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2.
Nat Commun ; 12(1): 5207, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34471127

RESUMO

Uropathogenic Escherichia coli assemble surface structures termed pili or fimbriae to initiate infection of the urinary tract. P pili facilitate bacterial colonization of the kidney and pyelonephritis. P pili are assembled through the conserved chaperone-usher pathway. Much of the structural and functional understanding of the chaperone-usher pathway has been gained through investigations of type 1 pili, which promote binding to the bladder and cystitis. In contrast, the structural basis for P pilus biogenesis at the usher has remained elusive. This is in part due to the flexible and variable-length P pilus tip fiber, creating structural heterogeneity, and difficulties isolating stable P pilus assembly intermediates. Here, we circumvent these hindrances and determine cryo-electron microscopy structures of the activated PapC usher in the process of secreting two- and three-subunit P pilus assembly intermediates, revealing processive steps in P pilus biogenesis and capturing new conformational dynamics of the usher assembly machine.


Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Fímbrias Bacterianas/química , Fímbrias Bacterianas/metabolismo , Escherichia coli Uropatogênica/metabolismo , Microscopia Crioeletrônica , Proteínas de Escherichia coli/genética , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/genética , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Ligação Proteica , Conformação Proteica , Escherichia coli Uropatogênica/genética
3.
Nat Commun ; 12(1): 5254, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34489436

RESUMO

Pdr5, a member of the extensive ABC transporter superfamily, is representative of a clinically relevant subgroup involved in pleiotropic drug resistance. Pdr5 and its homologues drive drug efflux through uncoupled hydrolysis of nucleotides, enabling organisms such as baker's yeast and pathogenic fungi to survive in the presence of chemically diverse antifungal agents. Here, we present the molecular structure of Pdr5 solved with single particle cryo-EM, revealing details of an ATP-driven conformational cycle, which mechanically drives drug translocation through an amphipathic channel, and a clamping switch within a conserved linker loop that acts as a nucleotide sensor. One half of the transporter remains nearly invariant throughout the cycle, while its partner undergoes changes that are transmitted across inter-domain interfaces to support a peristaltic motion of the pumped molecule. The efflux model proposed here rationalises the pleiotropic impact of Pdr5 and opens new avenues for the development of effective antifungal compounds.


Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Trifosfato de Adenosina/metabolismo , Domínio Catalítico , Microscopia Crioeletrônica , Detergentes/química , Farmacorresistência Fúngica/genética , Pleiotropia Genética , Hidrólise , Mutação , Conformação Proteica , Domínios Proteicos , Rodaminas/química , Rodaminas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Vanadatos/química , Vanadatos/metabolismo
4.
Nat Commun ; 12(1): 5277, 2021 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-34489474

RESUMO

The pyruvate dehydrogenase complex (PDHc) links glycolysis to the citric acid cycle by converting pyruvate into acetyl-coenzyme A. PDHc encompasses three enzymatically active subunits, namely pyruvate dehydrogenase, dihydrolipoyl transacetylase, and dihydrolipoyl dehydrogenase. Dihydrolipoyl transacetylase is a multidomain protein comprising a varying number of lipoyl domains, a peripheral subunit-binding domain, and a catalytic domain. It forms the structural core of the complex, provides binding sites for the other enzymes, and shuffles reaction intermediates between the active sites through covalently bound lipoyl domains. The molecular mechanism by which this shuttling occurs has remained elusive. Here, we report a cryo-EM reconstruction of the native E. coli dihydrolipoyl transacetylase core in a resting state. This structure provides molecular details of the assembly of the core and reveals how the lipoyl domains interact with the core at the active site.


