 3-helix TM repeats. vSGLT has a core structure of 10 TM helices organized in two inverted repeats (TM 1-5 and TM 6-10). In each case, a single sugar is bound in a central cavity and sugar selectivity is determined by hydrogen- and hydrophobic- bonding with side chains in the binding site. In vSGLT, the sodium-binding site is formed through coordination with carbonyl- and hydroxyl-oxygens from neighboring side chains, whereas in LacY the proton (H3O+) site is thought to be a single glutamate residue (Glu325). The remaining challenge for both transporters is to determine how ion electrochemical potential gradients drive uphill sugar transport.


Assuntos
Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Transporte de Sódio-Glucose/química , Proteínas de Transporte de Sódio-Glucose/metabolismo , Sítios de Ligação , Transporte Biológico Ativo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Glucose/metabolismo , Lactose/metabolismo , Modelos Moleculares , Proteínas de Transporte de Monossacarídeos/química , Proteínas de Transporte de Monossacarídeos/metabolismo , Conformação Proteica , Açúcares/metabolismo , Simportadores/química , Simportadores/metabolismo
8.
Nat Commun ; 12(1): 2056, 2021 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-33824341

RESUMO

Biotin is an essential micro-nutrient across the three domains of life. The paradigm earlier step of biotin synthesis denotes "BioC-BioH" pathway in Escherichia coli. Here we report that BioZ bypasses the canonical route to begin biotin synthesis. In addition to its origin of Rhizobiales, protein phylogeny infers that BioZ is domesticated to gain an atypical role of ß-ketoacyl-ACP synthase III. Genetic and biochemical characterization demonstrates that BioZ catalyzes the condensation of glutaryl-CoA (or ACP) with malonyl-ACP to give 5'-keto-pimeloyl ACP. This intermediate proceeds via type II fatty acid synthesis (FAS II) pathway, to initiate the formation of pimeloyl-ACP, a precursor of biotin synthesis. To further explore molecular basis of BioZ activity, we determine the crystal structure of Agrobacterium tumefaciens BioZ at 1.99 Å, of which the catalytic triad and the substrate-loading tunnel are functionally defined. In particular, we localize that three residues (S84, R147, and S287) at the distant bottom of the tunnel might neutralize the charge of free C-carboxyl group of the primer glutaryl-CoA. Taken together, this study provides molecular insights into the BioZ biotin synthesis pathway.


Assuntos
Vias Biossintéticas , Biotina/biossíntese , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteína de Transporte de Acila/metabolismo , Acil Coenzima A/metabolismo , Agrobacterium/crescimento & desenvolvimento , Sequência de Aminoácidos , Biocatálise , Cristalografia por Raios X , Simulação de Acoplamento Molecular , Filogenia , Multimerização Proteica , Homologia Estrutural de Proteína , Especificidade por Substrato
9.
Int J Mol Sci ; 22(5)2021 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-33803409

RESUMO

Protein engineering is actively pursued in industrial and laboratory settings for high thermostability. Among the many protein engineering methods, rational design by bioinformatics provides theoretical guidance without time-consuming experimental screenings. However, most rational design methods either rely on protein tertiary structure information or have limited accuracies. We proposed a primary-sequence-based algorithm for increasing the heat resistance of a protein while maintaining its functions. Using adenylate kinase (ADK) family as a model system, this method identified a series of amino acid sites closely related to thermostability. Single- and double-point mutants constructed based on this method increase the thermal denaturation temperature of the mesophilic Escherichia coli (E. coli) ADK by 5.5 and 8.3 °C, respectively, while preserving most of the catalytic function at ambient temperatures. Additionally, the constructed mutants have improved enzymatic activity at higher temperature.


Assuntos
Adenilato Quinase/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Temperatura Alta , Adenilato Quinase/genética , Adenilato Quinase/metabolismo , Estabilidade Enzimática , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo
10.
J Vis Exp ; (170)2021 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-33900288

