RESUMEN
The phospholipid bilayer has evolved to be a protective and selective barrier by which the cell maintains high concentrations of life sustaining organic and inorganic material. As gatekeepers responsible for an immense amount of bidirectional chemical traffic between the cytoplasm and extracellular milieu, ion channels have been studied in detail since their postulated existence nearly three-quarters of a century ago. Over the past fifteen years, we have begun to understand how selective permeability can be achieved for both cationic and anionic ions. Our mechanistic knowledge has expanded recently with studies of a large family of anion channels, the Formate Nitrite Transport (FNT) family. This family has proven amenable to structural studies at a resolution high enough to reveal intimate details of ion selectivity and gating. With five representative members having yielded a total of 15 crystal structures, this family represents one of the richest sources of structural information for anion channels.
Asunto(s)
Canales Aniónicos Dependientes del Voltaje/metabolismo , Canales Aniónicos Dependientes del Voltaje/ultraestructura , Proteínas de Transporte de Anión/metabolismo , Transporte Biológico , Cristalografía por Rayos X , Formiatos/metabolismo , Activación del Canal Iónico , Bombas Iónicas/metabolismo , Fosfolípidos/metabolismo , Conformación ProteicaRESUMEN
The hydrosulphide ion (HS(-)) and its undissociated form, hydrogen sulphide (H(2)S), which are believed to have been critical to the origin of life on Earth, remain important in physiology and cellular signalling. As a major metabolite in anaerobic bacterial growth, hydrogen sulphide is a product of both assimilatory and dissimilatory sulphate reduction. These pathways can reduce various oxidized sulphur compounds including sulphate, sulphite and thiosulphate. The dissimilatory sulphate reduction pathway uses this molecule as the terminal electron acceptor for anaerobic respiration, in which process it produces excess amounts of H(2)S (ref. 4). The reduction of sulphite is a key intermediate step in all sulphate reduction pathways. In Clostridium and Salmonella, an inducible sulphite reductase is directly linked to the regeneration of NAD(+), which has been suggested to have a role in energy production and growth, as well as in the detoxification of sulphite. Above a certain concentration threshold, both H(2)S and HS(-) inhibit cell growth by binding the metal centres of enzymes and cytochrome oxidase, necessitating a release mechanism for the export of this toxic metabolite from the cell. Here we report the identification of a hydrosulphide ion channel in the pathogen Clostridium difficile through a combination of genetic, biochemical and functional approaches. The HS(-) channel is a member of the formate/nitrite transport family, in which about 50 hydrosulphide ion channels form a third subfamily alongside those for formate (FocA) and for nitrite (NirC). The hydrosulphide ion channel is permeable to formate and nitrite as well as to HS(-) ions. Such polyspecificity can be explained by the conserved ion selectivity filter observed in the channel's crystal structure. The channel has a low open probability and is tightly regulated, to avoid decoupling of the membrane proton gradient.
Asunto(s)
Clostridioides difficile , Canales Iónicos/aislamiento & purificación , Canales Iónicos/metabolismo , Sulfuros/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Clostridioides difficile/química , Clostridioides difficile/efectos de los fármacos , Clostridioides difficile/genética , Cristalografía por Rayos X , Formiatos/metabolismo , Activación del Canal Iónico , Canales Iónicos/química , Canales Iónicos/genética , Transporte Iónico , Modelos Biológicos , Modelos Moleculares , Nitritos/metabolismo , Operón/genética , Proteolípidos/metabolismo , Fuerza Protón-Motriz , Relación Estructura-Actividad , Especificidad por Sustrato , Sulfuros/toxicidadRESUMEN
Biochemical and biophysical analysis on integral membrane proteins often requires monodisperse and stable protein samples. Here we describe a method to characterize protein thermostability by measuring its melting temperature in detergent using analytical size-exclusion chromatography. This quantitative method can be used to screen for compounds and conditions that stabilize the protein. With this technique we were able to assess and improve the thermostability of several membrane proteins. These conditions were in turn used to assist purification, to identify protein ligand and to improve crystal quality.
