Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 13 de 13
Filtrar
1.
Cell ; 163(5): 1138-1152, 2015 Nov 19.
Artículo en Inglés | MEDLINE | ID: mdl-26548953

RESUMEN

Diverse repertoires of antigen-receptor genes that result from combinatorial splicing of coding segments by V(D)J recombination are hallmarks of vertebrate immunity. The (RAG1-RAG2)2 recombinase (RAG) recognizes recombination signal sequences (RSSs) containing a heptamer, a spacer of 12 or 23 base pairs, and a nonamer (12-RSS or 23-RSS) and introduces precise breaks at RSS-coding segment junctions. RAG forms synaptic complexes only with one 12-RSS and one 23-RSS, a dogma known as the 12/23 rule that governs the recombination fidelity. We report cryo-electron microscopy structures of synaptic RAG complexes at up to 3.4 Å resolution, which reveal a closed conformation with base flipping and base-specific recognition of RSSs. Distortion at RSS-coding segment junctions and base flipping in coding segments uncover the two-metal-ion catalytic mechanism. Induced asymmetry involving tilting of the nonamer-binding domain dimer of RAG1 upon binding of HMGB1-bent 12-RSS or 23-RSS underlies the molecular mechanism for the 12/23 rule.


Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Homeodominio/química , Recombinación V(D)J , Secuencia de Aminoácidos , Animales , Microscopía por Crioelectrón , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/ultraestructura , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/ultraestructura , Humanos , Ratones , Datos de Secuencia Molecular , Complejos Multiproteicos/química , Complejos Multiproteicos/ultraestructura , Mutación , Alineación de Secuencia , Pez Cebra
2.
Nature ; 559(7715): 575-579, 2018 07.
Artículo en Inglés | MEDLINE | ID: mdl-29995856

RESUMEN

Mitochondrial calcium uptake is critical for regulating ATP production, intracellular calcium signalling, and cell death. This uptake is mediated by a highly selective calcium channel called the mitochondrial calcium uniporter (MCU). Here, we determined the structures of the pore-forming MCU proteins from two fungi by X-ray crystallography and single-particle cryo-electron microscopy. The stoichiometry, overall architecture, and individual subunit structure differed markedly from those described in the recent nuclear magnetic resonance structure of Caenorhabditis elegans MCU. We observed a dimer-of-dimer architecture across species and chemical environments, which was corroborated by biochemical experiments. Structural analyses and functional characterization uncovered the roles of key residues in the pore. These results reveal a new ion channel architecture, provide insights into calcium coordination, selectivity and conduction, and establish a structural framework for understanding the mechanism of mitochondrial calcium uniporter function.


Asunto(s)
Canales de Calcio/química , Canales de Calcio/ultraestructura , Microscopía por Crioelectrón , Fusarium/química , Metarhizium/química , Animales , Caenorhabditis elegans/química , Calcio/metabolismo , Canales de Calcio/metabolismo , Cristalografía por Rayos X , Activación del Canal Iónico , Modelos Moleculares , Dominios Proteicos , Multimerización de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Reproducibilidad de los Resultados , Solubilidad
3.
Nature ; 548(7667): 352-355, 2017 08 17.
Artículo en Inglés | MEDLINE | ID: mdl-28682307

