RESUMEN
The eIF4E-binding proteins (4E-BPs) represent a diverse class of translation inhibitors that are often deregulated in cancer cells. 4E-BPs inhibit translation by competing with eIF4G for binding to eIF4E through an interface that consists of canonical and non-canonical eIF4E-binding motifs connected by a linker. The lack of high-resolution structures including the linkers, which contain phosphorylation sites, limits our understanding of how phosphorylation inhibits complex formation. Furthermore, the binding mechanism of the non-canonical motifs is poorly understood. Here, we present structures of human eIF4E bound to 4E-BP1 and fly eIF4E bound to Thor, 4E-T, and eIF4G. These structures reveal architectural elements that are unique to 4E-BPs and provide insight into the consequences of phosphorylation. Guided by these structures, we designed and crystallized a 4E-BP mimic that shows increased repressive activity. Our studies pave the way for the rational design of 4E-BP mimics as therapeutic tools to decrease translation during oncogenic transformation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Proteínas de Drosophila/química , Factor 4E Eucariótico de Iniciación/química , Factor 4E Eucariótico de Iniciación/metabolismo , Factor 4G Eucariótico de Iniciación/química , Péptidos y Proteínas de Señalización Intracelular/química , Factores de Iniciación de Péptidos/química , Fosfoproteínas/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Secuencias de Aminoácidos , Animales , Sitios de Unión , Unión Competitiva , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular , Cristalografía por Rayos X , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Factor 4G Eucariótico de Iniciación/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Modelos Moleculares , Imitación Molecular , Factores de Iniciación de Péptidos/genética , Factores de Iniciación de Péptidos/metabolismo , Fosfoproteínas/metabolismo , Fosforilación , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
The eIF4E-binding proteins (4E-BPs) are a diverse class of translation regulators that share a canonical eIF4E-binding motif (4E-BM) with eIF4G. Consequently, they compete with eIF4G for binding to eIF4E, thereby inhibiting translation initiation. Mextli (Mxt) is an unusual 4E-BP that promotes translation by also interacting with eIF3. Here we present the crystal structures of the eIF4E-binding regions of the Drosophila melanogaster (Dm) and Caenorhabditis elegans (Ce) Mxt proteins in complex with eIF4E in the cap-bound and cap-free states. The structures reveal unexpected evolutionary plasticity in the eIF4E-binding mode, with a classical bipartite interface for Ce Mxt and a novel tripartite interface for Dm Mxt. Both interfaces comprise a canonical helix and a noncanonical helix that engage the dorsal and lateral surfaces of eIF4E, respectively. Remarkably, Dm Mxt contains a C-terminal auxiliary helix that lies anti-parallel to the canonical helix on the eIF4E dorsal surface. In contrast to the eIF4G and Ce Mxt complexes, the Dm eIF4E-Mxt complexes are resistant to competition by bipartite 4E-BPs, suggesting that Dm Mxt can bind eIF4E when eIF4G binding is inhibited. Our results uncovered unexpected diversity in the binding modes of 4E-BPs, resulting in eIF4E complexes that display differential sensitivity to 4E-BP regulation.
Asunto(s)
Proteínas de Caenorhabditis elegans/química , Proteínas de Drosophila/química , Regulación de la Expresión Génica/fisiología , Modelos Moleculares , Dominios y Motivos de Interacción de Proteínas/fisiología , Animales , Caenorhabditis elegans/química , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/química , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Evolución Molecular , Variación Genética , Unión Proteica , Dominios y Motivos de Interacción de Proteínas/genética , Estructura Terciaria de Proteína , Reproducibilidad de los ResultadosRESUMEN
The role of soil methylotrophs in methanol exchange with the atmosphere has been widely overlooked. Methanol can be derived from plant polymers and be consumed by soil microbial communities. In the current study, methanol-utilizing methylotrophs of 14 aerated soils were examined to resolve their comparative diversities and capacities to utilize ambient concentrations of methanol. Abundances of cultivable methylotrophs ranged from 10(6)-10(8) gsoilDW(-1). Methanol dissimilation was measured based on conversion of supplemented (14)C-methanol, and occurred at concentrations down to 0.002 µmol methanol gsoilDW(-1). Tested soils exhibited specific affinities to methanol (a(0)s=0.01 d(-1)) that were similar to those of other environments suggesting that methylotrophs with similar affinities were present. Two deep-branching alphaproteobacterial genotypes of mch responded to the addition of ambient concentrations of methanol (îº0.6 µmol methanol gsoilDW(-1)) in one of these soils. Methylotroph community structures were assessed by amplicon pyrosequencing of genes of mono carbon metabolism (mxaF, mch and fae). Alphaproteobacteria-affiliated genotypes were predominant in all investigated soils, and the occurrence of novel genotypes indicated a hitherto unveiled diversity of methylotrophs. Correlations between vegetation type, soil pH and methylotroph community structure suggested that plant-methylotroph interactions were determinative for soil methylotrophs.