RESUMEN
The gating ring-forming RCK domain regulates channel gating in response to various cellular chemical stimuli in eukaryotic Slo channel families and the majority of ligand-gated prokaryotic K(+) channels and transporters. Here we present structural and functional studies of a dual RCK-containing, multi-ligand gated K(+) channel from Geobacter sulfurreducens, named GsuK. We demonstrate that ADP and NAD(+) activate the GsuK channel, whereas Ca(2+) serves as an allosteric inhibitor. Multiple crystal structures elucidate the structural basis of multi-ligand gating in GsuK, and also reveal a unique ion conduction pore with segmented inner helices. Structural comparison leads us to propose a novel pore opening mechanics that is distinct from other K(+) channels.DOI:http://dx.doi.org/10.7554/eLife.00184.001.
Asunto(s)
Proteínas Bacterianas/química , Calcio/química , Geobacter/química , Canales Iónicos Activados por Ligandos/química , Canales de Potasio/química , Adenosina Difosfato , Regulación Alostérica , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Calcio/metabolismo , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Geobacter/metabolismo , Activación del Canal Iónico , Canales Iónicos Activados por Ligandos/genética , Canales Iónicos Activados por Ligandos/metabolismo , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , NAD/química , NAD/metabolismo , Canales de Potasio/genética , Canales de Potasio/metabolismo , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de SecuenciaRESUMEN
Eukaryotic translation termination is mediated by two interacting release factors, eRF1 and eRF3, which act cooperatively to ensure efficient stop codon recognition and fast polypeptide release. The crystal structures of human and Schizosaccharomyces pombe full-length eRF1 in complex with eRF3 lacking the GTPase domain revealed details of the interaction between these two factors and marked conformational changes in eRF1 that occur upon binding to eRF3, leading eRF1 to resemble a tRNA molecule. Small-angle X-ray scattering analysis of the eRF1/eRF3/GTP complex suggested that eRF1's M domain contacts eRF3's GTPase domain. Consistently, mutation of Arg192, which is predicted to come in close contact with the switch regions of eRF3, revealed its important role for eRF1's stimulatory effect on eRF3's GTPase activity. An ATP molecule used as a crystallization additive was bound in eRF1's putative decoding area. Mutational analysis of the ATP-binding site shed light on the mechanism of stop codon recognition by eRF1.
Asunto(s)
Codón de Terminación/metabolismo , Modelos Moleculares , Factores de Terminación de Péptidos/química , Proteínas de Schizosaccharomyces pombe/química , Schizosaccharomyces , Adenosina Trifosfato/metabolismo , GTP Fosfohidrolasas/metabolismo , Orden Génico , Humanos , Mutación , Factores de Terminación de Péptidos/genética , Unión Proteica , Biosíntesis de Proteínas/genética , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Reproducibilidad de los Resultados , Ribosomas/metabolismo , Dispersión del Ángulo Pequeño , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismoRESUMEN
Rhodocetin is a unique heterodimer consisting of alpha- and beta-subunits of 133 and 129 residues, respectively. The molecule, purified from the crude venom of the Malayan pit viper, Calloselasma rhodostoma, functions as an inhibitor of collagen-induced aggregation. Rhodocetin has been shown to have activity only when present as a dimer. The dimer is formed without an intersubunit disulfide bridge, unlike all the other Ca(2+)-dependent lectin-like proteins. We report here the 1.9 A resolution structure of rhodocetin, which reveals the compensatory interactions that occur in the absence of the disulfide bridge to preserve activity.
Asunto(s)
Venenos de Crotálidos/química , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X , Dimerización , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica/fisiología , Conformación Proteica , Estructura Terciaria de Proteína , Subunidades de Proteína , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Relación Estructura-ActividadRESUMEN
Translation termination in eukaryotes is governed by two interacting release factors, eRF1 and eRF3. The crystal structure of the eEF1alpha-like region of eRF3 from S. pombe determined in three states (free protein, GDP-, and GTP-bound forms) reveals an overall structure that is similar to EF-Tu, although with quite different domain arrangements. In contrast to EF-Tu, GDP/GTP binding to eRF3c does not induce dramatic conformational changes, and Mg(2+) is not required for GDP binding to eRF3c. Mg(2+) at higher concentration accelerates GDP release, suggesting a novel mechanism for nucleotide exchange on eRF3 from that of other GTPases. Mapping sequence conservation onto the molecular surface, combined with mutagenesis analysis, identified the eRF1 binding region, and revealed an essential function for the C terminus of eRF3. The N-terminal extension, rich in acidic amino acids, blocks the proposed eRF1 binding site, potentially regulating eRF1 binding to eRF3 in a competitive manner.
Asunto(s)
Cristalografía por Rayos X , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Factores de Terminación de Péptidos/química , Factores de Terminación de Péptidos/metabolismo , Schizosaccharomyces/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sitios de Unión , Unión Competitiva , Secuencia Conservada , Análisis Mutacional de ADN , Proteínas Fúngicas/genética , Prueba de Complementación Genética , Variación Genética , Guanosina Difosfato/química , Guanosina Trifosfato/química , Magnesio/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis , Fragmentos de Péptidos/metabolismo , Factores de Terminación de Péptidos/genética , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Schizosaccharomyces/crecimiento & desarrollo , Homología de Secuencia de Aminoácido , Técnicas del Sistema de Dos HíbridosRESUMEN
Purpureotin, a novel di-dimeric C-type lectin-like protein (CLP) from Trimeresurus purpureomaculatus, was purified and sequenced. While its native molecular mass was determined to be 63kDa, purpureotin showed a single band of 30kDa on nonreducing SDS-PAGE and two polypeptide chains (16.0 and 14.5kDa) under reducing condition. These results were subsequently confirmed by mass spectrometric analyses. Based on these results, we postulate that purpureotin is a dimer of the alpha,beta-heterodimer which is held together by noncovalent interactions. Molecular modeling studies indicate that a dimer of alpha,beta-heterodimers can be formed where the alpha chains are held together by electrostatic charges and beta chains via hydrophobic interactions. Functionally, purpureotin induced platelet aggregation without any cofactor in a dose-dependent manner. However, the platelet aggregation effect was blocked by echicetin. Therefore, purpureotin is assumed to be a GPIb-binding protein which binds to the same or a closely related GPIb site on platelets as echicetin.