RESUMEN
The initial step in the sensory transduction pathway underpinning hearing and balance in mammals involves the conversion of force into the gating of a mechanosensory transduction channel1. Despite the profound socioeconomic impacts of hearing disorders and the fundamental biological significance of understanding mechanosensory transduction, the composition, structure and mechanism of the mechanosensory transduction complex have remained poorly characterized. Here we report the single-particle cryo-electron microscopy structure of the native transmembrane channel-like protein 1 (TMC-1) mechanosensory transduction complex isolated from Caenorhabditis elegans. The two-fold symmetric complex is composed of two copies each of the pore-forming TMC-1 subunit, the calcium-binding protein CALM-1 and the transmembrane inner ear protein TMIE. CALM-1 makes extensive contacts with the cytoplasmic face of the TMC-1 subunits, whereas the single-pass TMIE subunits reside on the periphery of the complex, poised like the handles of an accordion. A subset of complexes additionally includes a single arrestin-like protein, arrestin domain protein (ARRD-6), bound to a CALM-1 subunit. Single-particle reconstructions and molecular dynamics simulations show how the mechanosensory transduction complex deforms the membrane bilayer and suggest crucial roles for lipid-protein interactions in the mechanism by which mechanical force is transduced to ion channel gating.
Asunto(s)
Caenorhabditis elegans , Microscopía por Crioelectrón , Canales Iónicos , Mecanotransducción Celular , Animales , Arrestinas/química , Arrestinas/metabolismo , Arrestinas/ultraestructura , Caenorhabditis elegans/química , Caenorhabditis elegans/ultraestructura , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/ultraestructura , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/metabolismo , Proteínas de Unión al Calcio/ultraestructura , Activación del Canal Iónico , Canales Iónicos/química , Canales Iónicos/metabolismo , Canales Iónicos/ultraestructura , LípidosRESUMEN
Mechanotransduction is the process by which a mechanical force, such as touch, is converted into an electrical signal. Transmembrane channel-like (TMC) proteins are an evolutionarily conserved family of membrane proteins whose function has been linked to a variety of mechanosensory processes, including hearing and balance sensation in vertebrates and locomotion in Drosophila. TMC1 and TMC2 are components of ion channel complexes, but the molecular features that tune these complexes to diverse mechanical stimuli are unknown. Caenorhabditis elegans express two TMC homologs, TMC-1 and TMC-2, both of which are the likely pore-forming subunits of mechanosensitive ion channels but differ in their expression pattern and functional role in the worm. Here, we present the single-particle cryo-electron microscopy structure of the native TMC-2 complex isolated from C. elegans. The complex is composed of two copies of the pore-forming TMC-2 subunit, the calcium and integrin binding protein CALM-1 and the transmembrane inner ear protein TMIE. Comparison of the TMC-2 complex to the recently published cryo-EM structure of the C. elegans TMC-1 complex highlights conserved protein-lipid interactions, as well as a π-helical structural motif in the pore-forming helices, that together suggest a mechanism for TMC-mediated mechanosensory transduction.
Asunto(s)
Proteínas de Caenorhabditis elegans , Mecanotransducción Celular , Animales , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Microscopía por Crioelectrón , Canales Iónicos/metabolismo , Lípidos , Mecanotransducción Celular/fisiología , Proteínas de la Membrana/metabolismoRESUMEN
EXD2 (3'-5' exonuclease domain-containing protein 2) is an essential protein with a conserved DEDDy superfamily 3'-5' exonuclease domain. Recent research suggests that EXD2 has two potential functions: as a component of the DNA double-strand break repair machinery and as a ribonuclease for the regulation of mitochondrial translation. Herein, electron microscope imaging analysis and proximity labeling revealed that EXD2 is anchored to the mitochondrial outer membrane through a conserved N-terminal transmembrane domain, while the C-terminal region is cytosolic. Crystal structures of the exonuclease domain in complex with Mn2+/Mg2+ revealed a domain-swapped dimer in which the central α5-α7 helices are mutually crossed over, resulting in chimeric active sites. Additionally, the C-terminal segments absent in other DnaQ family exonucleases enclose the central chimeric active sites. Combined structural and biochemical analyses demonstrated that the unusual dimeric organization stabilizes the active site, facilitates discrimination between DNA and RNA substrates based on divalent cation coordination and generates a positively charged groove that binds substrates.