Assuntos
Proteínas de Escherichia coli/química , Complexo Piruvato Desidrogenase/química , Complexo Piruvato Desidrogenase/metabolismo , Domínio Catalítico , Microscopia Crioeletrônica , Di-Hidrolipoil-Lisina-Resíduo Acetiltransferase/química , Di-Hidrolipoil-Lisina-Resíduo Acetiltransferase/metabolismo , Proteínas de Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Lisina/análogos & derivados , Lisina/química , Lisina/metabolismo , Modelos Moleculares , Domínios Proteicos , Complexo Piruvato Desidrogenase/isolamento & purificação , Ácido Tióctico/análogos & derivados , Ácido Tióctico/química , Ácido Tióctico/metabolismo
5.
Nat Commun ; 12(1): 5236, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34475399

RESUMO

New drugs are urgently needed to combat the global TB epidemic. Targeting simultaneously multiple respiratory enzyme complexes of Mycobacterium tuberculosis is regarded as one of the most effective treatment options to shorten drug administration regimes, and reduce the opportunity for the emergence of drug resistance. During infection and proliferation, the cytochrome bd oxidase plays a crucial role for mycobacterial pathophysiology by maintaining aerobic respiration at limited oxygen concentrations. Here, we present the cryo-EM structure of the cytochrome bd oxidase from M. tuberculosis at 2.5 Å. In conjunction with atomistic molecular dynamics (MD) simulation studies we discovered a previously unknown MK-9-binding site, as well as a unique disulfide bond within the Q-loop domain that defines an inactive conformation of the canonical quinol oxidation site in Actinobacteria. Our detailed insights into the long-sought atomic framework of the cytochrome bd oxidase from M. tuberculosis will form the basis for the design of highly specific drugs to act on this enzyme.


Assuntos
Grupo dos Citocromos b/química , Grupo dos Citocromos d/química , Complexo de Proteínas da Cadeia de Transporte de Elétrons/química , Mycobacterium tuberculosis/enzimologia , Proteínas de Bactérias/química , Sítios de Ligação , Microscopia Crioeletrônica , Simulação de Dinâmica Molecular , Oxirredutases/química , Conformação Proteica , Subunidades Proteicas , Vitamina K 2/análogos & derivados , Vitamina K 2/química
6.
Nat Commun ; 12(1): 4817, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34376662

RESUMO

Engineered ectodomain trimer immunogens based on BG505 envelope glycoprotein are widely utilized as components of HIV vaccine development platforms. In this study, we used rhesus macaques to evaluate the immunogenicity of several stabilized BG505 SOSIP constructs both as free trimers and presented on a nanoparticle. We applied a cryoEM-based method for high-resolution mapping of polyclonal antibody responses elicited in immunized animals (cryoEMPEM). Mutational analysis coupled with neutralization assays were used to probe the neutralization potential at each epitope. We demonstrate that cryoEMPEM data can be used for rapid, high-resolution analysis of polyclonal antibody responses without the need for monoclonal antibody isolation. This approach allowed to resolve structurally distinct classes of antibodies that bind overlapping sites. In addition to comprehensive mapping of commonly targeted neutralizing and non-neutralizing epitopes in BG505 SOSIP immunogens, our analysis revealed that epitopes comprising engineered stabilizing mutations and of partially occupied glycosylation sites can be immunogenic.


Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Formação de Anticorpos/imunologia , Anticorpos Anti-HIV/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/ultraestrutura , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/ultraestrutura , Microscopia Crioeletrônica/métodos , Epitopos/química , Epitopos/imunologia , Epitopos/metabolismo , Glicosilação , Anticorpos Anti-HIV/química , Anticorpos Anti-HIV/ultraestrutura , Infecções por HIV/imunologia , Infecções por HIV/prevenção & controle , Infecções por HIV/virologia , HIV-1/genética , HIV-1/imunologia , HIV-1/metabolismo , Humanos , Macaca mulatta , Modelos Moleculares , Conformação Proteica , Produtos do Gene env do Vírus da Imunodeficiência Humana/ultraestrutura
7.
Science ; 373(6556): 768-774, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34385391

RESUMO

CRISPR-associated transposition systems allow guide RNA-directed integration of a single DNA cargo in one orientation at a fixed distance from a programmable target sequence. We used cryo-electron microscopy (cryo-EM) to define the mechanism that underlies this process by characterizing the transposition regulator, TnsC, from a type V-K CRISPR-transposase system. In this scenario, polymerization of adenosine triphosphate-bound TnsC helical filaments could explain how polarity information is passed to the transposase. TniQ caps the TnsC filament, representing a universal mechanism for target information transfer in Tn7/Tn7-like elements. Transposase-driven disassembly establishes delivery of the element only to unused protospacers. Finally, TnsC transitions to define the fixed point of insertion, as revealed by structures with the transition state mimic ADP•AlF3 These mechanistic findings provide the underpinnings for engineering CRISPR-associated transposition systems for research and therapeutic applications.


