Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 31
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Ultramicroscopy ; 262: 113962, 2024 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-38642481

RESUMO

Ewald sphere curvature correction, which extends beyond the projection approximation, stretches the shallow depth of field in cryo-EM reconstructions of thick particles. Here we show that even for previously assumed thin particles, reconstruction artifacts which we refer to as ghosts can appear. By retrieving the lost phases of the electron exitwaves and accounting for the first Born approximation scattering within the particle, we show that these ghosts can be effectively eliminated. Our simulations demonstrate how such ghostbusting can improve reconstructions as compared to existing state-of-the-art software. Like ptychographic cryo-EM, our Ghostbuster algorithm uses phase retrieval to improve reconstructions, but unlike the former, we do not need to modify the existing data acquisition pipelines.

2.
Nat Commun ; 15(1): 389, 2024 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-38195598

RESUMO

Structural biology efforts using cryogenic electron microscopy are frequently stifled by specimens adopting "preferred orientations" on grids, leading to anisotropic map resolution and impeding structure determination. Tilting the specimen stage during data collection is a generalizable solution but has historically led to substantial resolution attenuation. Here, we develop updated data collection and image processing workflows and demonstrate, using multiple specimens, that resolution attenuation is negligible or significantly reduced across tilt angles. Reconstructions with and without the stage tilted as high as 60° are virtually indistinguishable. These strategies allowed the reconstruction to 3 Å resolution of a bacterial RNA polymerase with preferred orientation, containing an unnatural nucleotide for studying novel base pair recognition. Furthermore, we present a quantitative framework that allows cryo-EM practitioners to define an optimal tilt angle during data acquisition. These results reinforce the utility of employing stage tilt for data collection and provide quantitative metrics to obtain isotropic maps.


Assuntos
Benchmarking , Sistemas Computacionais , Microscopia Crioeletrônica , Anisotropia , Coleta de Dados
3.
Commun Chem ; 7(1): 7, 2024 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-38182798

RESUMO

Halogenation of pyrrole requires strong electrophilic reagents and often leads to undesired polyhalogenated products. Biocatalytic halogenation is a highly attractive approach given its chemoselectivity and benign reaction conditions. While there are several reports of enzymatic phenol and indole halogenation in organic synthesis, corresponding reports on enzymatic pyrrole halogenation have been lacking. Here we describe the in vitro functional and structural characterization of PrnC, a flavin-dependent halogenase that can act on free-standing pyrroles. Computational modeling and site mutagenesis studies identified three key residues in the catalytic pocket. A moderate resolution map using single-particle cryogenic electron microscopy reveals PrnC to be a dimer. This native PrnC can halogenate a library of structurally diverse pyrrolic heterocycles in a site-selective manner and be applied in the chemoenzymatic synthesis of a chlorinated analog of the agrochemical fungicide Fludioxonil.

5.
bioRxiv ; 2023 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-37503021

RESUMO

Structural biology efforts using cryogenic electron microscopy are frequently stifled by specimens adopting "preferred orientations" on grids, leading to anisotropic map resolution and impeding structure determination. Tilting the specimen stage during data collection is a generalizable solution but has historically led to substantial resolution attenuation. Here, we develop updated data collection and image processing workflows and demonstrate, using multiple specimens, that resolution attenuation is negligible or significantly reduced across tilt angles. Reconstructions with and without the stage tilted as high as 60° are virtually indistinguishable. These strategies allowed the reconstruction to 3 Å resolution of a bacterial RNA polymerase with preferred orientation. Furthermore, we present a quantitative framework that allows cryo-EM practitioners to define an optimal tilt angle for dataset acquisition. These data reinforce the utility of employing stage tilt for data collection and provide quantitative metrics to obtain isotropic maps.

6.
Subcell Biochem ; 99: 379-391, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36151383

RESUMO

The mycobacteria genus is responsible for numerous infectious diseases that have afflicted the human race since antiquity-tuberculosis and leprosy in particular. An important contributor to their evolutionary success is their unique cell envelope, which constitutes a quasi-impermeable barrier, protecting the microorganism from external threats, antibiotics included. The arabinofuranosyltransferases are a family of enzymes, unique to the Actinobacteria family that mycobacteria genus belongs to, that are critical to building of this cell envelope. In this chapter, we will analyze available structures of members of the mycobacterial arabinofuranosyltransferase, clarify their function, as well as explore the common themes present amongst this family of enzymes, as revealed by recent research.