Asunto(s)
Exodesoxirribonucleasas/química , Magnesio/metabolismo , Manganeso/metabolismo , Secuencia de Aminoácidos , Dominio Catalítico , Cristalografía por Rayos X , ADN/metabolismo , Roturas del ADN de Doble Cadena , Dimerización , Exodesoxirribonucleasas/metabolismo , Células HEK293 , Humanos , Membranas Mitocondriales/metabolismo , Modelos Moleculares , Dominios Proteicos , ARN/metabolismo , Proteínas Recombinantes/química , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
The endoplasmic reticulum (ER)-mitochondria encounter structure (ERMES) comprises mitochondrial distribution and morphology 12 (Mdm12), maintenance of mitochondrial morphology 1 (Mmm1), Mdm34, and Mdm10 and mediates physical membrane contact sites and nonvesicular lipid trafficking between the ER and mitochondria in yeast. Herein, we report two crystal structures of the synaptotagmin-like mitochondrial lipid-binding protein (SMP) domain of Mmm1 and the Mdm12-Mmm1 complex at 2.8 Å and 3.8 Å resolution, respectively. Mmm1 adopts a dimeric SMP structure augmented with two extra structural elements at the N and C termini that are involved in tight self-association and phospholipid coordination. Mmm1 binds two phospholipids inside the hydrophobic cavity, and the phosphate ion of the distal phospholipid is specifically recognized through extensive H-bonds. A positively charged concave surface on the SMP domain not only mediates ER membrane docking but also results in preferential binding to glycerophospholipids such as phosphatidylcholine (PC), phosphatidic acid (PA), phosphatidylglycerol (PG), and phosphatidylserine (PS), some of which are substrates for lipid-modifying enzymes in mitochondria. The Mdm12-Mmm1 structure reveals two Mdm12s binding to the SMP domains of the Mmm1 dimer in a pairwise head-to-tail manner. Direct association of Mmm1 and Mdm12 generates a 210-Å-long continuous hydrophobic tunnel that facilitates phospholipid transport. The Mdm12-Mmm1 complex binds all glycerophospholipids except for phosphatidylethanolamine (PE) in vitro.
Asunto(s)
Retículo Endoplásmico/metabolismo , Proteínas Fúngicas/metabolismo , Mitocondrias/metabolismo , Fosfolípidos/metabolismo , Transporte de Proteínas/fisiología , Levaduras/metabolismo , Transporte Biológico/fisiología , Glicerofosfolípidos/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Mitocondriales/metabolismoRESUMEN
Formation of the nucleus-vacuole junction (NVJ) is mediated by direct interaction between the vacuolar protein Vac8p and the outer nuclear endoplasmic reticulum membrane protein Nvj1p. Herein we report the crystal structure of Vac8p bound to Nvj1p at 2.4-Å resolution. Vac8p comprises a flexibly connected N-terminal H1 helix followed by 12 armadillo repeats (ARMs) that form a right-handed superhelical structure. The extended 80-Å-long loop of Nvj1p specifically binds the highly conserved inner groove formed from ARM1-12 of Vac8p. Disruption of the Nvj1p-Vac8p interaction results in the loss of tight NVJs, which impairs piecemeal microautophagy of the nucleus in Saccharomyces cerevisiae Vac8p cationic triad (Arg276, Arg317, and Arg359) motifs interacting with Nvj1p are also critical to the recognition of Atg13p, a key component of the cytoplasm-to-vacuole targeting (CVT) pathway, indicating competitive binding to Vac8p. Indeed, mutation of the cationic triad abolishes CVT of Ape1p in vivo. Combined with biochemical data, the crystal structure reveals a Vac8p homodimer formed from ARM1, and this self-association, likely regulated by the flexible H1 helix and the C terminus of Nvj1p, is critical for Vac8p cellular functions.