Assuntos
Proteínas de Bactérias/química , Proteínas Associadas a CRISPR/química , Cianobactérias/química , Elementos de DNA Transponíveis , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Microscopia Crioeletrônica , Cianobactérias/genética , Cianobactérias/metabolismo , DNA Bacteriano/metabolismo , Modelos Moleculares , Conformação Proteica , Dobramento de Proteína , RNA Bacteriano/metabolismo , Transposases/química , Transposases/metabolismo
8.
Nat Commun ; 12(1): 5098, 2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34429416

RESUMO

KdpFABC, a high-affinity K+ pump, combines the ion channel KdpA and the P-type ATPase KdpB to secure survival at K+ limitation. Here, we apply a combination of cryo-EM, biochemical assays, and MD simulations to illuminate the mechanisms underlying transport and the coupling to ATP hydrolysis. We show that ions are transported via an intersubunit tunnel through KdpA and KdpB. At the subunit interface, the tunnel is constricted by a phenylalanine, which, by polarized cation-π stacking, controls K+ entry into the canonical substrate binding site (CBS) of KdpB. Within the CBS, ATPase coupling is mediated by the charge distribution between an aspartate and a lysine. Interestingly, individual elements of the ion translocation mechanism of KdpFABC identified here are conserved among a wide variety of P-type ATPases from different families. This leads us to the hypothesis that KdpB might represent an early descendant of a common ancestor of cation pumps.


Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Proteínas de Transporte de Cátions/química , Proteínas de Transporte de Cátions/metabolismo , Transporte de Íons/fisiologia , Ácido Aspártico/metabolismo , Sítios de Ligação , Proteínas de Transporte de Cátions/genética , Microscopia Crioeletrônica , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Lisina/metabolismo , Simulação de Dinâmica Molecular , Mutação , Fenilalanina , Potássio/metabolismo , Subunidades Proteicas , ATPase Trocadora de Sódio-Potássio
9.
Science ; 373(6553): 413-419, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34437114

RESUMO

Adenosine monophosphate (AMP)-activated protein kinase (AMPK) regulates metabolism in response to the cellular energy states. Under energy stress, AMP stabilizes the active AMPK conformation, in which the kinase activation loop (AL) is protected from protein phosphatases, thus keeping the AL in its active, phosphorylated state. At low AMP:ATP (adenosine triphosphate) ratios, ATP inhibits AMPK by increasing AL dynamics and accessibility. We developed conformation-specific antibodies to trap ATP-bound AMPK in a fully inactive, dynamic state and determined its structure at 3.5-angstrom resolution using cryo-electron microscopy. A 180° rotation and 100-angstrom displacement of the kinase domain fully exposes the AL. On the basis of the structure and supporting biophysical data, we propose a multistep mechanism explaining how adenine nucleotides and pharmacological agonists modulate AMPK activity by altering AL phosphorylation and accessibility.


Assuntos
Proteínas Quinases Ativadas por AMP/química , Proteínas Quinases Ativadas por AMP/imunologia , Proteínas Quinases Ativadas por AMP/metabolismo , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Microscopia Crioeletrônica , Humanos , Fragmentos Fab das Imunoglobulinas , Modelos Moleculares , Fosforilação , Conformação Proteica , Domínios Proteicos , Engenharia de Proteínas
10.
Nat Commun ; 12(1): 5064, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34417468

RESUMO

Ghrelin, also called "the hunger hormone", is a gastric peptide hormone that regulates food intake, body weight, as well as taste sensation, reward, cognition, learning and memory. One unique feature of ghrelin is its acylation, primarily with an octanoic acid, which is essential for its binding and activation of the ghrelin receptor, a G protein-coupled receptor. The multifaceted roles of ghrelin make ghrelin receptor a highly attractive drug target for growth retardation, obesity, and metabolic disorders. Here we present two cryo-electron microscopy structures of Gq-coupled ghrelin receptor bound to ghrelin and a synthetic agonist, GHRP-6. Analysis of these two structures reveals a unique binding pocket for the octanoyl group, which guides the correct positioning of the peptide to initiate the receptor activation. Together with mutational and functional data, our structures define the rules for recognition of the acylated peptide hormone and activation of ghrelin receptor, and provide structural templates to facilitate drug design targeting ghrelin receptor.