Assuntos
Mycobacterium tuberculosis , Mycobacterium , Antibacterianos , Membrana Celular , Parede Celular , Humanos
7.
Structure ; 30(10): 1403-1410.e4, 2022 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-36041457

RESUMO

We used the Legionella pneumophila effector SidK to affinity purify the endogenous vacuolar-type ATPases (V-ATPases) from lemon fruit. The preparation was sufficient for cryoelectron microscopy, allowing structure determination of the enzyme in two rotational states. The structure defines the ATP:H+ ratio of the enzyme, demonstrating that it can establish a maximum ΔpH of ∼3, which is insufficient to maintain the low pH observed in the vacuoles of juice sac cells in lemons and other citrus fruit. Compared with yeast and mammalian enzymes, the membrane region of the plant V-ATPase lacks subunit f and possesses an unusual configuration of transmembrane α helices. Subunit H, which inhibits ATP hydrolysis in the isolated catalytic region of V-ATPase, adopts two different conformations in the intact complex, hinting at a role in modulating activity in the intact enzyme.


Assuntos
Citrus , ATPases Vacuolares Próton-Translocadoras , Trifosfato de Adenosina , Animais , Microscopia Crioeletrônica , Mamíferos/metabolismo , Saccharomyces cerevisiae/metabolismo , ATPases Vacuolares Próton-Translocadoras/química , ATPases Vacuolares Próton-Translocadoras/metabolismo , Vacúolos/metabolismo
8.
Life Sci Alliance ; 5(11)2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-35794005

RESUMO

V-ATPases are rotary proton pumps that serve as signaling hubs with numerous protein binding partners. CryoEM with exhaustive focused classification allowed detection of endogenous proteins associated with porcine kidney V-ATPase. An extra C subunit was found in ∼3% of complexes, whereas ∼1.6% of complexes bound mEAK-7, a protein with proposed roles in dauer formation in nematodes and mTOR signaling in mammals. High-resolution cryoEM of porcine kidney V-ATPase with recombinant mEAK-7 showed that mEAK-7's TLDc domain interacts with V-ATPase's stator, whereas its C-terminal α helix binds V-ATPase's rotor. This crosslink would be expected to inhibit rotary catalysis. However, unlike the yeast TLDc protein Oxr1p, exogenous mEAK-7 does not inhibit V-ATPase and mEAK-7 overexpression in cells does not alter lysosomal or phagosomal pH. Instead, cryoEM suggests that the mEAK-7:V-ATPase interaction is disrupted by ATP-induced rotation of the rotor. Comparison of Oxr1p and mEAK-7 binding explains this difference. These results show that V-ATPase binding by TLDc domain proteins can lead to effects ranging from strong inhibition to formation of labile interactions that are sensitive to the enzyme's activity.


Assuntos
ATPases Vacuolares Próton-Translocadoras , Animais , Microscopia Crioeletrônica , Mamíferos/metabolismo , Ligação Proteica , Subunidades Proteicas/química , Suínos , ATPases Vacuolares Próton-Translocadoras/química , ATPases Vacuolares Próton-Translocadoras/metabolismo
9.
Nat Biotechnol ; 40(12): 1834-1844, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-35879364

RESUMO

Mutations in Ras family proteins are implicated in 33% of human cancers, but direct pharmacological inhibition of Ras mutants remains challenging. As an alternative to direct inhibition, we screened for sensitivities in Ras-mutant cells and discovered 249C as a Ras-mutant selective cytotoxic agent with nanomolar potency against a spectrum of Ras-mutant cancers. 249C binds to vacuolar (V)-ATPase with nanomolar affinity and inhibits its activity, preventing lysosomal acidification and inhibiting autophagy and macropinocytosis pathways that several Ras-driven cancers rely on for survival. Unexpectedly, potency of 249C varies with the identity of the Ras driver mutation, with the highest potency for KRASG13D and G12V both in vitro and in vivo, highlighting a mutant-specific dependence on macropinocytosis and lysosomal pH. Indeed, 249C potently inhibits tumor growth without adverse side effects in mouse xenografts of KRAS-driven lung and colon cancers. A comparison of isogenic SW48 xenografts with different KRAS mutations confirmed that KRASG13D/+ (followed by G12V/+) mutations are especially sensitive to 249C treatment. These data establish proof-of-concept for targeting V-ATPase in cancers driven by specific KRAS mutations such as KRASG13D and G12V.