Asunto(s)
Núcleo Celular/metabolismo , Receptores Citoplasmáticos y Nucleares/química , Proteínas de Saccharomyces cerevisiae/química , Vacuolas/metabolismo , Proteínas de Transporte Vesicular/química , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Sustitución de Aminoácidos , Autofagia , Proteínas Relacionadas con la Autofagia/química , Proteínas Relacionadas con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/metabolismo , Unión Competitiva , Cristalografía por Rayos X , Citoplasma/metabolismo , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Dominios y Motivos de Interacción de Proteínas , Estructura Cuaternaria de Proteína , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Secuencias Repetitivas de Aminoácido , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMEN
The endoplasmic reticulum-mitochondria encounter structure (ERMES) is a protein complex that plays a tethering role in physically connecting ER and mitochondria membranes. The ERMES complex is composed of Mdm12, Mmm1, and Mdm34, which have a SMP domain in common, and Mdm10. Here, we report the crystal structure of S. cerevisiae Mdm12. The Mdm12 forms a dimeric SMP structure through domain swapping of the ß1-strand comprising residues 1-7. Biochemical experiments reveal a phospholipid-binding site located along a hydrophobic channel of the Mdm12 structure and that Mdm12 might have a binding preference for glycerophospholipids harboring a positively charged head group. Strikingly, both full-length Mdm12 and Mdm12 truncated to exclude the disordered region (residues 74-114) display the same organization in the asymmetric unit, although they crystallize as a tetramer and hexamer, respectively. Taken together, these studies provide a novel understanding of the overall organization of SMP domains in the ERMES complex, indicating that Mdm12 interacts with Mdm34 through head-to-head contact, and with Mmm1 through tail-to-tail contact of SMP domains.
Asunto(s)
Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/química , Mitocondrias/metabolismo , Dinámicas Mitocondriales , Proteínas Mitocondriales/química , Modelos Moleculares , Proteínas de Saccharomyces cerevisiae/química , Sitios de Unión , Cristalografía por Rayos X , Proteínas de la Membrana/metabolismo , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/metabolismo , Fosfolípidos/metabolismo , Dominios Proteicos , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The mitochondrial pool of Hsp90 and its mitochondrial paralogue, TRAP1, suppresses cell death and reprograms energy metabolism in cancer cells; therefore, Hsp90 and TRAP1 have been suggested as target proteins for anticancer drug development. Here, we report that the actual target protein in cancer cell mitochondria is TRAP1, and current Hsp90 inhibitors cannot effectively inactivate TRAP1 because of their insufficient accumulation in the mitochondria. To develop mitochondrial TRAP1 inhibitors, we determined the crystal structures of human TRAP1 complexed with Hsp90 inhibitors. The isopropyl amine of the Hsp90 inhibitor PU-H71 was replaced with the mitochondria-targeting moiety triphenylphosphonium to produce SMTIN-P01. SMTIN-P01 showed a different mode of action from the nontargeted PU-H71, as well as much improved cytotoxicity to cancer cells. In addition, we determined the structure of a TRAP1-adenylyl-imidodiphosphate (AMP-PNP) complex. On the basis of comparative analysis of TRAP1 structures, we propose a molecular mechanism of ATP hydrolysis that is crucial for chaperone function.
Asunto(s)
Benzodioxoles/química , Benzodioxoles/farmacología , Diseño de Fármacos , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP90 de Choque Térmico/química , Mitocondrias/efectos de los fármacos , Purinas/química , Purinas/farmacología , Aminas/química , Línea Celular Tumoral , Cristalografía por Rayos X , Humanos , Mitocondrias/metabolismo , Modelos Moleculares , Compuestos Organofosforados/química , Multimerización de Proteína , Estabilidad Proteica , Estructura Cuaternaria de ProteínaRESUMEN
Purification of membrane proteins for biochemical and structural studies is commonly achieved by recombinant overexpression in heterologous cell lines. However, many membrane proteins do not form a functional complex in a heterologous system, and few methods exist to purify sufficient protein from a native source for use in biochemical, biophysical and structural studies. Here, we provide a detailed protocol for the isolation of membrane protein complexes from transgenic Caenorhabditis elegans. We describe how to grow a genetically modified C. elegans line in abundance using standard laboratory equipment, and how to optimize purification conditions on a small scale using fluorescence-detection size-exclusion chromatography. Optimized conditions can then be applied to a large-scale preparation, enabling the purification of adequate quantities of a target protein for structural, biochemical and biophysical studies. Large-scale worm growth can be accomplished in ~9 d, and each optimization experiment can be completed in less than 1 d. We have used these methods to isolate the transmembrane channel-like protein 1 complex, as well as three additional protein complexes (transmembrane-like channel 2, lipid transfer protein and 'Protein S'), from transgenic C. elegans, demonstrating the utility of this approach in purifying challenging, low-abundance membrane protein complexes.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animales , Animales Modificados Genéticamente , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Cromatografía en Gel , Línea Celular , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismoRESUMEN
Mechanotransduction is the process by which a mechanical force, such as touch, is converted into an electrical signal. Transmembrane channel-like (TMC) proteins are an evolutionarily-conserved family of ion channels whose function has been linked to a variety of mechanosensory processes, including hearing and balance sensation in vertebrates and locomotion in Drosophila. The molecular features that tune homologous TMC ion channel complexes to diverse mechanical stimuli are unknown. Caenorhabditis elegans express two TMC homologs, TMC-1 and TMC-2, both of which are the likely pore-forming subunits of mechanosensitive ion channels but differ in their expression pattern and functional role in the worm. Here we present the single particle cryo-electron microscopy structure of the native TMC-2 complex isolated from C. elegans. The complex is composed of two copies each of the pore-forming TMC-2 subunit, the calcium and integrin binding protein CALM-1 and the transmembrane inner ear protein TMIE. Comparison of the TMC-2 complex to the recently published cryo-EM structure of the C. elegans TMC-1 complex reveals differences in subunit composition and highlights conserved protein-lipid interactions, as well as other structural features, that together suggest a mechanism for TMC-mediated mechanosensory transduction.
