RESUMO
CRISPR-associated transposons (CASTs) are mobile genetic elements that co-opt CRISPR-Cas systems for RNA-guided DNA transposition. CASTs integrate large DNA cargos into the attachment (att) site independently of homology-directed repair and thus hold promise for eukaryotic genome engineering. However, the functional diversity and complexity of CASTs hinder an understanding of their mechanisms. Here, we present the high-resolution cryoelectron microscopy (cryo-EM) structure of the reconstituted â¼1 MDa post-transposition complex of the type V-K CAST, together with different assembly intermediates and diverse TnsC filament lengths, thus enabling the recapitulation of the integration complex formation. The results of mutagenesis experiments probing the roles of specific residues and TnsB-binding sites show that transposition activity can be enhanced and suggest that the distance between the PAM and att sites is determined by the lengths of the TnsB C terminus and the TnsC filament. This singular model of RNA-guided transposition provides a foundation for repurposing the system for genome-editing applications.
Assuntos
Sistemas CRISPR-Cas , Microscopia Crioeletrônica , Elementos de DNA Transponíveis , Elementos de DNA Transponíveis/genética , Sítios de Ligação , Edição de Genes/métodos , Modelos Moleculares , RNA Guia de Sistemas CRISPR-Cas/genética , RNA Guia de Sistemas CRISPR-Cas/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Conformação Proteica , Conformação de Ácido NucleicoRESUMO
Retrons are toxin-antitoxin systems protecting bacteria against bacteriophages via abortive infection. The Retron-Eco1 antitoxin is formed by a reverse transcriptase (RT) and a non-coding RNA (ncRNA)/multi-copy single-stranded DNA (msDNA) hybrid that neutralizes an uncharacterized toxic effector. Yet, the molecular mechanisms underlying phage defense remain unknown. Here, we show that the N-glycosidase effector, which belongs to the STIR superfamily, hydrolyzes NAD+ during infection. Cryoelectron microscopy (cryo-EM) analysis shows that the msDNA stabilizes a filament that cages the effector in a low-activity state in which ADPr, a NAD+ hydrolysis product, is covalently linked to the catalytic E106 residue. Mutations shortening the msDNA induce filament disassembly and the effector's toxicity, underscoring the msDNA role in immunity. Furthermore, we discovered a phage-encoded Retron-Eco1 inhibitor (U56) that binds ADPr, highlighting the intricate interplay between retron systems and phage evolution. Our work outlines the structural basis of Retron-Eco1 defense, uncovering ADPr's pivotal role in immunity.
Assuntos
Bacteriófagos , Microscopia Crioeletrônica , NAD , NAD/metabolismo , Bacteriófagos/genética , Bacteriófagos/metabolismo , Bacteriófagos/imunologia , Hidrólise , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/imunologia , Sistemas Toxina-Antitoxina/genética , Escherichia coli/virologia , Escherichia coli/genética , Escherichia coli/imunologia , Escherichia coli/metabolismoRESUMO
The dopamine transporter (DAT) is crucial for regulating dopamine signalling and is the prime mediator for the rewarding and addictive effects of cocaine1. As part of the neurotransmitter sodium symporter family, DAT uses the Na+ gradient across cell membranes to transport dopamine against its chemical gradient2. The transport mechanism involves both intra- and extracellular gates that control substrate access to a central site. However, the molecular intricacies of this process and the inhibitory mechanism of cocaine have remained unclear. Here, we present the molecular structure of human DAT in complex with cocaine at a resolution of 2.66 Å. Our findings reveal that DAT adopts the expected LeuT-fold, posing in an outward-open conformation with cocaine bound at the central (S1) site. Notably, while an Na+ occupies the second Na+ site (Na2), the Na1 site seems to be vacant, with the side chain of Asn82 occupying the presumed Na+ space. This structural insight elucidates the mechanism for the cocaine inhibition of human DAT and deepens our understanding of neurotransmitter transport. By shedding light on the molecular underpinnings of how cocaine acts, our study lays a foundation for the development of targeted medications to combat addiction.