Asunto(s)
Proteínas de Transporte de Anión/química , Fosfatidato Fosfatasa/química , Proteínas de Transporte de Anión/aislamiento & purificación , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Cromatografía de Afinidad , Cromatografía en Gel , Cristalización , Cristalografía por Rayos X , Glucósidos/química , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/aislamiento & purificación , Fosfatidato Fosfatasa/aislamiento & purificación , Estabilidad Proteica , Solubilidad , Temperatura de TransiciónRESUMEN
Ultraviolet (UV) light-induced pyrimidine photodimers are repaired by the nucleotide excision repair pathway. Photolesions have biophysical parameters closely resembling undamaged DNA, impeding discovery through damage surveillance proteins. The DDB1-DDB2 complex serves in the initial detection of UV lesions in vivo. Here we present the structures of the DDB1-DDB2 complex alone and bound to DNA containing either a 6-4 pyrimidine-pyrimidone photodimer (6-4PP) lesion or an abasic site. The structure shows that the lesion is held exclusively by the WD40 domain of DDB2. A DDB2 hairpin inserts into the minor groove, extrudes the photodimer into a binding pocket, and kinks the duplex by approximately 40 degrees. The tightly localized probing of the photolesions, combined with proofreading in the photodimer pocket, enables DDB2 to detect lesions refractory to detection by other damage surveillance proteins. The structure provides insights into damage recognition in chromatin and suggests a mechanism by which the DDB1-associated CUL4 ubiquitin ligase targets proteins surrounding the site of damage.
Asunto(s)
Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Rayos Ultravioleta , Animales , Daño del ADN , Proteínas de Unión al ADN/química , Humanos , Modelos Moleculares , Dímeros de Pirimidina/química , Dímeros de Pirimidina/metabolismo , Xerodermia Pigmentosa/genética , Xerodermia Pigmentosa/metabolismo , Pez Cebra , Proteínas de Pez Cebra/metabolismoRESUMEN
SWI2/SNF2 chromatin-remodeling proteins mediate the mobilization of nucleosomes and other DNA-associated proteins. SWI2/SNF2 proteins contain sequence motifs characteristic of SF2 helicases but do not have helicase activity. Instead, they couple ATP hydrolysis with the generation of superhelical torsion in DNA. The structure of the nucleosome-remodeling domain of zebrafish Rad54, a protein involved in Rad51-mediated homologous recombination, reveals that the core of the SWI2/SNF2 enzymes consist of two alpha/beta-lobes similar to SF2 helicases. The Rad54 helicase lobes contain insertions that form two helical domains, one within each lobe. These insertions contain SWI2/SNF2-specific sequence motifs likely to be central to SWI2/SNF2 function. A broad cleft formed by the two lobes and flanked by the helical insertions contains residues conserved in SWI2/SNF2 proteins and motifs implicated in DNA-binding by SF2 helicases. The Rad54 structure suggests that SWI2/SNF2 proteins use a mechanism analogous to helicases to translocate on dsDNA.
Asunto(s)
Cromatina/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/metabolismo , Pez Cebra/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Línea Celular , Cristalografía , ADN Helicasas/química , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/genética , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Alineación de Secuencia , Factores de Transcripción/química , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
Werner syndrome (WS) is a premature aging syndrome caused by mutations in the WS gene (WRN) and a deficiency in the function of the Werner protein (WRN). WRN is a multifunctional nuclear protein that catalyzes three DNA-dependent reactions: a 3'-5'-exonuclease, an ATPase, and a 3'-5'-helicase. Deficiency in WRN results in a cellular phenotype of genomic instability. The biochemical characteristics of WRN and the cellular phenotype of WRN mutants suggest that WRN plays an important role in DNA metabolic pathways such as recombination, transcription, replication, and repair. The catalytic activities of WRN have been extensively studied and are fairly well understood. However, much less is known about the domain-specific interactions between WRN and its DNA substrates. This study identifies and characterizes three distinct WRN DNA binding domains using recombinant truncated fragments of WRN and five DNA substrates (long forked duplex, blunt-ended duplex, single-stranded DNA, 5'-overhang duplex, and Holliday junction). Substrate-specific DNA binding activity was detected in three domains, one N-terminal and two different C-terminal WRN fragments (RecQ conserved domain and helicase RNase D conserved domain-containing domains). The substrate specificity of each DNA binding domain may indicate that each protein domain has a distinct biological function. The importance of these results is discussed with respect to proposed roles for WRN in distinct DNA metabolic pathways.