Assuntos
Antineoplásicos , Neoplasias , ATPases Vacuolares Próton-Translocadoras , Humanos , Camundongos , Animais , Linhagem Celular Tumoral , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Vacuolares Próton-Translocadoras/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas ras/genética , Proteínas ras/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Mutação/genética , Neoplasias/tratamento farmacológico , Neoplasias/genética
10.
Annu Rev Biochem ; 91: 1-32, 2022 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-35320683

RESUMO

Cryo-electron microscopy (cryo-EM) continues its remarkable growth as a method for visualizing biological objects, which has been driven by advances across the entire pipeline. Developments in both single-particle analysis and in situ tomography have enabled more structures to be imaged and determined to better resolutions, at faster speeds, and with more scientists having improved access. This review highlights recent advances at each stageof the cryo-EM pipeline and provides examples of how these techniques have been used to investigate real-world problems, including antibody development against the SARS-CoV-2 spike during the recent COVID-19 pandemic.


Assuntos
COVID-19 , Pandemias , Microscopia Crioeletrônica/métodos , Humanos , SARS-CoV-2 , Imagem Individual de Molécula
11.
Proc Natl Acad Sci U S A ; 118(51)2021 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-34916296

RESUMO

The human extracellular calcium-sensing (CaS) receptor controls plasma Ca2+ levels and contributes to nutrient-dependent maintenance and metabolism of diverse organs. Allosteric modulation of the CaS receptor corrects disorders of calcium homeostasis. Here, we report the cryogenic-electron microscopy reconstructions of a near-full-length CaS receptor in the absence and presence of allosteric modulators. Activation of the homodimeric CaS receptor requires a break in the transmembrane 6 (TM6) helix of each subunit, which facilitates the formation of a TM6-mediated homodimer interface and expansion of homodimer interactions. This transformation in TM6 occurs without a positive allosteric modulator. Two modulators with opposite functional roles bind to overlapping sites within the transmembrane domain through common interactions, acting to stabilize distinct rotamer conformations of key residues on the TM6 helix. The positive modulator reinforces TM6 distortion and maximizes subunit contact to enhance receptor activity, while the negative modulator strengthens an intact TM6 to dampen receptor function. In both active and inactive states, the receptor displays symmetrical transmembrane conformations that are consistent with its homodimeric assembly.


Assuntos
Cálcio/metabolismo , Regulação da Expressão Gênica/fisiologia , Homeostase/fisiologia , Receptores de Detecção de Cálcio/metabolismo , Microscopia Crioeletrônica , Células HEK293 , Humanos , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Receptores de Detecção de Cálcio/genética , Transdução de Sinais
12.
Nat Commun ; 12(1): 3661, 2021 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-34135340

RESUMO

SARS-CoV-2, the virus responsible for COVID-19, has caused a global pandemic. Antibodies can be powerful biotherapeutics to fight viral infections. Here, we use the human apoferritin protomer as a modular subunit to drive oligomerization of antibody fragments and transform antibodies targeting SARS-CoV-2 into exceptionally potent neutralizers. Using this platform, half-maximal inhibitory concentration (IC50) values as low as 9 × 10-14 M are achieved as a result of up to 10,000-fold potency enhancements compared to corresponding IgGs. Combination of three different antibody specificities and the fragment crystallizable (Fc) domain on a single multivalent molecule conferred the ability to overcome viral sequence variability together with outstanding potency and IgG-like bioavailability. The MULTi-specific, multi-Affinity antiBODY (Multabody or MB) platform thus uniquely leverages binding avidity together with multi-specificity to deliver ultrapotent and broad neutralizers against SARS-CoV-2. The modularity of the platform also makes it relevant for rapid evaluation against other infectious diseases of global health importance. Neutralizing antibodies are a promising therapeutic for SARS-CoV-2.