Assuntos
Cocaína , Proteínas da Membrana Plasmática de Transporte de Dopamina , Humanos , Sítios de Ligação , Cocaína/metabolismo , Cocaína/química , Cocaína/farmacologia , Microscopia Crioeletrônica , Dopamina/metabolismo , Dopamina/química , Proteínas da Membrana Plasmática de Transporte de Dopamina/química , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/ultraestrutura , Modelos Moleculares , Neurotransmissores/metabolismo , Ligação Proteica , Conformação Proteica/efeitos dos fármacos , Sódio/química , Sódio/metabolismoRESUMO
Cmr-ß is a type III-B CRISPR-Cas complex that, upon target RNA recognition, unleashes a multifaceted immune response against invading genetic elements, including single-stranded DNA (ssDNA) cleavage, cyclic oligoadenylate synthesis, and also a unique UA-specific single-stranded RNA (ssRNA) hydrolysis by the Cmr2 subunit. Here, we present the structure-function relationship of Cmr-ß, unveiling how binding of the target RNA regulates the Cmr2 activities. Cryoelectron microscopy (cryo-EM) analysis revealed the unique subunit architecture of Cmr-ß and captured the complex in different conformational stages of the immune response, including the non-cognate and cognate target-RNA-bound complexes. The binding of the target RNA induces a conformational change of Cmr2, which together with the complementation between the 5' tag in the CRISPR RNAs (crRNA) and the 3' antitag of the target RNA activate different configurations in a unique loop of the Cmr3 subunit, which acts as an allosteric sensor signaling the self- versus non-self-recognition. These findings highlight the diverse defense strategies of type III complexes.
Assuntos
Imunidade Adaptativa/fisiologia , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/fisiologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Proteínas Arqueais/química , Proteínas Arqueais/fisiologia , Proteínas Arqueais/ultraestrutura , Proteínas Associadas a CRISPR/ultraestrutura , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/fisiologia , Microscopia Crioeletrônica , DNA de Cadeia Simples/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica , RNA Mensageiro/metabolismo , Relação Estrutura-Atividade , Sulfolobus/genética , Sulfolobus/fisiologiaRESUMO
Transcription initiation involves the conversion from closed promoter complexes, comprising RNA polymerase (RNAP) and double-stranded promoter DNA, to open complexes, in which the enzyme is able to access the DNA template in a single-stranded form. The complex between bacterial RNAP and its major variant sigma factor sigma(54) remains as a closed complex until ATP hydrolysis-dependent remodeling by activator proteins occurs. This remodeling facilitates DNA melting and allows the transition to the open complex. Here we present cryoelectron microscopy reconstructions of bacterial RNAP in complex with sigma(54) alone, and of RNAP-sigma(54) with an AAA+ activator. Together with photo-crosslinking data that establish the location of promoter DNA within the complexes, we explain why the RNAP-sigma(54) closed complex is unable to access the DNA template and propose how the structural changes induced by activator binding can initiate conformational changes that ultimately result in formation of the open complex.
Assuntos
RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Ativação Enzimática , Archaea/genética , Archaea/metabolismo , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Bactérias/genética , Bactérias/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Holoenzimas/genética , Holoenzimas/metabolismo , Processamento de Imagem Assistida por Computador , Modelos Moleculares , Regiões Promotoras Genéticas , Conformação Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Polimerase Sigma 54/química , RNA Polimerase Sigma 54/genética , RNA Polimerase Sigma 54/metabolismo , Moldes GenéticosRESUMO
In higher plants a variable number of peripheral LHCII trimers can strongly (S), moderately (M) or loosely (L) associate with the dimeric PSII core (C2) complex via monomeric Lhcb proteins to form PSII-LHCII supercomplexes with different structural organizations. By solubilizing isolated stacked pea thylakoid membranes either with the α or ß isomeric forms of the detergent n-dodecyl-D-maltoside, followed by sucrose density ultracentrifugation, we previously showed that PSII-LHCII supercomplexes of types C2S2M2 and C2S2, respectively, can be isolated [S. Barera et al., Phil. Trans. R Soc. B 67 (2012) 3389-3399]. Here we analysed their protein composition by applying extensive bottom-up and top-down mass spectrometry on the two forms of the isolated supercomplexes. In this way, we revealed the presence of the antenna proteins Lhcb3 and Lhcb6 and of the extrinsic polypeptides PsbP, PsbQ and PsbR exclusively in the C2S2M2 supercomplex. Other proteins of the PSII core complex, common to the C2S2M2 and C2S2 supercomplexes, including the low molecular mass subunits, were also detected and characterized. To complement the proteomic study with structural information, we performed negative stain transmission electron microscopy and single particle analysis on the PSII-LHCII supercomplexes isolated from pea thylakoid membranes solubilized with n-dodecyl-α-D-maltoside. We observed the C2S2M2 supercomplex in its intact form as the largest PSII complex in our preparations. Its dataset was further analysed in silico, together with that of the second largest identified sub-population, corresponding to its C2S2 subcomplex. In this way, we calculated 3D electron density maps for the C2S2M2 and C2S2 supercomplexes, approaching respectively 30 and 28Å resolution, extended by molecular modelling towards the atomic level. This article is part of a special issue entitled: photosynthesis research for sustainability: keys to produce clean energy.
Assuntos
Complexos de Proteínas Captadores de Luz/química , Complexo de Proteína do Fotossistema II/química , Proteômica/métodos , Microscopia Eletrônica de Transmissão , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tilacoides/químicaRESUMO
Bacteria have evolved a variety of mechanisms for developing community-based biofilms. These bacterial aggregates are of clinical importance, as they are a major source of recurrent disease. Bacterial surface fibers (pili) permit adherence to biotic and abiotic substrates, often in a highly specific manner. The Escherichia coli common pilus (ECP) represents a remarkable family of extracellular fibers that are associated with both disease-causing and commensal strains. ECP plays a dual role in early-stage biofilm development and host cell recognition. Despite being the most common fimbrial structure, relatively little is known regarding its biogenesis, architecture, and function. Here we report atomic-resolution insight into the biogenesis and architecture of ECP. We also derive a structural model for entwined ECP fibers that not only illuminates interbacteria communication during biofilm formation but also provides a useful foundation for the design of novel nanofibers.
Assuntos
Biofilmes , Escherichia coli/crescimento & desenvolvimento , Fímbrias Bacterianas/metabolismo , Fímbrias Bacterianas/fisiologia , Adesinas Bacterianas , Fenômenos Fisiológicos Bacterianos , Cristalografia por Raios X/métodos , Escherichia coli/fisiologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/fisiologia , Proteínas de Fímbrias/química , Proteínas de Fímbrias/fisiologia , Variação Genética , Microscopia Eletrônica/métodos , Modelos Genéticos , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/fisiologia , Conformação Molecular , Nanotecnologia/métodosRESUMO
Protein secretion systems in Gram-negative bacteria evolved into a variety of molecular nanomachines. They are related to cell envelope complexes, which are involved in assembly of surface appendages or transport of solutes. They are classified as types, the most recent addition being the type VI secretion system (T6SS). The T6SS displays similarities to bacteriophage tail, which drives DNA injection into bacteria. The Hcp protein is related to the T4 bacteriophage tail tube protein gp19, whereas VgrG proteins structurally resemble the gp27/gp5 puncturing device of the phage. The tube and spike of the phage are pushed through the bacterial envelope upon contraction of a tail sheath composed of gp18. In Vibrio cholerae it was proposed that VipA and VipB assemble into a tail sheathlike structure. Here we confirm these previous data by showing that HsiB1 and HsiC1 of the Pseudomonas aeruginosa H1-T6SS assemble into tubules resulting from stacking of cogwheel-like structures showing predominantly 12-fold symmetry. The internal diameter of the cogwheels is ~100 Å, which is large enough to accommodate an Hcp tube whose external diameter has been reported to be 85 Å. The N-terminal 212 residues of HsiC1 are sufficient to form a stable complex with HsiB1, but the C terminus of HsiC1 is essential for the formation of the tubelike structure. Bioinformatics analysis suggests that HsiC1 displays similarities to gp18-like proteins in its C-terminal region. In conclusion, we provide further structural and mechanistic insights into the T6SS and show that a phage sheathlike structure is likely to be a conserved element across all T6SSs.
Assuntos
Proteínas de Bactérias/fisiologia , Sistemas de Secreção Bacterianos/fisiologia , Bacteriófagos/metabolismo , Regulação Bacteriana da Expressão Gênica , Pseudomonas aeruginosa/metabolismo , Sequência de Aminoácidos , Biologia Computacional/métodos , Deleção de Genes , Microscopia Eletrônica/métodos , Dados de Sequência Molecular , Plasmídeos/metabolismo , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Técnicas do Sistema de Duplo-HíbridoRESUMO
Multidrug resistance-associated protein 4 (MRP4) is an ATP-binding cassette (ABC) transporter expressed at multiple tissue barriers where it actively extrudes a wide variety of drug compounds. Overexpression of MRP4 provides resistance to clinically used antineoplastic agents, making it a highly attractive therapeutic target for countering multidrug resistance. Here, we report cryo-EM structures of multiple physiologically relevant states of lipid bilayer-embedded human MRP4, including complexes between MRP4 and two widely used chemotherapeutic agents and a complex between MRP4 and its native substrate. The structures display clear similarities and distinct differences in the coordination of these chemically diverse substrates and, in combination with functional and mutational analysis, reveal molecular details of the transport mechanism. Our study provides key insights into the unusually broad substrate specificity of MRP4 and constitutes an important contribution toward a general understanding of multidrug transporters.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Antineoplásicos , Humanos , Antineoplásicos/farmacologia , Resistência a Múltiplos Medicamentos , Proteínas de Membrana Transportadoras , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismoRESUMO
CRISPR-Cas12j is a recently identified family of miniaturized RNA-guided endonucleases from phages. These ribonucleoproteins provide a compact scaffold gathering all key activities of a genome editing tool. We provide the first structural insight into the Cas12j family by determining the cryoEM structure of Cas12j3/R-loop complex after DNA cleavage. The structure reveals the machinery for PAM recognition, hybrid assembly and DNA cleavage. The crRNA-DNA hybrid is directed to the stop domain that splits the hybrid, guiding the T-strand towards the catalytic site. The conserved RuvC insertion is anchored in the stop domain and interacts along the phosphate backbone of the crRNA in the hybrid. The assembly of a hybrid longer than 12-nt activates catalysis through key functional residues in the RuvC insertion. Our findings suggest why Cas12j unleashes unspecific ssDNA degradation after activation. A site-directed mutagenesis analysis supports the DNA cutting mechanism, providing new avenues to redesign CRISPR-Cas12j nucleases for genome editing.
Assuntos
Sistemas CRISPR-Cas , Endodesoxirribonucleases/química , Edição de Genes , Bacteriófagos/enzimologia , Bacteriófagos/genética , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Domínio Catalítico , Microscopia Crioeletrônica , Clivagem do DNA , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação Proteica , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , RNA Viral/genética , RNA Viral/metabolismoRESUMO
Bacterial sigma (sigma) factors confer gene specificity upon the RNA polymerase, the central enzyme that catalyses gene transcription. The binding of the alternative sigma factor sigma(54) confers upon the RNA polymerase special functional and regulatory properties, making it suited for control of several major adaptive responses. Here, we summarize our current understanding of the interactions the sigma(54) factor makes with the bacterial transcription machinery.