Assuntos
Oligopeptídeos/química , Receptores de Grelina/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular , Microscopia Crioeletrônica , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/química , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Grelina/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Oligopeptídeos/metabolismo , Ligação Proteica , Receptores de Grelina/metabolismo , Receptores de Grelina/ultraestrutura
11.
Nat Commun ; 12(1): 4754, 2021 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-34362932

RESUMO

Chaperonins are homo- or hetero-oligomeric complexes that use ATP binding and hydrolysis to facilitate protein folding. ATP hydrolysis exhibits both positive and negative cooperativity. The mechanism by which chaperonins coordinate ATP utilization in their multiple subunits remains unclear. Here we use cryoEM to study ATP binding in the homo-oligomeric archaeal chaperonin from Methanococcus maripaludis (MmCpn), consisting of two stacked rings composed of eight identical subunits each. Using a series of image classification steps, we obtained different structural snapshots of individual chaperonins undergoing the nucleotide binding process. We identified nucleotide-bound and free states of individual subunits in each chaperonin, allowing us to determine the ATP occupancy state of each MmCpn particle. We observe distinctive tertiary and quaternary structures reflecting variations in nucleotide occupancy and subunit conformations in each chaperonin complex. Detailed analysis of the nucleotide distribution in each MmCpn complex indicates that individual ATP binding events occur in a statistically random manner for MmCpn, both within and across the rings. Our findings illustrate the power of cryoEM to characterize a biochemical property of multi-subunit ligand binding cooperativity at the individual particle level.


Assuntos
Trifosfato de Adenosina/metabolismo , Microscopia Crioeletrônica , Chaperoninas do Grupo II/química , Chaperoninas do Grupo II/metabolismo , Chaperoninas/metabolismo , Hidrólise , Mathanococcus/metabolismo , Modelos Moleculares , Conformação Proteica , Dobramento de Proteína , Subunidades Proteicas/metabolismo
12.
Nature ; 596(7871): 296-300, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34349264

RESUMO

During the splicing of introns from precursor messenger RNAs (pre-mRNAs), the U2 small nuclear ribonucleoprotein (snRNP) must undergo stable integration into the spliceosomal A complex-a poorly understood, multistep process that is facilitated by the DEAD-box helicase Prp5 (refs. 1-4). During this process, the U2 small nuclear RNA (snRNA) forms an RNA duplex with the pre-mRNA branch site (the U2-BS helix), which is proofread by Prp5 at this stage through an unclear mechanism5. Here, by deleting the branch-site adenosine (BS-A) or mutating the branch-site sequence of an actin pre-mRNA, we stall the assembly of spliceosomes in extracts from the yeast Saccharomyces cerevisiae directly before the A complex is formed. We then determine the three-dimensional structure of this newly identified assembly intermediate by cryo-electron microscopy. Our structure indicates that the U2-BS helix has formed in this pre-A complex, but is not yet clamped by the HEAT domain of the Hsh155 protein (Hsh155HEAT), which exhibits an open conformation. The structure further reveals a large-scale remodelling/repositioning of the U1 and U2 snRNPs during the formation of the A complex that is required to allow subsequent binding of the U4/U6.U5 tri-snRNP, but that this repositioning is blocked in the pre-A complex by the presence of Prp5. Our data suggest that binding of Hsh155HEAT to the bulged BS-A of the U2-BS helix triggers closure of Hsh155HEAT, which in turn destabilizes Prp5 binding. Thus, Prp5 proofreads the branch site indirectly, hindering spliceosome assembly if branch-site mutations prevent the remodelling of Hsh155HEAT. Our data provide structural insights into how a spliceosomal helicase enhances the fidelity of pre-mRNA splicing.