RESUMO

Chromatin-associated condensates are implicated in many nuclear processes, but the underlying mechanisms remain elusive. This protocol describes a chemically-induced protein dimerization system to create condensates on telomeres. The chemical dimerizer consists of two linked ligands that can each bind to a protein: Halo ligand to Halo-enzyme and trimethoprim (TMP) to E. coli dihydrofolate reductase (eDHFR), respectively. Fusion of Halo enzyme to a telomere protein anchors dimerizers to telomeres through covalent Halo ligand-enzyme binding. Binding of TMP to eDHFR recruits eDHFR-fused phase separating proteins to telomeres and induces condensate formation. Because TMP-eDHFR interaction is non-covalent, condensation can be reversed by using excess free TMP to compete with the dimerizer for eDHFR binding. An example of inducing promyelocytic leukemia (PML) nuclear body formation on telomeres and determining condensate growth, dissolution, localization and composition is shown. This method can be easily adapted to induce condensates at other genomic locations by fusing Halo to a protein that directly binds to the local chromatin or to dCas9 that is targeted to the genomic locus with a guide RNA. By offering the temporal resolution required for single cell live imaging while maintaining phase separation in a population of cells for biochemical assays, this method is suitable for probing both the formation and function of chromatin-associated condensates.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Multimerização Proteica , Telômero/metabolismo , Tetra-Hidrofolato Desidrogenase/metabolismo , Trimetoprima/metabolismo , Proteínas de Escherichia coli/química , Humanos , Ligantes , Ligação Proteica , Tetra-Hidrofolato Desidrogenase/química , Trimetoprima/química
11.
Methods Mol Biol ; 2281: 67-80, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33847952

RESUMO

Single-stranded DNA (ssDNA)-binding protein (SSB) is essential for DNA metabolic processes. SSB also binds to many DNA-binding proteins that constitute the SSB interactome. The mechanism through which PriA helicase, an initiator protein in the DNA replication restart process, is stimulated by SSB in Escherichia coli (EcSSB) has been established. However, some Gram-positive bacterial SSBs such as Bacillus subtilis SsbA (a counterpart of EcSSB), Staphylococcus aureus SsbA, SsbB, and SsbC do not activate PriA helicase. Here, we describe some of the methods used in our laboratory to compare SSB-PriA functional and physical interactions in Gram-positive and -negative bacteria.


Assuntos
Bacillus subtilis/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/metabolismo , Staphylococcus aureus/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , DNA Helicases/química , Proteínas de Ligação a DNA/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Ligação Proteica , Análise de Sequência de Proteína , Especificidade da Espécie , Ressonância de Plasmônio de Superfície
12.
Methods Mol Biol ; 2281: 81-91, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33847953

RESUMO

Understanding protein-protein interactions is key to unraveling protein function in vivo. Here we describe a dual/triple-plasmid system that enables co-expression of two, or three, recombinant proteins harboring different affinity tags in the same Escherichia coli cell. This novel protein expression system provides a platform to understand protein-protein interactions and enables researchers to study protein complex formation and in vivo localization.


Assuntos
DNA Helicases/química , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Sítios de Ligação , DNA Helicases/genética , Replicação do DNA , DNA Bacteriano/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/genética , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo
13.
Methods Mol Biol ; 2281: 93-115, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33847954

RESUMO

The ability of magnetic tweezers to apply forces and measure molecular displacements has resulted in its extensive use to study the activity of enzymes involved in various aspects of nucleic acid metabolism. These studies have led to the discovery of key aspects of protein-protein and protein-nucleic acid interaction, uncovering dynamic heterogeneities that are lost to ensemble averaging in bulk experiments. The versatility of magnetic tweezers lies in the possibility and ease of tracking multiple parallel single-molecule events to yield statistically relevant single-molecule data. Moreover, they allow tracking both fast millisecond dynamics and slow processes (spanning several hours). In this chapter, we present the protocols used to study the interaction between E. coli SSB, single-stranded DNA (ssDNA), and E. coli RecQ helicase using magnetic tweezers. In particular, we propose constant force and force modulation assays to investigate SSB binding to DNA, as well as to characterize various facets of RecQ helicase activity stimulation by SSB.


Assuntos
DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , RecQ Helicases/metabolismo , Imagem Individual de Molécula/instrumentação , Proteínas de Ligação a DNA/química , Escherichia coli/genética , Proteínas de Escherichia coli/química , Fenômenos Magnéticos , Ligação Proteica , Fatores de Tempo
14.
Molecules ; 26(9)2021 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-33924762

RESUMO

Currently, the detection of pathogens such as Escherichia coli through instrumental alternatives with fast response and excellent sensitivity and selectivity are being studied. Biosensors are systems consisting of nanomaterials and biomolecules that exhibit remarkable properties such as simplicity, portable, affordable, user­friendly, and deliverable to end­users. For this, in this work we report for the first time, to our knowledge, the bioinformatic design of a new peptide based on TIR protein, a receptor of Intimin membrane protein which is characteristic of E. coli. This peptide (named PEPTIR­1.0) was used as recognition element in a biosensor based on AuNPs­modified screen­printed electrodes for the detection of E. coli. The morphological and electrochemical characteristics of the biosensor obtained were studied. Results show that the biosensor can detect the bacteria with limits of detection and quantification of 2 and 6 CFU/mL, respectively. Moreover, the selectivity of the system is statistically significant towards the detection of the pathogen in the presence of other microorganisms such as P. aeruginosa and S. aureus. This makes this new PEPTIR­1.0 based biosensor can be used in the rapid, sensitive, and selective detection of E. coli in aqueous matrices.


Assuntos
Técnicas Biossensoriais , Técnicas Eletroquímicas , Escherichia coli O157/química , Proteínas de Escherichia coli/química , Peptídeos/química , Receptores de Superfície Celular/química , Microbiologia da Água , Sequência de Aminoácidos , Biologia Computacional/métodos , Microbiologia de Alimentos , Ouro/química , Ligantes , Nanopartículas Metálicas/química , Nanopartículas Metálicas/ultraestrutura , Modelos Moleculares , Peptídeos/análise , Conformação Proteica , Sensibilidade e Especificidade
15.
Int J Mol Sci ; 22(6)2021 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-33799639

RESUMO

Cas3 is a ssDNA-targeting nuclease-helicase essential for class 1 prokaryotic CRISPR immunity systems, which has been utilized for genome editing in human cells. Cas3-DNA crystal structures show that ssDNA follows a pathway from helicase domains into a HD-nuclease active site, requiring protein conformational flexibility during DNA translocation. In genetic studies, we had noted that the efficacy of Cas3 in CRISPR immunity was drastically reduced when temperature was increased from 30 °C to 37 °C, caused by an unknown mechanism. Here, using E. coli Cas3 proteins, we show that reduced nuclease activity at higher temperature corresponds with measurable changes in protein structure. This effect of temperature on Cas3 was alleviated by changing a single highly conserved tryptophan residue (Trp-406) into an alanine. This Cas3W406A protein is a hyperactive nuclease that functions independently from temperature and from the interference effector module Cascade. Trp-406 is situated at the interface of Cas3 HD and RecA1 domains that is important for maneuvering DNA into the nuclease active site. Molecular dynamics simulations based on the experimental data showed temperature-induced changes in positioning of Trp-406 that either blocked or cleared the ssDNA pathway. We propose that Trp-406 forms a 'gate' for controlling Cas3 nuclease activity via access of ssDNA to the nuclease active site. The effect of temperature in these experiments may indicate allosteric control of Cas3 nuclease activity caused by changes in protein conformations. The hyperactive Cas3W406A protein may offer improved Cas3-based genetic editing in human cells.


Assuntos
Proteínas Associadas a CRISPR/metabolismo , DNA Helicases/metabolismo , DNA de Cadeia Simples/metabolismo , DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Triptofano/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Alanina/química , Alanina/genética , Alanina/metabolismo , Sequência de Aminoácidos , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas , Domínio Catalítico/genética , Dicroísmo Circular , DNA/química , DNA/genética , DNA Helicases/química , DNA Helicases/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Edição de Genes/métodos , Humanos , Mutação de Sentido Incorreto , Conformação Proteica , Homologia de Sequência de Aminoácidos , Temperatura , Triptofano/química , Triptofano/genética
16.
Nat Commun ; 12(1): 2162, 2021 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-33846343

RESUMO

Diguanylate cyclases synthesising the bacterial second messenger c-di-GMP are found to be regulated by a variety of sensory input domains that control the activity of their catalytical GGDEF domain, but how activation proceeds mechanistically is, apart from a few examples, still largely unknown. As part of two-component systems, they are activated by cognate histidine kinases that phosphorylate their Rec input domains. DgcR from Leptospira biflexa is a constitutively dimeric prototype of this class of diguanylate cyclases. Full-length crystal structures reveal that BeF3- pseudo-phosphorylation induces a relative rotation of two rigid halves in the Rec domain. This is coupled to a reorganisation of the dimeric structure with concomitant switching of the coiled-coil linker to an alternative heptad register. Finally, the activated register allows the two substrate-loaded GGDEF domains, which are linked to the end of the coiled-coil via a localised hinge, to move into a catalytically competent dimeric arrangement. Bioinformatic analyses suggest that the binary register switch mechanism is utilised by many diguanylate cyclases with N-terminal coiled-coil linkers.


Assuntos
Proteínas de Escherichia coli/metabolismo , Leptospira/enzimologia , Fósforo-Oxigênio Liases/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Ácido Aspártico/metabolismo , Berílio/química , Ativação Enzimática , Proteínas de Escherichia coli/química , Retroalimentação Fisiológica , Fluoretos/química , Cinética , Modelos Moleculares , Fósforo-Oxigênio Liases/química , Fosforilação , Domínios Proteicos , Multimerização Proteica , Estrutura Secundária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Rotação
17.
Nat Commun ; 12(1): 2200, 2021 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-33850130

RESUMO

Split inteins are powerful tools for seamless ligation of synthetic split proteins. Yet, their use remains limited because the already intricate split site identification problem is often complicated by the requirement of extein junction sequences. To address this, we augment a mini-Mu transposon-based screening approach and devise the intein-assisted bisection mapping (IBM) method. IBM robustly reveals clusters of split sites on five proteins, converting them into AND or NAND logic gates. We further show that the use of inteins expands functional sequence space for splitting a protein. We also demonstrate the utility of our approach over rational inference of split sites from secondary structure alignment of homologous proteins, and that basal activities of highly active proteins can be mitigated by splitting them. Our work offers a generalizable and systematic route towards creating split protein-intein fusions for synthetic biology.


Assuntos
Inteínas/fisiologia , Engenharia de Proteínas/métodos , Proteínas/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Sequenciamento de Nucleotídeos em Larga Escala , Inteínas/genética , Modelos Moleculares , Conformação Proteica , Processamento de Proteína , Proteínas/química , Proteínas/genética , Biologia Sintética/métodos
19.
Nat Struct Mol Biol ; 28(3): 310-318, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33712813

RESUMO

Cellulose is frequently found in communities of sessile bacteria called biofilms. Escherichia coli and other enterobacteriaceae modify cellulose with phosphoethanolamine (pEtN) to promote host tissue adhesion. The E. coli pEtN cellulose biosynthesis machinery contains the catalytic BcsA-B complex that synthesizes and secretes cellulose, in addition to five other subunits. The membrane-anchored periplasmic BcsG subunit catalyzes pEtN modification. Here we present the structure of the roughly 1 MDa E. coli Bcs complex, consisting of one BcsA enzyme associated with six copies of BcsB, determined by single-particle cryo-electron microscopy. BcsB homo-oligomerizes primarily through interactions between its carbohydrate-binding domains as well as intermolecular beta-sheet formation. The BcsB hexamer creates a half spiral whose open side accommodates two BcsG subunits, directly adjacent to BcsA's periplasmic channel exit. The cytosolic BcsE and BcsQ subunits associate with BcsA's regulatory PilZ domain. The macrocomplex is a fascinating example of cellulose synthase specification.


Assuntos
Celulose/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Biocatálise , Microscopia Crioeletrônica , Proteínas de Escherichia coli/ultraestrutura , Modelos Moleculares , Complexos Multienzimáticos/ultraestrutura , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Reprodutibilidade dos Testes
20.
Int J Mol Sci ; 22(4)2021 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-33672263

RESUMO

The 70 kDa and 90 kDa heat shock proteins Hsp70 and Hsp90 are two abundant and highly conserved ATP-dependent molecular chaperones that participate in the maintenance of cellular homeostasis. In Escherichia coli, Hsp90 (Hsp90Ec) and Hsp70 (DnaK) directly interact and collaborate in protein remodeling. Previous work has produced a model of the direct interaction of both chaperones. The locations of the residues involved have been confirmed and the model has been validated. In this study, we investigate the allosteric communication between Hsp90Ec and DnaK and how the chaperones couple their conformational cycles. Using elastic network models (ENM), normal mode analysis (NMA), and a structural perturbation method (SPM) of asymmetric and symmetric DnaK-Hsp90Ec, we extract biologically relevant vibrations and identify residues involved in allosteric signaling. When one DnaK is bound, the dominant normal modes favor biological motions that orient a substrate protein bound to DnaK within the substrate/client binding site of Hsp90Ec and release the substrate from the DnaK substrate binding domain. The presence of one DnaK molecule stabilizes the entire Hsp90Ec protomer to which it is bound. Conversely, the symmetric model of DnaK binding results in steric clashes of DnaK molecules and suggests that the Hsp90Ec and DnaK chaperone cycles operate independently. Together, this data supports an asymmetric binding of DnaK to Hsp90Ec.


Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/química , Proteínas de Choque Térmico HSP90/metabolismo , Regulação Alostérica , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo
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