RESUMEN
Phosphorus is required for all life and microorganisms can extract it from their environment through several metabolic pathways. When phosphate is in limited supply, some bacteria are able to use phosphonate compounds, which require specialized enzymatic machinery to break the stable carbon-phosphorus (C-P) bond. Despite its importance, the details of how this machinery catabolizes phosphonates remain unknown. Here we determine the crystal structure of the 240-kilodalton Escherichia coli C-P lyase core complex (PhnG-PhnH-PhnI-PhnJ; PhnGHIJ), and show that it is a two-fold symmetric hetero-octamer comprising an intertwined network of subunits with unexpected self-homologies. It contains two potential active sites that probably couple phosphonate compounds to ATP and subsequently hydrolyse the C-P bond. We map the binding site of PhnK on the complex using electron microscopy, and show that it binds to a conserved insertion domain of PhnJ. Our results provide a structural basis for understanding microbial phosphonate breakdown.
Asunto(s)
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Liasas/química , Liasas/metabolismo , Adenosina Trifosfato/metabolismo , Sitios de Unión , Biocatálisis , Carbono/química , Carbono/metabolismo , Secuencia Conservada , Cristalografía por Rayos X , Proteínas de Escherichia coli/ultraestructura , Hidrólisis , Hierro/química , Hierro/metabolismo , Liasas/ultraestructura , Microscopía Electrónica , Modelos Moleculares , Organofosfonatos/metabolismo , Fósforo/química , Fósforo/metabolismo , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Azufre/química , Azufre/metabolismoRESUMEN
Emphysema and liver cirrhosis can be caused by the Z mutation (Glu342Lys) in the serine protease inhibitor α1-antitrypsin (α1AT), which is found in more than 4% of the Northern European population. Homozygotes experience deficiency in the lung concomitantly with a massive accumulation of polymers within hepatocytes, causing their destruction. Recently, it was proposed that Z-α1AT polymerizes by a C-terminal domain swap. In this study, small-angle x-ray scattering (SAXS) was used to characterize Z-α1AT polymers in solution. The data show that the Z-α1AT trimer, tetramer, and pentamer all form ring-like structures in strong support of a common domain-swap polymerization mechanism that can lead to self-terminating polymers.
Asunto(s)
Simulación de Dinámica Molecular , Multimerización de Proteína , alfa 1-Antitripsina/química , Secuencia de Aminoácidos , Humanos , Datos de Secuencia Molecular , Mutación , Unión Proteica , Estructura Terciaria de Proteína , Dispersión del Ángulo Pequeño , Difracción de Rayos X , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismoAsunto(s)
Caenorhabditis elegans/metabolismo , Factor Tu de Elongación Peptídica/química , Factor Tu de Elongación Peptídica/metabolismo , ARN de Transferencia/metabolismo , Animales , Modelos Moleculares , Unión Proteica , Conformación Proteica , ARN de Transferencia/química , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
In an attempt to understand ribosome-induced GTP hydrolysis on eEF2, we determined a 12.6-A cryo-electron microscopy reconstruction of the eEF2-bound 80S ribosome in the presence of aluminum tetrafluoride and GDP, with aluminum tetrafluoride mimicking the gamma-phosphate during hydrolysis. This is the first visualization of a structure representing a transition-state complex on the ribosome. Tight interactions are observed between the factor's G domain and the large ribosomal subunit, as well as between domain IV and an intersubunit bridge. In contrast, some of the domains of eEF2 implicated in small subunit binding display a large degree of flexibility. Furthermore, we find support for a transition-state model conformation of the switch I region in this complex where the reoriented switch I region interacts with a conserved rRNA region of the 40S subunit formed by loops of the 18S RNA helices 8 and 14. This complex is structurally distinct from the eEF2-bound 80S ribosome complexes previously reported, and analysis of this map sheds light on the GTPase-coupled translocation mechanism.
Asunto(s)
Microscopía por Crioelectrón , Factor 2 de Elongación Peptídica/metabolismo , Factor 2 de Elongación Peptídica/ultraestructura , Ribosomas/metabolismo , Ribosomas/ultraestructura , Compuestos de Aluminio/farmacología , Fluoruros/farmacología , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/ultraestructura , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Hidrólisis/efectos de los fármacos , Modelos Moleculares , Factor 2 de Elongación Peptídica/química , Estructura Terciaria de Proteína , Ribosomas/efectos de los fármacosRESUMEN
Two recent cryo-EM reconstructions of the ribosome-bound release factor RF2 reveal an open, tri-lobed shape of RF2, in contrast to the comma-shaped molecule seen in the crystal structure. This indicates that RF2 undergoes a conformational change upon binding to the ribosome. Moreover, RF2 does not seem to be a molecular mimic of tRNA as is the case for elongation factor G.