Assuntos
RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/metabolismo , Precursores de RNA/química , Precursores de RNA/genética , Splicing de RNA , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae , Spliceossomos/enzimologia , Actinas/genética , Adenosina/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , RNA Helicases DEAD-box/ultraestrutura , Modelos Moleculares , Mutação , Domínios Proteicos , Precursores de RNA/metabolismo , Precursores de RNA/ultraestrutura , Splicing de RNA/genética , Ribonucleoproteína Nuclear Pequena U1/metabolismo , Ribonucleoproteína Nuclear Pequena U2/química , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Spliceossomos/química , Spliceossomos/metabolismo
13.
Nat Commun ; 12(1): 4983, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34404783

RESUMO

Parasites of the phylum Apicomplexa cause important diseases including malaria, cryptosporidiosis and toxoplasmosis. These intracellular pathogens inject the contents of an essential organelle, the rhoptry, into host cells to facilitate invasion and infection. However, the structure and mechanism of this eukaryotic secretion system remain elusive. Here, using cryo-electron tomography and subtomogram averaging, we report the conserved architecture of the rhoptry secretion system in the invasive stages of two evolutionarily distant apicomplexans, Cryptosporidium parvum and Toxoplasma gondii. In both species, we identify helical filaments, which appear to shape and compartmentalize the rhoptries, and an apical vesicle (AV), which facilitates docking of the rhoptry tip at the parasite's apical region with the help of an elaborate ultrastructure named the rhoptry secretory apparatus (RSA); the RSA anchors the AV at the parasite plasma membrane. Depletion of T. gondii Nd9, a protein required for rhoptry secretion, disrupts the RSA ultrastructure and AV-anchoring. Moreover, T. gondii contains a line of AV-like vesicles, which interact with a pair of microtubules and accumulate towards the AV, leading to a working model for AV-reloading and discharging of multiple rhoptries. Together, our analyses provide an ultrastructural framework to understand how these important parasites deliver effectors into host cells.


Assuntos
Organelas/metabolismo , Organelas/ultraestrutura , Parasitos/metabolismo , Parasitos/ultraestrutura , Proteínas de Protozoários/química , Animais , Evolução Biológica , Membrana Celular/metabolismo , Microscopia Crioeletrônica , Criptosporidiose , Cryptosporidium , Cryptosporidium parvum/citologia , Cryptosporidium parvum/efeitos dos fármacos , Cryptosporidium parvum/metabolismo , Interações Hospedeiro-Parasita , Microtúbulos/ultraestrutura , Proteínas de Protozoários/metabolismo , Toxoplasma/citologia , Toxoplasma/efeitos dos fármacos , Toxoplasma/metabolismo , Toxoplasmose
14.
Nat Commun ; 12(1): 4996, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34404793

RESUMO

Between 10 and 20 million people worldwide are infected with the human T-cell lymphotropic virus type 1 (HTLV-1). Despite causing life-threatening pathologies there is no therapeutic regimen for this deltaretrovirus. Here, we screened a library of integrase strand transfer inhibitor (INSTI) candidates built around several chemical scaffolds to determine their effectiveness in limiting HTLV-1 infection. Naphthyridines with substituents in position 6 emerged as the most potent compounds against HTLV-1, with XZ450 having highest efficacy in vitro. Using single-particle cryo-electron microscopy we visualised XZ450 as well as the clinical HIV-1 INSTIs raltegravir and bictegravir bound to the active site of the deltaretroviral intasome. The structures reveal subtle differences in the coordination environment of the Mg2+ ion pair involved in the interaction with the INSTIs. Our results elucidate the binding of INSTIs to the HTLV-1 intasome and support their use for pre-exposure prophylaxis and possibly future treatment of HTLV-1 infection.