RESUMEN

Misfolded endoplasmic reticulum proteins are retro-translocated through the membrane into the cytosol, where they are poly-ubiquitinated, extracted from the membrane, and degraded by the proteasome-a pathway termed endoplasmic reticulum-associated protein degradation (ERAD). Proteins with misfolded domains in the endoplasmic reticulum lumen or membrane are discarded through the ERAD-L and ERAD-M pathways, respectively. In Saccharomyces cerevisiae, both pathways require the ubiquitin ligase Hrd1, a multi-spanning membrane protein with a cytosolic RING finger domain. Hrd1 is the crucial membrane component for retro-translocation, but it is unclear whether it forms a protein-conducting channel. Here we present a cryo-electron microscopy structure of S. cerevisiae Hrd1 in complex with its endoplasmic reticulum luminal binding partner, Hrd3. Hrd1 forms a dimer within the membrane with one or two Hrd3 molecules associated at its luminal side. Each Hrd1 molecule has eight transmembrane segments, five of which form an aqueous cavity extending from the cytosol almost to the endoplasmic reticulum lumen, while a segment of the neighbouring Hrd1 molecule forms a lateral seal. The aqueous cavity and lateral gate are reminiscent of features of protein-conducting conduits that facilitate polypeptide movement in the opposite direction-from the cytosol into or across membranes. Our results suggest that Hrd1 forms a retro-translocation channel for the movement of misfolded polypeptides through the endoplasmic reticulum membrane.


Asunto(s)
Microscopía por Crioelectrón , Degradación Asociada con el Retículo Endoplásmico , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/ultraestructura , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestructura , Saccharomyces cerevisiae/química , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/ultraestructura , Interacciones Hidrofóbicas e Hidrofílicas , Glicoproteínas de Membrana/química , Modelos Moleculares , Conformación Proteica , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/química , Ubiquitina-Proteína Ligasas/química
4.
J Am Chem Soc ; 142(29): 12715-12729, 2020 07 22.
Artículo en Inglés | MEDLINE | ID: mdl-32575981

RESUMEN

How the distinctive lipid composition of mammalian plasma membranes impacts membrane protein structure is largely unexplored, partly because of the dearth of isotropic model membrane systems that contain abundant sphingolipids and cholesterol. This gap is addressed by showing that sphingomyelin and cholesterol-rich (SCOR) lipid mixtures with phosphatidylcholine can be cosolubilized by n-dodecyl-ß-melibioside to form bicelles. Small-angle X-ray and neutron scattering, as well as cryo-electron microscopy, demonstrate that these assemblies are stable over a wide range of conditions and exhibit the bilayered-disc morphology of ideal bicelles even at low lipid-to-detergent mole ratios. SCOR bicelles are shown to be compatible with a wide array of experimental techniques, as applied to the transmembrane human amyloid precursor C99 protein in this medium. These studies reveal an equilibrium between low-order oligomer structures that differ significantly from previous experimental structures of C99, providing an example of how ordered membranes alter membrane protein structure.


Asunto(s)
Colesterol/química , Proteínas de la Membrana/química , Esfingolípidos/química , Microscopía por Crioelectrón , Humanos
5.
J Struct Biol ; 199(3): 225-236, 2017 09.
Artículo en Inglés | MEDLINE | ID: mdl-28827185

RESUMEN

This paper provides an overview of the discussion and presentations from the Workshop on the Management of Large CryoEM Facilities held at the New York Structural Biology Center, New York, NY on February 6-7, 2017. A major objective of the workshop was to discuss best practices for managing cryoEM facilities. The discussions were largely focused on supporting single-particle methods for cryoEM and topics included: user access, assessing projects, workflow, sample handling, microscopy, data management and processing, and user training.


Asunto(s)
Microscopía por Crioelectrón , Investigación/organización & administración , Microscopía por Crioelectrón/instrumentación , Flujo de Trabajo
6.
Blood ; 126(8): 935-8, 2015 Aug 20.
Artículo en Inglés | MEDLINE | ID: mdl-26065652

RESUMEN

Association with the D'D3 domain of von Willebrand factor (VWF) stabilizes factor VIII (FVIII) in the circulation and maintains it at a level sufficient to prevent spontaneous bleeding. We used negative-stain electron microscopy (EM) to visualize complexes of FVIII with dimeric and monomeric forms of the D'D3 domain. The EM averages show that FVIII interacts with the D'D3 domain primarily through its C1 domain, with the C2 domain providing a secondary attachment site. Hydrogen-deuterium exchange mass spectrometry corroborated the importance of the C1 domain in D'D3 binding and implicates additional surface regions on FVIII in the interaction. Together, our results establish that the C1 domain is the major binding site on FVIII for VWF, reiterate the importance of the a3 acidic peptide in VWF binding, and suggest that the A3 and C2 domains play ancillary roles in this interaction.