Assuntos
Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Regiões Promotoras Genéticas , RNA Polimerase Sigma 54/química , Transcrição Gênica , Bactérias/enzimologia , Bactérias/genética , RNA Polimerase Sigma 54/metabolismo , RNA Bacteriano/genéticaRESUMO
The MCM complex from the archaeon Methanother-mobacter thermautotrophicus is a model for the eukaryotic MCM2-7 helicase. We present electron-microscopy single-particle reconstructions of a DNA treated M.thermautotrophicus MCM sample and a ADP.AlF(x) treated sample, respectively assembling as double hexamers and double heptamers. The electron-density maps display an unexpected asymmetry between the two rings, suggesting that large conformational changes can occur within the complex. The structure of the MCM N-terminal domain, as well as the AAA+ and the C-terminal HTH dom-ains of ZraR can be fitted into the reconstructions. Distinct configurations can be modelled for the AAA+ and the HTH domains, suggesting the nature of the conformational change within the complex. The pre-sensor 1 and the helix 2 insertions, important for the activity, can be located pointing towards the centre of the channel in the presence of DNA. We propose a mechanistic model for the helicase activity, based on a ligand-controlled rotation of the AAA+ subunits.
Assuntos
Proteínas Arqueais/química , DNA Helicases/química , Methanobacteriaceae/enzimologia , Modelos Moleculares , Adenosina Trifosfatases/química , Proteínas Arqueais/metabolismo , Proteínas Arqueais/ultraestrutura , DNA/química , DNA Helicases/metabolismo , DNA Helicases/ultraestrutura , Processamento de Imagem Assistida por Computador , Estrutura Terciária de ProteínaRESUMO
α-synuclein (αS) is an intrinsically disordered protein whose fibrillar aggregates are the major constituents of Lewy bodies in Parkinson's disease. Although the specific function of αS is still unclear, a general consensus is forming that it has a key role in regulating the process of neurotransmitter release, which is associated with the mediation of synaptic vesicle interactions and assembly. Here we report the analysis of wild-type αS and two mutational variants linked to familial Parkinson's disease to describe the structural basis of a molecular mechanism enabling αS to induce the clustering of synaptic vesicles. We provide support for this 'double-anchor' mechanism by rationally designing and experimentally testing a further mutational variant of αS engineered to promote stronger interactions between synaptic vesicles. Our results characterize the nature of the active conformations of αS that mediate the clustering of synaptic vesicles, and indicate their relevance in both functional and pathological contexts.
Assuntos
Vesículas Sinápticas/metabolismo , alfa-Sinucleína/metabolismo , Animais , Membrana Celular/metabolismo , Escherichia coli , Simulação de Dinâmica Molecular , Ratos , Vesículas Sinápticas/ultraestrutura , alfa-Sinucleína/genética , alfa-Sinucleína/isolamento & purificaçãoRESUMO
It was the work of Jan Anderson, together with Keith Boardman, that showed it was possible to physically separate photosystem I (PSI) from photosystem II (PSII), and it was Jan Anderson who realized the importance of this work in terms of the fluid-mosaic model as applied to the thylakoid membrane. Since then, there has been a steady progress in the development of biochemical procedures to isolate PSII and PSI both for physical and structural studies. Dodecylmaltoside (DM) has emerged as an effective mild detergent for this purpose. DM is a glucoside-based surfactant with a bulky hydrophilic head group composed of two sugar rings and a non-charged alkyl glycoside chain. Two isomers of this molecule exist, differing only in the configuration of the alkyl chain around the anomeric centre of the carbohydrate head group, axial in α-DM and equatorial in ß-DM. We have compared the use of α-DM and ß-DM for the isolation of supramolecular complexes of PSII by a single-step solubilization of stacked thylakoid membranes isolated from peas. As a result, we have optimized conditions to obtain homogeneous preparations of the C(2)S(2)M(2) and C(2)S(2) supercomplexes following the nomenclature of Dekker & Boekema (2005 Biochim. Biophys. Acta 1706, 12-39). These PSII-LHCII supercomplexes were subjected to biochemical and structural analyses.