Assuntos
Antivirais/química , Antivirais/farmacologia , Microscopia Crioeletrônica , Infecções por HTLV-I/tratamento farmacológico , Vírus Linfotrópico T Tipo 1 Humano/efeitos dos fármacos , Amidas , Domínio Catalítico , Deltaretrovirus , Farmacorresistência Viral/efeitos dos fármacos , Integrase de HIV/efeitos dos fármacos , HIV-1 , Compostos Heterocíclicos com 3 Anéis , Vírus Linfotrópico T Tipo 1 Humano/genética , Humanos , Naftiridinas/farmacologia , Piperazinas , Piridonas , Proteínas Recombinantes
15.
Nat Commun ; 12(1): 4972, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34404795

RESUMO

A variety of artificial cells springs from the functionalization of liposomes with proteins. However, these models suffer from low durability without repair and replenishment mechanisms, which can be partly addressed by replacing the lipids with polymers. Yet natural membranes are also dynamically remodeled in multiple cellular processes. Here, we show that synthetic amphiphile membranes also undergo fusion, mediated by the protein machinery for synaptic secretion. We integrated fusogenic SNAREs in polymer and hybrid vesicles and observed efficient membrane and content mixing. We determined bending rigidity and pore edge tension as key parameters for fusion and described its plausible progression through cryo-EM snapshots. These findings demonstrate that dynamic membrane phenomena can be reconstituted in synthetic materials, thereby providing new tools for the assembly of synthetic protocells.


Assuntos
Fusão de Membrana/fisiologia , Membranas/metabolismo , Polímeros/metabolismo , Proteínas SNARE/química , Proteínas SNARE/metabolismo , Animais , Microscopia Crioeletrônica , Lipossomos/metabolismo , Proteínas do Tecido Nervoso , Ligação Proteica , Proteínas R-SNARE , Ratos , Proteína 25 Associada a Sinaptossoma , Sintaxina 1 , Proteína 2 Associada à Membrana da Vesícula
16.
Mol Cell ; 81(15): 3038-3040, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34358455

RESUMO

We talk to Chisae Nagiri and Wataru Shihoya about their paper, "Cryo-EM structure of the ß3 adrenergic receptor reveals the molecular basis of subtype selectivity," and last author Osamu Nureki tells us about the research in his lab in Tokyo.


Assuntos
Microscopia Crioeletrônica , Receptores Adrenérgicos beta 3 , Humanos , Laboratórios , Biologia Molecular , Receptores Adrenérgicos beta 3/química
17.
Mol Cell ; 81(16): 3400-3409.e3, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34352203

RESUMO

Non-homologous end joining (NHEJ) is one of two critical mechanisms utilized in humans to repair DNA double-strand breaks (DSBs). Unrepaired or incorrect repair of DSBs can lead to apoptosis or cancer. NHEJ involves several proteins, including the Ku70/80 heterodimer, DNA-dependent protein kinase catalytic subunit (DNA-PKcs), X-ray cross-complementing protein 4 (XRCC4), XRCC4-like factor (XLF), and ligase IV. These core proteins bind DSBs and ligate the damaged DNA ends. However, details of the structural assembly of these proteins remain unclear. Here, we present cryo-EM structures of NHEJ supercomplexes that are composed of these core proteins and DNA, revealing the detailed structural architecture of this assembly. We describe monomeric and dimeric forms of this supercomplex and also propose the existence of alternate dimeric forms of long-range synaptic complexes. Finally, we show that mutational disruption of several structural features within these NHEJ complexes negatively affects DNA repair.


Assuntos
DNA Ligase Dependente de ATP/ultraestrutura , Enzimas Reparadoras do DNA/ultraestrutura , Proteína Quinase Ativada por DNA/ultraestrutura , Proteínas de Ligação a DNA/ultraestrutura , Complexos Multiproteicos/ultraestrutura , Apoptose/genética , Microscopia Crioeletrônica , Quebras de DNA de Cadeia Dupla , Dano ao DNA/genética , Reparo do DNA por Junção de Extremidades/genética , DNA Ligase Dependente de ATP/genética , Reparo do DNA/genética , Enzimas Reparadoras do DNA/genética , Proteína Quinase Ativada por DNA/genética , Proteínas de Ligação a DNA/genética , Humanos , Autoantígeno Ku/genética , Autoantígeno Ku/ultraestrutura , Complexos Multiproteicos/genética , Fosforilação/genética
18.
Int J Mol Sci ; 22(16)2021 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-34445650

RESUMO

Cryo-electron microscopy (Cryo-EM) has become a routine technology for resolving the structure of biological macromolecules due to the resolution revolution in recent years. The specimens are typically prepared in a very thin layer of vitrified ice suspending in the holes of the perforated amorphous carbon film. However, the samples prepared by directly applying to the conventional support membranes may suffer from partial or complete denaturation caused by sticking to the air-water interface (AWI). With the application in materials, graphene has also been used recently to improve frozen sample preparation instead of a suspended conventional amorphous thin carbon. It has been proven that graphene or graphene oxide and various chemical modifications on its surface can effectively prevent particles from adsorbing to the AWI, which improves the dispersion, adsorbed number, and orientation preference of frozen particles in the ice layer. Their excellent properties and thinner thickness can significantly reduce the background noise, allowing high-resolution three-dimensional reconstructions using a minimum data set.


Assuntos
Microscopia Crioeletrônica/métodos , Grafite/química , Substâncias Macromoleculares/química , Manejo de Espécimes/métodos , Água/química
19.
Molecules ; 26(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34443547

RESUMO

Phosphatidylglycerols represent a large share of the lipids in the plasmamembrane of procaryotes. Therefore, this study investigates the role of charged lipids in the plasma membrane with respect to the interaction of the antiviral saponin glycyrrhizin with such membranes. Glycyrrhizin is a natural triterpenic-based surfactant found in licorice. Vesicles made of 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1'-glycerol) (DOPG)/glycyrrhizin are characterized by small-angle scattering with neutrons and X-rays (SANS and SAXS). Small-angle scattering data are first evaluated by the model-independent modified Kratky-Porod method and afterwards fitted by a model describing the shape of small unilamellar vesicles (SUV) with an internal head-tail contrast. Complete miscibility of DOPG and glycyrrhizin was revealed even at a ratio of lipid:saponin of 1:1. Additional information about the chain-chain correlation distance of the lipid/saponin mixtures in the SUV structures is obtained from wide-angle X-ray scattering (WAXS).


Assuntos
Microscopia Crioeletrônica , Ácido Glicirrízico/química , Fosfatidilgliceróis/química , Espalhamento a Baixo Ângulo , Difração de Nêutrons , Difração de Raios X
20.
Int J Mol Sci ; 22(15)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34360907

RESUMO

The superfamily of P-loop channels includes various potassium channels, voltage-gated sodium and calcium channels, transient receptor potential channels, and ionotropic glutamate receptors. Despite huge structural and functional diversity of the channels, their pore-forming domain has a conserved folding. In the past two decades, scores of atomic-scale structures of P-loop channels with medically important drugs in the inner pore have been published. High structural diversity of these complexes complicates the comparative analysis of these structures. Here we 3D-aligned structures of drug-bound P-loop channels, compared their geometric characteristics, and analyzed the energetics of ligand-channel interactions. In the superimposed structures drugs occupy most of the sterically available space in the inner pore and subunit/repeat interfaces. Cationic groups of some drugs occupy vacant binding sites of permeant ions in the inner pore and selectivity-filter region. Various electroneutral drugs, lipids, and detergent molecules are seen in the interfaces between subunits/repeats. In many structures the drugs strongly interact with lipid and detergent molecules, but physiological relevance of such interactions is unclear. Some eukaryotic sodium and calcium channels have state-dependent or drug-induced π-bulges in the inner helices, which would be difficult to predict. The drug-induced π-bulges may represent a novel mechanism of gating modulation.


Assuntos
Domínio AAA , Canais de Cálcio/metabolismo , Microscopia Crioeletrônica/métodos , Preparações Farmacêuticas/metabolismo , Canais de Potássio/metabolismo , Receptores Ionotrópicos de Glutamato/metabolismo , Canais de Potencial de Receptor Transitório/metabolismo , Canais de Sódio Disparados por Voltagem/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Canais de Cálcio/química , Biologia Computacional/métodos , Eucariotos/metabolismo , Ligantes , Modelos Moleculares , Canais de Potássio/química , Conformação Proteica em alfa-Hélice , Receptores Ionotrópicos de Glutamato/química , Alinhamento de Sequência , Canais de Potencial de Receptor Transitório/química , Canais de Sódio Disparados por Voltagem/química
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