Asunto(s)
Factor VIII/química , Factor VIII/metabolismo , Factor de von Willebrand/química , Factor de von Willebrand/metabolismo , Sitios de Unión , Factor VIII/ultraestructura , Células HEK293 , Humanos , Espectrometría de Masas , Microscopía Electrónica , Estructura Terciaria de Proteína , Factor de von Willebrand/ultraestructura
7.
Proc Natl Acad Sci U S A ; 107(30): 13467-72, 2010 Jul 27.
Artículo en Inglés | MEDLINE | ID: mdl-20624955

RESUMEN

Clostridium difficile toxins A and B are members of an important class of virulence factors known as large clostridial toxins (LCTs). Toxin action involves four major steps: receptor-mediated endocytosis, translocation of a catalytic glucosyltransferase domain across the membrane, release of the enzymatic moiety by autoproteolytic processing, and a glucosyltransferase-dependent inactivation of Rho family proteins. We have imaged toxin A (TcdA) and toxin B (TcdB) holotoxins by negative stain electron microscopy to show that these molecules are similar in structure. We then determined a 3D structure for TcdA and mapped the organization of its functional domains. The molecule has a "pincher-like" head corresponding to the delivery domain and two tails, long and short, corresponding to the receptor-binding and glucosyltransferase domains, respectively. A second structure, obtained at the acidic pH of an endosome, reveals a significant structural change in the delivery and glucosyltransferase domains, and thus provides a framework for understanding the molecular mechanism of LCT cellular intoxication.


Asunto(s)
Proteínas Bacterianas/química , Toxinas Bacterianas/química , Clostridioides difficile/metabolismo , Enterotoxinas/química , Estructura Terciaria de Proteína , Animales , Proteínas Bacterianas/ultraestructura , Sitios de Unión , Células CHO , Cricetinae , Cricetulus , Electroforesis en Gel de Poliacrilamida , Glucosiltransferasas/química , Glucosiltransferasas/metabolismo , Concentración de Iones de Hidrógeno , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica , Modelos Moleculares , Conformación Proteica
8.
Biochemistry ; 49(27): 5743-52, 2010 Jul 13.
Artículo en Inglés | MEDLINE | ID: mdl-20527875

RESUMEN

Helicobacter pylori VacA is a pore-forming toxin that causes multiple alterations in human cells and contributes to the pathogenesis of peptic ulcer disease and gastric cancer. The toxin is secreted by H. pylori as an 88 kDa monomer (p88) consisting of two domains (p33 and p55). While an X-ray crystal structure for p55 exists and p88 oligomers have been visualized by cryo-electron microscopy, a detailed analysis of p33 has been hindered by an inability to purify this domain in an active form. In this study, we expressed and purified a recombinant form of p33 under denaturing conditions and optimized conditions for the refolding of the soluble protein. We show that refolded p33 can be added to purified p55 in trans to cause vacuolation of HeLa cells and inhibition of IL-2 production by Jurkat cells, effects identical to those produced by the p88 toxin from H. pylori. The p33 protein markedly enhances the cell binding properties of p55. Size exclusion chromatography experiments suggest that p33 and p55 assemble into a complex consistent with the size of a p88 monomer. Electron microscopy of these p33/p55 complexes reveals small rod-shaped structures that can convert to oligomeric flower-shaped structures in the presence of detergent. We propose that the oligomerization observed in these experiments mimics the process by which VacA oligomerizes when in contact with membranes of host cells.


