RESUMO
Thermostable clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas9) enzymes could improve genome-editing efficiency and delivery due to extended protein lifetimes. However, initial experimentation demonstrated Geobacillus stearothermophilus Cas9 (GeoCas9) to be virtually inactive when used in cultured human cells. Laboratory-evolved variants of GeoCas9 overcome this natural limitation by acquiring mutations in the wedge (WED) domain that produce >100-fold-higher genome-editing levels. Cryoelectron microscopy (cryo-EM) structures of the wild-type and improved GeoCas9 (iGeoCas9) enzymes reveal extended contacts between the WED domain of iGeoCas9 and DNA substrates. Biochemical analysis shows that iGeoCas9 accelerates DNA unwinding to capture substrates under the magnesium-restricted conditions typical of mammalian but not bacterial cells. These findings enabled rational engineering of other Cas9 orthologs to enhance genome-editing levels, pointing to a general strategy for editing enzyme improvement. Together, these results uncover a new role for the Cas9 WED domain in DNA unwinding and demonstrate how accelerated target unwinding dramatically improves Cas9-induced genome-editing activity.
Assuntos
Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Microscopia Crioeletrônica , DNA , Edição de Genes , Humanos , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/química , Proteína 9 Associada à CRISPR/metabolismo , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , DNA/metabolismo , DNA/genética , Edição de Genes/métodos , Geobacillus stearothermophilus/genética , Geobacillus stearothermophilus/metabolismo , Células HEK293 , Domínios Proteicos , Genoma Humano , Modelos Moleculares , Estrutura Terciária de Proteína , Conformação de Ácido Nucleico , Biocatálise , Magnésio/química , Magnésio/metabolismoRESUMO
The emergence of hypervirulent clade 2 Clostridioides difficile is associated with severe symptoms and accounts for >20% of global infections. TcdB is a dominant virulence factor of C. difficile, and clade 2 strains exclusively express two TcdB variants (TcdB2 and TcdB4) that use unknown receptors distinct from the classic TcdB. Here, we performed CRISPR/Cas9 screens for TcdB4 and identified tissue factor pathway inhibitor (TFPI) as its receptor. Using cryo-EM, we determined a complex structure of the full-length TcdB4 with TFPI, defining a common receptor-binding region for TcdB. Residue variations within this region divide major TcdB variants into 2 classes: one recognizes Frizzled (FZD), and the other recognizes TFPI. TFPI is highly expressed in the intestinal glands, and recombinant TFPI protects the colonic epithelium from TcdB2/4. These findings establish TFPI as a colonic crypt receptor for TcdB from clade 2 C. difficile and reveal new mechanisms for CDI pathogenesis.
Assuntos
Toxinas Bacterianas , Clostridioides difficile , Proteínas de Bactérias/química , Toxinas Bacterianas/química , Clostridioides difficile/genética , Lipoproteínas/genéticaRESUMO
Optobiochemical control of protein activities allows the investigation of protein functions in living cells with high spatiotemporal resolution. Over the last two decades, numerous natural photosensory domains have been characterized and synthetic domains engineered and assembled into photoregulatory systems to control protein function with light. Here, we review the field of optobiochemistry, categorizing photosensory domains by chromophore, describing photoregulatory systems by mechanism of action, and discussing protein classes frequently investigated using optical methods. We also present examples of how spatial or temporal control of proteins in living cells has provided new insights not possible with traditional biochemical or cell biological techniques.
Assuntos
Bioquímica/métodos , Proteínas/química , Proteínas/genética , Proteínas/metabolismo , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Criptocromos/química , Criptocromos/metabolismo , Flavina-Adenina Dinucleotídeo/química , Flavina-Adenina Dinucleotídeo/metabolismo , Luz , Optogenética/métodos , Processos Fotoquímicos , Fotorreceptores Microbianos/química , Fotorreceptores Microbianos/metabolismo , Fitocromo/química , Fitocromo/metabolismo , Domínios Proteicos , Engenharia de Proteínas/métodos , Vitamina B 12/metabolismoRESUMO
Membrane remodeling and repair are essential for all cells. Proteins that perform these functions include Vipp1/IM30 in photosynthetic plastids, PspA in bacteria, and ESCRT-III in eukaryotes. Here, using a combination of evolutionary and structural analyses, we show that these protein families are homologous and share a common ancient evolutionary origin that likely predates the last universal common ancestor. This homology is evident in cryo-electron microscopy structures of Vipp1 rings from the cyanobacterium Nostoc punctiforme presented over a range of symmetries. Each ring is assembled from rungs that stack and progressively tilt to form dome-shaped curvature. Assembly is facilitated by hinges in the Vipp1 monomer, similar to those in ESCRT-III proteins, which allow the formation of flexible polymers. Rings have an inner lumen that is able to bind and deform membranes. Collectively, these data suggest conserved mechanistic principles that underlie Vipp1, PspA, and ESCRT-III-dependent membrane remodeling across all domains of life.