RESUMEN
Armadillo (ARM) repeat proteins constitute a large protein family with diverse and fundamental functions in all organisms, and armadillo repeat domains share high structural similarity. However, exactly how these structurally similar proteins can mediate diverse functions remains a long-standing question. Vac8 (vacuole related 8) is a multifunctional protein that plays pivotal roles in various autophagic pathways, including piecemeal microautophagy of the nucleus (PMN) and cytoplasm-to-vacuole targeting (Cvt) pathways in the budding yeast Saccharomyces cerevisiae. Vac8 comprises an H1 helix at the N terminus, followed by 12 armadillo repeats. Herein, we report the crystal structure of Vac8 bound to Atg13, a key component of autophagic machinery. The 70-Å extended loop of Atg13 binds to the ARM domain of Vac8 in an antiparallel manner. Structural, biochemical, and in vivo experiments demonstrated that the H1 helix of Vac8 intramolecularly associates with the first ARM and regulates its self-association, which is crucial for Cvt and PMN pathways. The structure of H1 helix-deleted Vac8 complexed with Atg13 reveals that Vac8[Δ19-33]-Atg13 forms a heterotetramer and adopts an extended superhelical structure exclusively employed in the Cvt pathway. Most importantly, comparison of Vac8-Nvj1 and Vac8-Atg13 provides a molecular understanding of how a single ARM domain protein adopts different quaternary structures depending on its associated proteins to differentially regulate 2 closely related but distinct cellular pathways. ABBREVIATIONS: Ape1: aminopeptidase I; ARM: armadillo repeat; Atg: autophagy-related; AUC: analytical ultracentrifugation; Cvt: cytoplasm-to-vacuole targeting; DIC: differential interference contrast; GFP: green fluorescent protein; GST: glutathione-S-transferase; ITC: isothermal titration calorimetry; NVJ: nucleus-vacuole junction; PDB: protein data bank; PMN: piecemeal microautophagy of the nucleus; prApe1: precursor Ape1; RMSD: root-mean-square deviation; SAXS: small-angle X-ray scattering; SD-N: nitrogen starvation medium; SEC: size-exclusion chromatography; tAtg13: Atg13 construct comprising residues 567-695; tNvj1: Nvj1 construct comprising residues 229-321; tVac8: Vac8 construct comprising residues 10-515; Vac8: vacuole related 8.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Proteínas del Dominio Armadillo/química , Proteínas Relacionadas con la Autofagia/química , Microautofagia/genética , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Vacuolas/metabolismo , Proteínas de Transporte Vesicular/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas del Dominio Armadillo/metabolismo , Proteínas Relacionadas con la Autofagia/metabolismo , Cromatografía Liquida , Reactivos de Enlaces Cruzados/química , Cristalografía por Rayos X , Citoplasma/metabolismo , Dimerización , Enlace de Hidrógeno , Microautofagia/efectos de los fármacos , Conformación Proteica en Hélice alfa , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/genética , Receptores Citoplasmáticos y Nucleares/química , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Sirolimus/farmacología , Espectrometría de Masas en Tándem , Vacuolas/efectos de los fármacos , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Kohlschutter-Tönz syndrome (KTS) is a rare autosomal-recessive disorder of childhood onset characterized by global developmental delay, spasticity, epilepsy, and amelogenesis imperfecta. Rogdi, an essential protein, is highly conserved across metazoans, and mutations in Rogdi are linked to KTS. However, how certain mutations in Rogdi abolish its physiological functions and cause KTS is not known. In this study, we determined the crystal structure of human Rogdi protein at atomic resolution. Rogdi forms a novel elongated curved structure comprising the α domain, a leucine-zipper-like four-helix bundle, and a characteristic ß-sheet domain. Within the α domain, the N-terminal H1 helix (residues 19-45) pairs with the C-terminal H6 helix (residues 252-287) in an antiparallel manner, indicating that the integrity of the four-helix bundle requires both N- and C-terminal residues. The crystal structure, in conjunction with biochemical data, indicates that the α domain might undergo a conformational change and provide a structural platform for protein-protein interactions. Disruption of the four-helix bundle by mutation results in significant destabilization of the structure. This study provides structural insights into how certain mutations in Rogdi affect its structure and cause KTS, which has important implications for the development of pharmaceutical agents against this debilitating neurological disease.