Assuntos
Glucosídeos/química , Complexos de Proteínas Captadores de Luz/química , Complexo de Proteína do Fotossistema II/química , Pisum sativum/química , Proteínas das Membranas dos Tilacoides/isolamento & purificação , Tilacoides/química , Cloreto de Cálcio/química , Cristalografia por Raios X , Cianobactérias/química , Eletroforese em Gel de Poliacrilamida , Complexos de Proteínas Captadores de Luz/isolamento & purificação , Modelos Moleculares , Oxigênio/química , Complexo de Proteína do Fotossistema II/isolamento & purificação , Ligação Proteica , Proteínas das Membranas dos Tilacoides/química , Ultracentrifugação/métodosRESUMO
Bacteria produce functional amyloid fibers called curli in a controlled, noncytotoxic manner. These extracellular fimbriae enable biofilm formation and promote pathogenicity. Understanding curli biogenesis is important for appreciating microbial lifestyles and will offer clues as to how disease-associated human amyloid formation might be ameliorated. Proteins encoded by the curli specific genes (csgA-G) are required for curli production. We have determined the structure of CsgC and derived the first structural model of the outer-membrane subunit translocator CsgG. Unexpectedly, CsgC is related to the N-terminal domain of DsbD, both in structure and oxido-reductase capability. Furthermore, we show that CsgG belongs to the nascent class of helical outer-membrane macromolecular exporters. A cysteine in a CsgG transmembrane helix is a potential target of CsgC, and mutation of this residue influences curli assembly. Our study provides the first high-resolution structural insights into curli biogenesis.
Assuntos
Biofilmes , Escherichia coli O157/fisiologia , Proteínas de Escherichia coli/química , Fímbrias Bacterianas/química , Lipoproteínas/química , Multimerização Proteica , Motivos de Aminoácidos , Sequência de Aminoácidos , Cristalografia por Raios X , Escherichia coli O157/crescimento & desenvolvimento , Escherichia coli O157/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fímbrias Bacterianas/metabolismo , Lipoproteínas/genética , Lipoproteínas/metabolismo , Microscopia Eletrônica , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Oxirredução , Estrutura Terciária de ProteínaRESUMO
bEBPs (bacterial enhancer-binding proteins) are AAA+ (ATPase associated with various cellular activities) transcription activators that activate gene transcription through a specific bacterial sigma factor, sigma(54). Sigma(54)-RNAP (RNA polymerase) binds to promoter DNA sites and forms a stable closed complex, unable to proceed to transcription. The closed complex must be remodelled using energy from ATP hydrolysis provided by bEBPs to melt DNA and initiate transcription. Recently, large amounts of structural and biochemical data have produced insights into how ATP hydrolysis within the active site of bEBPs is coupled to the re-modelling of the closed complex. In the present article, we review some of the key nucleotides, mutations and techniques used and how they have contributed towards our understanding of the function of bEBPs.
Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , Hidrólise , Nucleotídeos/metabolismo , Conformação ProteicaRESUMO
The primary candidate for the eukaryotic replicative helicase is the MCM2-7 complex, a hetero-oligomer formed by six AAA+ paralogous polypeptides. A simplified model for structure-function studies is the homo-oligomeric orthologue from the archaeon Methanothermobacter thermoautotrophicus. The crystal structure of the DNA-interacting N-terminal domain of this homo-oligomer revealed a double hexamer in a head-to-head configuration; single-particle electron microscopy studies have shown that the full-length protein complex can form both single and double rings, in which each ring can consist of a cyclical arrangement of six or seven subunits. Using single-particle techniques and especially multivariate statistical symmetry analysis, we have assessed the changes in stoichiometry that the complex undergoes when treated with various nucleotide analogues or when binding a double-stranded DNA fragment. We found that the binding of nucleotides or of double-stranded DNA leads to the preferred formation of double-ring structures. Specifically, the protein complex is present as a double heptamer when treated with a nucleotide analogue, but it is rather found as a double hexamer when complexed with double-stranded DNA. The possible physiological role of the various stoichiometries of the complex is discussed in the light of the proposed mechanisms of helicase activity.
Assuntos
Proteínas Arqueais/metabolismo , Cromossomos de Archaea/metabolismo , DNA Helicases/química , DNA Helicases/metabolismo , Difosfato de Adenosina/farmacologia , Proteínas Arqueais/química , Proteínas Arqueais/isolamento & purificação , Proteínas Arqueais/ultraestrutura , Cromossomos de Archaea/química , DNA/metabolismo , DNA Helicases/genética , Escherichia coli/genética , Methanobacteriaceae/enzimologia , Modelos Biológicos , Nucleotídeos/metabolismo , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Especificidade por Substrato , Transformação GenéticaRESUMO
In eukaryotes, a family of six homologous minichromosome maintenance (MCM) proteins has a key function in ensuring that DNA replication occurs only once before cell division. Whereas all eukaryotes have six paralogues, in some Archaea a single protein forms a homomeric assembly. The complex is likely to function as a helicase during DNA replication. We have used electron microscopy to obtain a three-dimensional reconstruction of the full-length MCM from Methanobacterium thermoautotrophicum. Six monomers are arranged around a sixfold axis, generating a ring-shaped molecule with a large central cavity and lateral holes. The channel running through the molecule can easily accommodate double-stranded DNA. The crystal structure of the amino-terminal fragment of MCM and a model for the AAA+ hexamer have been docked into the map, whereas additional electron density suggests that the carboxy-terminal domain is located at the interface between the two domains. The structure suggests that the MCM complex is likely to act in a different manner to traditional hexameric helicases and is likely to bear more similarity to the SV40 large T antigen or to double-stranded DNA translocases.
Assuntos
Archaea/química , Proteínas Arqueais/química , DNA Helicases/química , Estrutura Quaternária de Proteína , Microscopia Eletrônica , Mitocôndrias/genética , Mitocôndrias/metabolismo , Modelos Moleculares , Estrutura Terciária de ProteínaRESUMO
Termination of protein synthesis occurs when the messenger RNA presents a stop codon in the ribosomal aminoacyl (A) site. Class I release factor proteins (RF1 or RF2) are believed to recognize stop codons via tripeptide motifs, leading to release of the completed polypeptide chain from its covalent attachment to transfer RNA in the ribosomal peptidyl (P) site. Class I RFs possess a conserved GGQ amino-acid motif that is thought to be involved directly in protein-transfer-RNA bond hydrolysis. Crystal structures of bacterial and eukaryotic class I RFs have been determined, but the mechanism of stop codon recognition and peptidyl-tRNA hydrolysis remains unclear. Here we present the structure of the Escherichia coli ribosome in a post-termination complex with RF2, obtained by single-particle cryo-electron microscopy (cryo-EM). Fitting the known 70S and RF2 structures into the electron density map reveals that RF2 adopts a different conformation on the ribosome when compared with the crystal structure of the isolated protein. The amino-terminal helical domain of RF2 contacts the factor-binding site of the ribosome, the 'SPF' loop of the protein is situated close to the mRNA, and the GGQ-containing domain of RF2 interacts with the peptidyl-transferase centre (PTC). By connecting the ribosomal decoding centre with the PTC, RF2 functionally mimics a tRNA molecule in the A site. Translational termination in eukaryotes is likely to be based on a similar mechanism.