Assuntos
Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/química , SARS-CoV-2/imunologia , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/química , Anticorpos Antivirais/imunologia , Especificidade de Anticorpos , Apoferritinas/química , Disponibilidade Biológica , Mapeamento de Epitopos , Humanos , Imunoglobulina G/imunologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Engenharia de Proteínas/métodos , Subunidades Proteicas/química , Glicoproteína da Espícula de Coronavírus/imunologia , Distribuição Tecidual
13.
Mol Cell ; 80(6): 938-939, 2020 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-33338409

RESUMO

The goal of structural biology is to understand biological macromolecules in as much detail as possible. Depending on the resolution of the structure obtained, insights will range from understanding interactions at the level of proteins, domains, or atoms. The three mainstay structural biology techniques are X-ray crystallography, nuclear magnetic resonance (NMR) imaging, and cryogenic electron microscopy (cryo-EM). Cryo-EM has rapidly gained popularity in recent years due to a combination of hardware and software advances, leading to the so-called Resolution Revolution (Kühlbrandt, 2014).


Assuntos
Microscopia Crioeletrônica , Biologia Molecular/métodos , Proteínas/ultraestrutura , Cristalografia por Raios X , Imageamento Tridimensional , Software
14.
Acta Crystallogr D Struct Biol ; 76(Pt 11): 1092-1103, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-33135680

RESUMO

Blotting times for conventional cryoEM specimen preparation complicate time-resolved studies and lead to some specimens adopting preferred orientations or denaturing at the air-water interface. Here, it is shown that solution sprayed onto one side of a holey cryoEM grid can be wicked through the grid by a glass-fiber filter held against the opposite side, often called the `back', of the grid, producing a film suitable for vitrification. This process can be completed in tens of milliseconds. Ultrasonic specimen application and through-grid wicking were combined in a high-speed specimen-preparation device that was named `Back-it-up' or BIU. The high liquid-absorption capacity of the glass fiber compared with self-wicking grids makes the method relatively insensitive to the amount of sample applied. Consequently, through-grid wicking produces large areas of ice that are suitable for cryoEM for both soluble and detergent-solubilized protein complexes. The speed of the device increases the number of views for a specimen that suffers from preferred orientations.


Assuntos
Apoferritinas/química , Microscopia Crioeletrônica/métodos , Hemaglutininas/química , ATPases Translocadoras de Prótons/química , Manejo de Espécimes , Ação Capilar , Humanos , Mycobacterium smegmatis/enzimologia , Saccharomyces cerevisiae/enzimologia , Manejo de Espécimes/instrumentação , Manejo de Espécimes/métodos , Vitrificação
15.
IUCrJ ; 7(Pt 5): 860-869, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32939278

RESUMO

Direct detector device (DDD) cameras have revolutionized electron cryomicroscopy (cryoEM) with their high detective quantum efficiency (DQE) and output of movie data. A high ratio of camera frame rate (frames per second) to camera exposure rate (electrons per pixel per second) allows electron counting, which further improves the DQE and enables the recording of super-resolution information. Movie output also allows the correction of specimen movement and compensation for radiation damage. However, these movies come at the cost of producing large volumes of data. It is common practice to sum groups of successive camera frames to reduce the final frame rate, and therefore the file size, to one suitable for storage and image processing. This reduction in the temporal resolution of the camera requires decisions to be made during data acquisition that may result in the loss of information that could have been advantageous during image analysis. Here, experimental analysis of a new electron-event representation (EER) data format for electron-counting DDD movies is presented, which is enabled by new hardware developed by Thermo Fisher Scientific for their Falcon DDD cameras. This format enables the recording of DDD movies at the raw camera frame rate without sacrificing either spatial or temporal resolution. Experimental data demonstrate that the method retains super-resolution information and allows the correction of specimen movement at the physical frame rate of the camera while maintaining manageable file sizes. The EER format will enable the development of new methods that can utilize the full spatial and temporal resolution of DDD cameras.

16.
Nat Commun ; 11(1): 3396, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32636380

RESUMO

Arabinosyltransferase B (EmbB) belongs to a family of membrane-bound glycosyltransferases that build the lipidated polysaccharides of the mycobacterial cell envelope, and are targets of anti-tuberculosis drug ethambutol. We present the 3.3 Å resolution single-particle cryo-electron microscopy structure of Mycobacterium smegmatis EmbB, providing insights on substrate binding and reaction mechanism. Mutations that confer ethambutol resistance map mostly around the putative active site, suggesting this to be the location of drug binding.


Assuntos
Mycobacterium smegmatis/enzimologia , Pentosiltransferases/química , Pentosiltransferases/ultraestrutura , Antituberculosos/farmacologia , Domínio Catalítico , Microscopia Crioeletrônica , Farmacorresistência Bacteriana , Etambutol/farmacologia , Lipídeos/química , Mutação , Mycobacterium tuberculosis/enzimologia , Polissacarídeos/química , Ligação Proteica
17.
Mol Cell ; 78(4): 683-699.e11, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32386575

RESUMO

Mycobacterium tuberculosis causes tuberculosis, a disease that kills over 1 million people each year. Its cell envelope is a common antibiotic target and has a unique structure due, in part, to two lipidated polysaccharides-arabinogalactan and lipoarabinomannan. Arabinofuranosyltransferase D (AftD) is an essential enzyme involved in assembling these glycolipids. We present the 2.9-Å resolution structure of M. abscessus AftD, determined by single-particle cryo-electron microscopy. AftD has a conserved GT-C glycosyltransferase fold and three carbohydrate-binding modules. Glycan array analysis shows that AftD binds complex arabinose glycans. Additionally, AftD is non-covalently complexed with an acyl carrier protein (ACP). 3.4- and 3.5-Å structures of a mutant with impaired ACP binding reveal a conformational change, suggesting that ACP may regulate AftD function. Mutagenesis experiments using a conditional knockout constructed in M. smegmatis confirm the essentiality of the putative active site and the ACP binding for AftD function.


Assuntos
Proteína de Transporte de Acila/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Microscopia Crioeletrônica/métodos , Glicosiltransferases/metabolismo , Mycobacterium smegmatis/enzimologia , Proteína de Transporte de Acila/genética , Proteínas de Bactérias/genética , Domínio Catalítico , Parede Celular/metabolismo , Galactanos/metabolismo , Glicosiltransferases/genética , Lipopolissacarídeos/metabolismo , Mutação , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/crescimento & desenvolvimento , Filogenia , Conformação Proteica , Especificidade por Substrato
18.
Nature ; 577(7790): 426-431, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31775157

RESUMO

The organization of genomic DNA into nucleosomes profoundly affects all DNA-related processes in eukaryotes. The histone chaperone known as 'facilitates chromatin transcription' (FACT1) (consisting of subunits SPT16 and SSRP1) promotes both disassembly and reassembly of nucleosomes during gene transcription, DNA replication and DNA repair2. However, the mechanism by which FACT causes these opposing outcomes is unknown. Here we report two cryo-electron-microscopic structures of human FACT in complex with partially assembled subnucleosomes, with supporting biochemical and hydrogen-deuterium exchange data. We find that FACT is engaged in extensive interactions with nucleosomal DNA and all histone variants. The large DNA-binding surface on FACT appears to be protected by the carboxy-terminal domains of both of its subunits, and this inhibition is released by interaction with H2A-H2B, allowing FACT-H2A-H2B to dock onto a complex containing DNA and histones H3 and H4 (ref. 3). SPT16 binds nucleosomal DNA and tethers H2A-H2B through its carboxy-terminal domain by acting as a placeholder for DNA. SSRP1 also contributes to DNA binding, and can assume two conformations, depending on whether a second H2A-H2B dimer is present. Our data suggest a compelling mechanism for how FACT maintains chromatin integrity during polymerase passage, by facilitating removal of the H2A-H2B dimer, stabilizing intermediate subnucleosomal states and promoting nucleosome reassembly. Our findings reconcile discrepancies regarding the many roles of FACT and underscore the dynamic interactions between histone chaperones and nucleosomes.


Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Grupo de Alta Mobilidade/química , Proteínas de Grupo de Alta Mobilidade/metabolismo , Nucleossomos/química , Nucleossomos/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Fatores de Elongação da Transcrição/química , Fatores de Elongação da Transcrição/metabolismo , Microscopia Crioeletrônica , DNA/química , DNA/metabolismo , Histonas/química , Histonas/metabolismo , Humanos , Modelos Moleculares , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína
19.
Nat Struct Mol Biol ; 26(12): 1123-1131, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31740855

RESUMO

Classically, G-protein-coupled receptors (GPCRs) are thought to activate G protein from the plasma membrane and are subsequently desensitized by ß-arrestin (ß-arr). However, some GPCRs continue to signal through G protein from internalized compartments, mediated by a GPCR-G protein-ß-arr 'megaplex'. Nevertheless, the molecular architecture of the megaplex remains unknown. Here, we present its cryo-electron microscopy structure, which shows simultaneous engagement of human G protein and bovine ß-arr to the core and phosphorylated tail, respectively, of a single active human chimeric ß2-adrenergic receptor with the C-terminal tail of the arginine vasopressin type 2 receptor (ß2V2R). All three components adopt their canonical active conformations, suggesting that a single megaplex GPCR is capable of simultaneously activating G protein and ß-arr. Our findings provide a structural basis for GPCR-mediated sustained internalized G protein signaling.


Assuntos
Proteínas de Ligação ao GTP/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , beta-Arrestinas/metabolismo , Animais , Bovinos , Microscopia Crioeletrônica , Endossomos/metabolismo , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/ultraestrutura , Humanos , Modelos Moleculares , Conformação Proteica , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/metabolismo , Receptores Adrenérgicos beta 2/ultraestrutura , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/ultraestrutura , Receptores de Vasopressinas/química , Receptores de Vasopressinas/metabolismo , Receptores de Vasopressinas/ultraestrutura , beta-Arrestinas/química , beta-Arrestinas/ultraestrutura
20.
Nature ; 576(7786): 315-320, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31776516

RESUMO

The emergence and spread of drug-resistant Plasmodium falciparum impedes global efforts to control and eliminate malaria. For decades, treatment of malaria has relied on chloroquine (CQ), a safe and affordable 4-aminoquinoline that was highly effective against intra-erythrocytic asexual blood-stage parasites, until resistance arose in Southeast Asia and South America and spread worldwide1. Clinical resistance to the chemically related current first-line combination drug piperaquine (PPQ) has now emerged regionally, reducing its efficacy2. Resistance to CQ and PPQ has been associated with distinct sets of point mutations in the P. falciparum CQ-resistance transporter PfCRT, a 49-kDa member of the drug/metabolite transporter superfamily that traverses the membrane of the acidic digestive vacuole of the parasite3-9. Here we present the structure, at 3.2 Å resolution, of the PfCRT isoform of CQ-resistant, PPQ-sensitive South American 7G8 parasites, using single-particle cryo-electron microscopy and antigen-binding fragment technology. Mutations that contribute to CQ and PPQ resistance localize primarily to moderately conserved sites on distinct helices that line a central negatively charged cavity, indicating that this cavity is the principal site of interaction with the positively charged CQ and PPQ. Binding and transport studies reveal that the 7G8 isoform binds both drugs with comparable affinities, and that these drugs are mutually competitive. The 7G8 isoform transports CQ in a membrane potential- and pH-dependent manner, consistent with an active efflux mechanism that drives CQ resistance5, but does not transport PPQ. Functional studies on the newly emerging PfCRT F145I and C350R mutations, associated with decreased PPQ susceptibility in Asia and South America, respectively6,9, reveal their ability to mediate PPQ transport in 7G8 variant proteins and to confer resistance in gene-edited parasites. Structural, functional and in silico analyses suggest that distinct mechanistic features mediate the resistance to CQ and PPQ in PfCRT variants. These data provide atomic-level insights into the molecular mechanism of this key mediator of antimalarial treatment failures.


Assuntos
Microscopia Crioeletrônica , Resistência a Medicamentos/efeitos dos fármacos , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/ultraestrutura , Plasmodium falciparum/química , Proteínas de Protozoários/química , Proteínas de Protozoários/ultraestrutura , Cloroquina/metabolismo , Cloroquina/farmacologia , Resistência a Medicamentos/genética , Concentração de Íons de Hidrogênio , Malária Falciparum/tratamento farmacológico , Malária Falciparum/parasitologia , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Modelos Moleculares , Mutação , Plasmodium falciparum/genética , Plasmodium falciparum/ultraestrutura , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Quinolinas/metabolismo , Quinolinas/farmacologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...