Asunto(s)
Amelogénesis Imperfecta/genética , Demencia/genética , Epilepsia/genética , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Secuencia de Aminoácidos , Cristalografía por Rayos X , Humanos , Leucina Zippers , Mutación , Dominios ProteicosRESUMEN
Although Hsp90 inhibitors can inhibit multiple tumorigenic pathways in cancer cells, their anticancer activity has been disappointingly modest. However, by forcing Hsp90 inhibitors into the mitochondria with mitochondrial delivery vehicles, they were converted into potent drugs targeting the mitochondrial Hsp90 paralog TRAP1. Here, to improve mitochondrial drug accumulation without using the mitochondrial delivery vehicle, we increased freely available drug concentrations in the cytoplasm by reducing the binding of the drugs to the abundant cytoplasmic Hsp90. After analyzing X-ray cocrystal structures, the purine ring of the Hsp90 inhibitor 2 (BIIB021) was modified to pyrazolopyrimidine scaffolds. One pyrazolopyrimidine, 12b (DN401), bound better to TRAP1 than to Hsp90, inactivated the mitochondrial TRAP1 in vivo, and it exhibited potent anticancer activity. Therefore, the rationale and feasible guidelines for developing 12b can potentially be exploited to design a potent TRAP1 inhibitor.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Pirazoles/química , Pirazoles/farmacología , Pirimidinas/química , Pirimidinas/farmacología , Animales , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapéutico , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Cristalografía por Rayos X , Proteínas HSP90 de Choque Térmico/metabolismo , Células HeLa , Humanos , Ratones Desnudos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/patología , Simulación del Acoplamiento Molecular , Neoplasias/metabolismo , Neoplasias/patología , Pirazoles/farmacocinética , Pirazoles/uso terapéutico , Pirimidinas/farmacocinética , Pirimidinas/uso terapéuticoRESUMEN
A processing additive dripping (PAD) approach to forming highly efficient (CH3 NH3 )PbI3 (MAPbI3 ) perovskite layers was investigated. A MAPbI3 (CB/DIO) perovskite film fabricated by this approach, which included briefly dripping chlorobenzene incorporating a small amount of diiodooctane (DIO) during casting of a MAPbI3 perovskite precursor dissolved in dimethylformamide, exhibited superior smooth, uniform morphologies with high crystallinity and large grains and revealed completely homogeneous surface coverage. The surface coverage and morphology of the substrate significantly affected the photovoltaic performance of planar heterojunction (PHJ) perovskite solar cells (PrSCs), resulting in a power conversion efficiency of 11.45 % with high open-circuit voltage of 0.91â V and the highest fill factor of 80.87 %. Moreover, the PAD approach could effectively provide efficient MAPbI3 (CB/DIO) perovskite layers for highly efficient, reproducible, uniform PHJ PrSC devices without performance loss or variation even over larger active areas.