Asunto(s)
Helicobacter pylori/genética , Helicobacter pylori/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica , Microscopía por Crioelectrón , Ciclofosfamida , Dactinomicina , Doxorrubicina , Células HeLa , Humanos , Interleucina-2/genética , Interleucina-2/metabolismo , Toxinas Biológicas/genética , Toxinas Biológicas/metabolismo , Vincristina , Rayos X
9.
Elife ; 82019 07 08.
Artículo en Inglés | MEDLINE | ID: mdl-31282860

RESUMEN

Iron storage proteins are essential for cellular iron homeostasis and redox balance. Ferritin proteins are the major storage units for bioavailable forms of iron. Some organisms lack ferritins, and it is not known how they store iron. Encapsulins, a class of protein-based organelles, have recently been implicated in microbial iron and redox metabolism. Here, we report the structural and mechanistic characterization of a 42 nm two-component encapsulin-based iron storage compartment from Quasibacillus thermotolerans. Using cryo-electron microscopy and x-ray crystallography, we reveal the assembly principles of a thermostable T = 4 shell topology and its catalytic ferroxidase cargo and show interactions underlying cargo-shell co-assembly. This compartment has an exceptionally large iron storage capacity storing over 23,000 iron atoms. Our results reveal a new approach for survival in diverse habitats with limited or fluctuating iron availability via an iron storage system able to store 10 to 20 times more iron than ferritin.


Asunto(s)
Bacillaceae/metabolismo , Proteínas Bacterianas/metabolismo , Hierro/metabolismo , Orgánulos/metabolismo , Bacillaceae/ultraestructura , Proteínas Bacterianas/química , Ceruloplasmina/química , Ceruloplasmina/metabolismo , Microscopía por Crioelectrón , Cristalografía por Rayos X , Ferritinas/química , Ferritinas/metabolismo , Homeostasis , Modelos Moleculares , Orgánulos/ultraestructura , Conformación Proteica
10.
Science ; 360(6389)2018 05 11.
Artículo en Inglés | MEDLINE | ID: mdl-29650704

RESUMEN

Mitochondrial adenosine triphosphate (ATP) synthase comprises a membrane embedded Fo motor that rotates to drive ATP synthesis in the F1 subunit. We used single-particle cryo-electron microscopy (cryo-EM) to obtain structures of the full complex in a lipid bilayer in the absence or presence of the inhibitor oligomycin at 3.6- and 3.8-angstrom resolution, respectively. To limit conformational heterogeneity, we locked the rotor in a single conformation by fusing the F6 subunit of the stator with the δ subunit of the rotor. Assembly of the enzyme with the F6-δ fusion caused a twisting of the rotor and a 9° rotation of the Fo c10-ring in the direction of ATP synthesis, relative to the structure of isolated Fo Our cryo-EM structures show how F1 and Fo are coupled, give insight into the proton translocation pathway, and show how oligomycin blocks ATP synthesis.


Asunto(s)
Membranas Mitocondriales/enzimología , ATPasas de Translocación de Protón Mitocondriales/química , Proteínas Motoras Moleculares/química , Proteínas de Saccharomyces cerevisiae/química , Adenosina Trifosfato/biosíntesis , Microscopía por Crioelectrón , Lípidos de la Membrana/química , Membranas Mitocondriales/química , ATPasas de Translocación de Protón Mitocondriales/ultraestructura , Proteínas Motoras Moleculares/ultraestructura , Oligomicinas/química , Conformación Proteica , Subunidades de Proteína , Proteínas de Saccharomyces cerevisiae/ultraestructura , Imagen Individual de Molécula
11.
Nat Commun ; 9(1): 1199, 2018 03 23.
Artículo en Inglés | MEDLINE | ID: mdl-29572442

RESUMEN

The B cell survival factor (TNFSF13B/BAFF) is often elevated in autoimmune diseases and is targeted in the clinic for the treatment of systemic lupus erythematosus. BAFF contains a loop region designated the flap, which is dispensable for receptor binding. Here we show that the flap of BAFF has two functions. In addition to facilitating the formation of a highly active BAFF 60-mer as shown previously, it also converts binding of BAFF to TNFRSF13C (BAFFR) into a signaling event via oligomerization of individual BAFF-BAFFR complexes. Binding and activation of BAFFR can therefore be targeted independently to inhibit or activate the function of BAFF. Moreover, structural analyses suggest that the flap of BAFF 60-mer temporarily prevents binding of an anti-BAFF antibody (belimumab) but not of a decoy receptor (atacicept). The observed differences in profiles of BAFF inhibition may confer distinct biological and clinical efficacies to these therapeutically relevant inhibitors.