Assuntos
Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteínas de Choque Térmico/metabolismo , Família Multigênica , Nostoc/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/ultraestrutura , Galinhas , Microscopia Crioeletrônica , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Evolução Molecular , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/ultraestrutura , Humanos , Modelos Moleculares , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos , TermodinâmicaRESUMO
Vesicle-inducing protein in plastids 1 (VIPP1) is essential for the biogenesis and maintenance of thylakoid membranes, which transform light into life. However, it is unknown how VIPP1 performs its vital membrane-remodeling functions. Here, we use cryo-electron microscopy to determine structures of cyanobacterial VIPP1 rings, revealing how VIPP1 monomers flex and interweave to form basket-like assemblies of different symmetries. Three VIPP1 monomers together coordinate a non-canonical nucleotide binding pocket on one end of the ring. Inside the ring's lumen, amphipathic helices from each monomer align to form large hydrophobic columns, enabling VIPP1 to bind and curve membranes. In vivo mutations in these hydrophobic surfaces cause extreme thylakoid swelling under high light, indicating an essential role of VIPP1 lipid binding in resisting stress-induced damage. Using cryo-correlative light and electron microscopy (cryo-CLEM), we observe oligomeric VIPP1 coats encapsulating membrane tubules within the Chlamydomonas chloroplast. Our work provides a structural foundation for understanding how VIPP1 directs thylakoid biogenesis and maintenance.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Chlamydomonas/metabolismo , Multimerização Proteica , Synechocystis/metabolismo , Tilacoides/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/ultraestrutura , Sítios de Ligação , Membrana Celular/metabolismo , Chlamydomonas/ultraestrutura , Microscopia Crioeletrônica , Proteínas de Fluorescência Verde/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Luz , Lipídeos/química , Modelos Moleculares , Nucleotídeos/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Estresse Fisiológico/efeitos da radiação , Synechocystis/ultraestrutura , Tilacoides/ultraestruturaRESUMO
PspA is the main effector of the phage shock protein (Psp) system and preserves the bacterial inner membrane integrity and function. Here, we present the 3.6 Å resolution cryoelectron microscopy (cryo-EM) structure of PspA assembled in helical rods. PspA monomers adopt a canonical ESCRT-III fold in an extended open conformation. PspA rods are capable of enclosing lipids and generating positive membrane curvature. Using cryo-EM, we visualized how PspA remodels membrane vesicles into µm-sized structures and how it mediates the formation of internalized vesicular structures. Hotspots of these activities are zones derived from PspA assemblies, serving as lipid transfer platforms and linking previously separated lipid structures. These membrane fusion and fission activities are in line with the described functional properties of bacterial PspA/IM30/LiaH proteins. Our structural and functional analyses reveal that bacterial PspA belongs to the evolutionary ancestry of ESCRT-III proteins involved in membrane remodeling.
Assuntos
Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteínas de Choque Térmico/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/ultraestrutura , Microscopia Crioeletrônica , Endocitose , Complexos Endossomais de Distribuição Requeridos para Transporte/química , Escherichia coli/metabolismo , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/ultraestrutura , Bicamadas Lipídicas/metabolismo , Modelos Moleculares , Domínios Proteicos , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos , Lipossomas Unilamelares/metabolismoRESUMO
Complex carbohydrates are essential for many biological processes, from protein quality control to cell recognition, energy storage, and cell wall formation. Many of these processes are performed in topologically extracellular compartments or on the cell surface; hence, diverse secretion systems evolved to transport the hydrophilic molecules to their sites of action. Polyprenyl lipids serve as ubiquitous anchors and facilitators of these transport processes. Here, we summarize and compare bacterial biosynthesis pathways relying on the recognition and transport of lipid-linked complex carbohydrates. In particular, we compare transporters implicated in O antigen and capsular polysaccharide biosyntheses with those facilitating teichoic acid and N-linked glycan transport. Further, we discuss recent insights into the generation, recognition, and recycling of polyprenyl lipids.
Assuntos
Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Glicolipídeos/biossíntese , Antígenos O/biossíntese , Poliprenois/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/química , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transporte Biológico , Carbono-Oxigênio Ligases/química , Carbono-Oxigênio Ligases/genética , Carbono-Oxigênio Ligases/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Glicosiltransferases/química , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Modelos Moleculares , Estrutura Secundária de Proteína , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Ácidos Teicoicos/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismoRESUMO
Many bacteria use the flagellum for locomotion and chemotaxis. Its bidirectional rotation is driven by a membrane-embedded motor, which uses energy from the transmembrane ion gradient to generate torque at the interface between stator units and rotor. The structural organization of the stator unit (MotAB), its conformational changes upon ion transport, and how these changes power rotation of the flagellum remain unknown. Here, we present ~3 Å-resolution cryoelectron microscopy reconstructions of the stator unit in different functional states. We show that the stator unit consists of a dimer of MotB surrounded by a pentamer of MotA. Combining structural data with mutagenesis and functional studies, we identify key residues involved in torque generation and present a detailed mechanistic model for motor function and switching of rotational direction.
Assuntos
Proteínas de Bactérias/ultraestrutura , Flagelos/ultraestrutura , Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Microscopia Crioeletrônica/métodos , Flagelos/metabolismo , Conformação Proteica , TorqueRESUMO
cGAS/DncV-like nucleotidyltransferase (CD-NTase) enzymes are immune sensors that synthesize nucleotide second messengers and initiate antiviral responses in bacterial and animal cells. Here, we discover Enterobacter cloacae CD-NTase-associated protein 4 (Cap4) as a founding member of a diverse family of >2,000 bacterial receptors that respond to CD-NTase signals. Structures of Cap4 reveal a promiscuous DNA endonuclease domain activated through ligand-induced oligomerization. Oligonucleotide recognition occurs through an appended SAVED domain that is an unexpected fusion of two CRISPR-associated Rossman fold (CARF) subunits co-opted from type III CRISPR immunity. Like a lock and key, SAVED effectors exquisitely discriminate 2'-5'- and 3'-5'-linked bacterial cyclic oligonucleotide signals and enable specific recognition of at least 180 potential nucleotide second messenger species. Our results reveal SAVED CARF family proteins as major nucleotide second messenger receptors in CBASS and CRISPR immune defense and extend the importance of linkage specificity beyond mammalian cGAS-STING signaling.
Assuntos
Bactérias/virologia , Bacteriófagos/metabolismo , Sistemas CRISPR-Cas , Imunidade , Oligonucleotídeos/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Desoxirribonuclease I/metabolismo , Ligantes , Mutagênese/genética , Nucleotidiltransferases/metabolismo , Ligação Proteica , Sistemas do Segundo MensageiroRESUMO
Aerobic life is possible because the molecular structure of oxygen (O2) makes direct reaction with most organic materials at ambient temperatures an exceptionally slow process. Of course, these reactions are inherently very favorable, and they occur rapidly with the release of a great deal of energy at high temperature. Nature has been able to tap this sequestered reservoir of energy with great spatial and temporal selectivity at ambient temperatures through the evolution of oxidase and oxygenase enzymes. One mechanism used by these enzymes for O2 activation has been studied in detail for the soluble form of the enzyme methane monooxygenase. These studies have revealed the step-by-step process of O2 activation and insertion into the ultimately stable C-H bond of methane. Additionally, an elegant regulatory mechanism has been defined that enlists size selection and quantum tunneling to allow methane oxidation to occur specifically in the presence of more easily oxidized substrates.
Assuntos
Bactérias/enzimologia , Metano/metabolismo , Oxigênio/metabolismo , Oxigenases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cristalografia , Cinética , Methylococcus capsulatus/enzimologia , Methylosinus trichosporium/enzimologia , Oxigenases/química , Conformação ProteicaRESUMO
Energy-coupling factor (ECF)-type ATP-binding cassette (ABC) transporters catalyze membrane transport of micronutrients in prokaryotes. Crystal structures and biochemical characterization have revealed that ECF transporters are mechanistically distinct from other ABC transport systems. Notably, ECF transporters make use of small integral membrane subunits (S-components) that are predicted to topple over in the membrane when carrying the bound substrate from the extracellular side of the bilayer to the cytosol. Here, we review the phylogenetic diversity of ECF transporters as well as recent structural and biochemical advancements that have led to the postulation of conceptually different mechanistic models. These models can be described as power stroke and thermal ratchet. Structural data indicate that the lipid composition and bilayer structure are likely to have great impact on the transport function. We argue that study of ECF transporters could lead to generic insight into membrane protein structure, dynamics, and interaction.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/genética , Trifosfato de Adenosina/metabolismo , Animais , Archaea/metabolismo , Proteínas Arqueais/química , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transporte Biológico , Cristalografia por Raios X , Humanos , Modelos Moleculares , Filogenia , Conformação ProteicaRESUMO
During cell division, newly replicated DNA is actively segregated to the daughter cells. In most bacteria, this process involves the DNA-binding protein ParB, which condenses the centromeric regions of sister DNA molecules into kinetochore-like structures that recruit the DNA partition ATPase ParA and the prokaroytic SMC/condensin complex. Here, we report the crystal structure of a ParB-like protein (PadC) that emerges to tightly bind the ribonucleotide CTP. The CTP-binding pocket of PadC is conserved in ParB and composed of signature motifs known to be essential for ParB function. We find that ParB indeed interacts with CTP and requires nucleotide binding for DNA condensation in vivo. We further show that CTP-binding modulates the affinity of ParB for centromeric parS sites, whereas parS recognition stimulates its CTPase activity. ParB proteins thus emerge as a new class of CTP-dependent molecular switches that act in concert with ATPases and GTPases to control fundamental cellular functions.
Assuntos
Proteínas de Bactérias/química , Citidina Trifosfato/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Myxococcus xanthus/genética , Myxococcus xanthus/metabolismo , Motivos de Nucleotídeos , Ligação ProteicaRESUMO
The molecular chaperone HSP90 facilitates the folding of several client proteins, including innate immune receptors and protein kinases. HSP90 is an essential component of plant and animal immunity, yet pathogenic strategies that directly target the chaperone have not been described. Here, we identify the HopBF1 family of bacterial effectors as eukaryotic-specific HSP90 protein kinases. HopBF1 adopts a minimal protein kinase fold that is recognized by HSP90 as a host client. As a result, HopBF1 phosphorylates HSP90 to completely inhibit the chaperone's ATPase activity. We demonstrate that phosphorylation of HSP90 prevents activation of immune receptors that trigger the hypersensitive response in plants. Consequently, HopBF1-dependent phosphorylation of HSP90 is sufficient to induce severe disease symptoms in plants infected with the bacterial pathogen, Pseudomonas syringae. Collectively, our results uncover a family of bacterial effector kinases with toxin-like properties and reveal a previously unrecognized betrayal mechanism by which bacterial pathogens modulate host immunity.
Assuntos
Proteínas de Arabidopsis/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Mimetismo Molecular/imunologia , Imunidade Vegetal/fisiologia , Adenosina Trifosfatases/metabolismo , Arabidopsis/imunologia , Arabidopsis/metabolismo , Arabidopsis/microbiologia , Proteínas de Bactérias/química , Células HEK293 , Proteínas de Choque Térmico HSP90/química , Células HeLa , Interações entre Hospedeiro e Microrganismos/imunologia , Humanos , Fosforilação , Plasmídeos/genética , Ligação Proteica , Dobramento de Proteína , Proteínas Quinases/metabolismo , Pseudomonas syringae/metabolismo , Saccharomyces cerevisiae/metabolismoRESUMO
Multicellular lifestyle requires cell-cell connections. In multicellular cyanobacteria, septal junctions enable molecular exchange between sister cells and are required for cellular differentiation. The structure of septal junctions is poorly understood, and it is unknown whether they are capable of controlling intercellular communication. Here, we resolved the in situ architecture of septal junctions by electron cryotomography of cryo-focused ion beam-milled cyanobacterial filaments. Septal junctions consisted of a tube traversing the septal peptidoglycan. Each tube end comprised a FraD-containing plug, which was covered by a cytoplasmic cap. Fluorescence recovery after photobleaching showed that intercellular communication was blocked upon stress. Gating was accompanied by a reversible conformational change of the septal junction cap. We provide the mechanistic framework for a cell junction that predates eukaryotic gap junctions by a billion years. The conservation of a gated dynamic mechanism across different domains of life emphasizes the importance of controlling molecular exchange in multicellular organisms.
Assuntos
Junções Comunicantes/metabolismo , Anabaena/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Carbonil Cianeto m-Clorofenil Hidrazona/análogos & derivados , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Comunicação Celular/efeitos dos fármacos , Microscopia Crioeletrônica , Junções Comunicantes/química , Junções Comunicantes/ultraestrutura , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , MutagêneseRESUMO
Copper-binding metallophores, or chalkophores, play a role in microbial copper homeostasis that is analogous to that of siderophores in iron homeostasis. The best-studied chalkophores are members of the methanobactin (Mbn) family-ribosomally produced, posttranslationally modified natural products first identified as copper chelators responsible for copper uptake in methane-oxidizing bacteria. To date, Mbns have been characterized exclusively in those species, but there is genomic evidence for their production in a much wider range of bacteria. This review addresses the current state of knowledge regarding the function, biosynthesis, transport, and regulation of Mbns. While the roles of several proteins in these processes are supported by substantial genetic and biochemical evidence, key aspects of Mbn manufacture, handling, and regulation remain unclear. In addition, other natural products that have been proposed to mediate copper uptake as well as metallophores that have biologically relevant roles involving copper binding, but not copper uptake, are discussed.
Assuntos
Proteínas de Bactérias/metabolismo , Quelantes/metabolismo , Cobre/metabolismo , Imidazóis/metabolismo , Oligopeptídeos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Fenômenos Biofísicos , Quelantes/química , Genoma Bacteriano , Homeostase , Imidazóis/química , Methylosinus trichosporium/genética , Methylosinus trichosporium/metabolismo , Modelos Biológicos , Estrutura Molecular , Oligopeptídeos/química , Oligopeptídeos/genética , Óperon , Transporte ProteicoRESUMO
CRISPR-Cas systems confer an adaptive immunity against viruses. Following viral injection, Cas1-Cas2 integrates segments of the viral genome (spacers) into the CRISPR locus. In type I CRISPR-Cas systems, efficient "primed" spacer acquisition and viral degradation (interference) require both the Cascade complex and the Cas3 helicase/nuclease. Here, we present single-molecule characterization of the Thermobifida fusca (Tfu) primed acquisition complex (PAC). We show that TfuCascade rapidly samples non-specific DNA via facilitated one-dimensional diffusion. Cas3 loads at target-bound Cascade and the Cascade/Cas3 complex translocates via a looped DNA intermediate. Cascade/Cas3 complexes stall at diverse protein roadblocks, resulting in a double strand break at the stall site. In contrast, Cas1-Cas2 samples DNA transiently via 3D collisions. Moreover, Cas1-Cas2 associates with Cascade and translocates with Cascade/Cas3, forming the PAC. PACs can displace different protein roadblocks, suggesting a mechanism for long-range spacer acquisition. This work provides a molecular basis for the coordinated steps in CRISPR-based adaptive immunity.
Assuntos
Actinomycetales/enzimologia , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/química , DNA Viral/metabolismo , Multimerização Proteica , Imagem Individual de MoléculaRESUMO
Ubiquitination constitutes one of the most important signaling mechanisms in eukaryotes. Conventional ubiquitination is catalyzed by the universally conserved E1-E2-E3 three-enzyme cascade in an ATP-dependent manner. The newly identified SidE family effectors of the pathogen Legionella pneumophila ubiquitinate several human proteins by a different mechanism without engaging any of the conventional ubiquitination machinery. We now report the crystal structures of SidE alone and in complex with ubiquitin, NAD, and ADP-ribose, thereby capturing different conformations of SidE before and after ubiquitin and ligand binding. The structures of ubiquitin bound to both mART and PDE domains reveal several unique features of the two reaction steps catalyzed by SidE. Further, the structural and biochemical results demonstrate that SidE family members do not recognize specific structural folds of the substrate proteins. Our studies provide both structural explanations for the functional observations and new insights into the molecular mechanisms of this non-canonical ubiquitination machinery.
Assuntos
Proteínas de Bactérias/química , Legionella pneumophila/metabolismo , Diester Fosfórico Hidrolases/química , Ubiquitina/química , Proteínas de Bactérias/metabolismo , Biocatálise , Cristalografia por Raios X , Dimerização , Diester Fosfórico Hidrolases/metabolismo , Ligação Proteica , Domínios Proteicos , Estrutura Quaternária de Proteína , Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
Cas12a, also known as Cpf1, is a type V-A CRISPR-Cas RNA-guided endonuclease that is used for genome editing based on its ability to generate specific dsDNA breaks. Here, we show cryo-EM structures of intermediates of the cleavage reaction, thus visualizing three protein regions that sense the crRNA-DNA hybrid assembly triggering the catalytic activation of Cas12a. Single-molecule FRET provides the thermodynamics and kinetics of the conformational activation leading to phosphodiester bond hydrolysis. These findings illustrate why Cas12a cuts its target DNA and unleashes unspecific cleavage activity, degrading ssDNA molecules after activation. In addition, we show that other crRNAs are able to displace the R-loop inside the protein after target DNA cleavage, terminating indiscriminate ssDNA degradation. We propose a model whereby the conformational activation of the enzyme results in indiscriminate ssDNA cleavage. The displacement of the R-loop by a new crRNA molecule will reset Cas12a specificity, targeting new DNAs.
Assuntos
Proteínas de Bactérias/química , Sistemas CRISPR-Cas , Clivagem do DNA , DNA de Cadeia Simples/química , Francisella/química , RNA Guia de Cinetoplastídeos/química , Proteínas de Bactérias/genética , Catálise , DNA de Cadeia Simples/genética , Francisella/genética , Edição de Genes , RNA Guia de Cinetoplastídeos/genéticaRESUMO
Hydrogen gas-evolving membrane-bound hydrogenase (MBH) and quinone-reducing complex I are homologous respiratory complexes with a common ancestor, but a structural basis for their evolutionary relationship is lacking. Here, we report the cryo-EM structure of a 14-subunit MBH from the hyperthermophile Pyrococcus furiosus. MBH contains a membrane-anchored hydrogenase module that is highly similar structurally to the quinone-binding Q-module of complex I while its membrane-embedded ion-translocation module can be divided into a H+- and a Na+-translocating unit. The H+-translocating unit is rotated 180° in-membrane with respect to its counterpart in complex I, leading to distinctive architectures for the two respiratory systems despite their largely conserved proton-pumping mechanisms. The Na+-translocating unit, absent in complex I, resembles that found in the Mrp H+/Na+ antiporter and enables hydrogen gas evolution by MBH to establish a Na+ gradient for ATP synthesis near 100°C. MBH also provides insights into Mrp structure and evolution of MBH-based respiratory enzymes.
Assuntos
Proteínas Arqueais/metabolismo , Hidrogenase/metabolismo , Pyrococcus furiosus/metabolismo , Sequência de Aminoácidos , Proteínas Arqueais/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Membrana Celular/química , Membrana Celular/metabolismo , Microscopia Crioeletrônica , Complexo I de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/metabolismo , Evolução Molecular , Hidrogênio/metabolismo , Hidrogenase/química , Hidrogenase/genética , Mutagênese , Estrutura Quaternária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Sódio/química , Sódio/metabolismo , Trocadores de Sódio-Hidrogênio/química , Trocadores de Sódio-Hidrogênio/metabolismoRESUMO
Ubiquitin E3 ligases control every aspect of eukaryotic biology by promoting protein ubiquitination and degradation. At the end of a three-enzyme cascade, ubiquitin ligases mediate the transfer of ubiquitin from an E2 ubiquitin-conjugating enzyme to specific substrate proteins. Early investigations of E3s of the RING (really interesting new gene) and HECT (homologous to the E6AP carboxyl terminus) types shed light on their enzymatic activities, general architectures, and substrate degron-binding modes. Recent studies have provided deeper mechanistic insights into their catalysis, activation, and regulation. In this review, we summarize the current progress in structure-function studies of ubiquitin ligases as well as exciting new discoveries of novel classes of E3s and diverse substrate recognition mechanisms. Our increased understanding of ubiquitin ligase function and regulation has provided the rationale for developing E3-targeting therapeutics for the treatment of human diseases.