RESUMEN
Terminally misfolded proteins are selectively recognized and cleared by the endoplasmic reticulum-associated degradation (ERAD) pathway. SEL1L, a component of the ERAD machinery, plays an important role in selecting and transporting ERAD substrates for degradation. We have determined the crystal structure of the mouse SEL1L central domain comprising five Sel1-Like Repeats (SLR motifs 5 to 9; hereafter called SEL1L(cent)). Strikingly, SEL1L(cent) forms a homodimer with two-fold symmetry in a head-to-tail manner. Particularly, the SLR motif 9 plays an important role in dimer formation by adopting a domain-swapped structure and providing an extensive dimeric interface. We identified that the full-length SEL1L forms a self-oligomer through the SEL1L(cent) domain in mammalian cells. Furthermore, we discovered that the SLR-C, comprising SLR motifs 10 and 11, of SEL1L directly interacts with the N-terminus luminal loops of HRD1. Therefore, we propose that certain SLR motifs of SEL1L play a unique role in membrane bound ERAD machinery.
Asunto(s)
Degradación Asociada con el Retículo Endoplásmico , Proteínas/química , Proteínas/metabolismo , Secuencias de Aminoácidos , Animales , Cristalografía por Rayos X , Péptidos y Proteínas de Señalización Intracelular , Ratones , Conformación Proteica , Secuencias Repetitivas de AminoácidoRESUMEN
A new organic-inorganic ternary bulk heterojunction (TBHJ) hybrid configuration comprised of nanostructured (CH3)3NHPbI3 (MAPbI3) perovskite-low bandgap PCPDTBT-PCBM was investigated. Well-organized TBHJ films were readily prepared by sequential spin-casting of sparsely covered MAPbI3 nano dots and PCPDTBT-PCBM bulk heterojunction (BHJ) composites on ITO/PEDOT:PSS substrates. The TBHJ hybrid device configuration comprising diiooctane (DIO) treated MAPbI3 perovskite nano dots and a PCPDTBT-PCBM BHJ composite processed with DIO additive exhibited excellent performances. The DIO additive played a key role in developing perovskite structures of MAPbI3 nano dots and induced the (110) directional crystallinity growth of longitudinal constructive morphologies such as nano rods. The improved photocurrent and fill factor compared to those of conventional BHJ devices led to an increase in efficiency of â¼28%. This improved photovoltaic performance originated from the higher quantum efficiencies contributed by the charge transfer from nanostructured MAPbI3 perovskite to PCBM. These TBHJs composed of nanostructured MAPbI3 perovskite, PCPDTBT, and PCBM also facilitated the exciton dissociation in the multi-BHJ system between MAPbI3 perovskite, PCPDTBT, and PCBM.
RESUMEN
Terminally misfolded or unassembled proteins are selectively recognized and cleared by the ER-associated degradation (ERAD) pathway. Suppressor/enhancer of lin-12-like (SEL1L), a component of the dislocation machinery containing the E3 ubiquitin ligase Hrd1, plays an important role in selecting and transporting ERAD substrates for degradation in the endoplasmic reticulum. In this study, the purification, crystallization and preliminary X-ray diffraction analysis of recombinant mouse SEL1L (residues 348-533) are reported. The crystals were obtained by the hanging-drop vapour-diffusion method at pH 8.5 and 277 K using 30% 2-propanol as a precipitant. Optimized crystals diffracted to 3.3 Å resolution at a synchrotron-radiation source. Preliminary X-ray diffraction analysis revealed that the crystals belonged to space group P21 and contained four molecules per asymmetric unit, with a solvent content of 44%.
Asunto(s)
Proteínas/química , Animales , Secuencia de Bases , Cristalización , Cristalografía por Rayos X , Cartilla de ADN , Péptidos y Proteínas de Señalización Intracelular , Ratones , Reacción en Cadena de la PolimerasaRESUMEN
Hsp90 is a molecular chaperone responsible for the assembly and regulation of many cellular client proteins. In particular, Trap1, a mitochondrial Hsp90 homologue, plays a pivotal role in maintaining mitochondrial integrity, protecting against apoptosis in cancer cells. The N (N-terminal)-M (middle) domain of human Trap1 was crystallized in complex with Hsp90 inhibitors (PU-H71 and BIIB-021) by the hanging-drop vapour-diffusion method at pH 6.5 and 293â K using 15% PEG 8K as a precipitant. Diffraction data were collected from crystals of the Trap1-PU-H71 (2.7â Å) and Trap1-BIIB-021 (3.1â Å) complexes to high resolution at a synchrotron-radiation source. Preliminary X-ray diffraction analysis revealed that both crystals belonged to space group P41212 or P43212, with unit-cell parameters a = b = 69.2, c = 252.5â Å, and contained one molecule per asymmetric unit according to Matthews coefficient calculations.