Asunto(s)
Factor Activador de Células B/química , Factor Activador de Células B/fisiología , Receptor del Factor Activador de Células B/química , Linfocitos B/citología , Animales , Anticuerpos Monoclonales Humanizados/farmacología , Factor Activador de Células B/genética , Diferenciación Celular , Supervivencia Celular , Reactivos de Enlaces Cruzados/química , Femenino , Técnicas de Sustitución del Gen , Células HEK293 , Humanos , Fragmentos de Inmunoglobulinas/química , Linfopenia/metabolismo , Masculino , Ratones , Ratones Transgénicos , Mutación , Unión Proteica , Dominios Proteicos , Proteínas Recombinantes de Fusión/farmacología
12.
Nat Struct Mol Biol ; 23(8): 761-3, 2016 08.
Artículo en Inglés | MEDLINE | ID: mdl-27428774

RESUMEN

We show here that the Saccharomyces cerevisiae GARP complex and the Cog1-4 subcomplex of the COG complex, both members of the complexes associated with tethering containing helical rods (CATCHR) family of multisubunit tethering complexes, share the same subunit organization. We also show that HOPS, a tethering complex acting in the endolysosomal pathway, shares a similar architecture, thus suggesting that multisubunit tethering complexes use related structural frameworks.


Asunto(s)
Saccharomyces cerevisiae/ultraestructura , Proteínas Adaptadoras del Transporte Vesicular/ultraestructura , Proteínas de Transporte de Membrana/ultraestructura , Microscopía Electrónica , Modelos Moleculares , Complejos Multiproteicos/ultraestructura , Estructura Cuaternaria de Proteína , Subunidades de Proteína/química , Proteínas de Saccharomyces cerevisiae/ultraestructura , Proteínas de Transporte Vesicular/ultraestructura
13.
J Mol Biol ; 425(3): 524-35, 2013 Feb 08.
Artículo en Inglés | MEDLINE | ID: mdl-23178866

RESUMEN

Helicobacter pylori is a Gram-negative bacterium that colonizes the human stomach and contributes to peptic ulceration and gastric adenocarcinoma. H. pylori secretes a pore-forming exotoxin known as vacuolating toxin (VacA). VacA contains two distinct domains, designated p33 and p55, and assembles into large "snowflake"-shaped oligomers. Thus far, no structural data are available for the p33 domain, which is essential for membrane channel formation. Using single-particle electron microscopy and the random conical tilt approach, we have determined the three-dimensional structures of six VacA oligomeric conformations at ~15-Å resolution. The p55 domain, composed primarily of ß-helical structures, localizes to the peripheral arms, while the p33 domain consists of two globular densities that localize within the center of the complexes. By fitting the VacA p55 crystal structure into the electron microscopy densities, we have mapped inter-VacA interactions that support oligomerization. In addition, we have examined VacA variants/mutants that differ from wild-type (WT) VacA in toxin activity and/or oligomeric structural features. Oligomers formed by VacA∆6-27, a mutant that fails to form membrane channels, lack an organized p33 central core. Mixed oligomers containing both WT and VacA∆6-27 subunits also lack an organized core. Oligomers formed by a VacA s2m1 chimera (which lacks cell-vacuolating activity) and VacAΔ301-328 (which retains vacuolating activity) each contain p33 central cores similar to those of WT oligomers. By providing the most detailed view of the VacA structure to date, these data offer new insights into the toxin's channel-forming component and the intermolecular interactions that underlie oligomeric assembly.


Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Multimerización de Proteína , Microscopía Electrónica/métodos , Modelos Moleculares , Mapeo de Interacción de Proteínas
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA