RESUMEN
Protein transport into the nucleus is mediated by transport receptors. Import of highly charged proteins, such as histone H1 and ribosomal proteins, requires a dimer of two transport receptors. In this study, we determined the cryo-EM structure of the Imp7:Impß:H1.0 complex, showing that the two importins form a cradle that accommodates the linker histone. The H1.0 globular domain is bound to Impß, whereas the acidic loops of Impß and Imp7 chaperone the positively charged C-terminal tail. Although it remains disordered, the H1 tail serves as a zipper that closes and stabilizes the structure through transient non-specific interactions with importins. Moreover, we found that the GGxxF and FxFG motifs in the Imp7 C-terminal tail are essential for Imp7:Impß dimerization and H1 import, resembling importin interaction with nucleoporins, which, in turn, promote complex disassembly. The architecture of many other complexes might be similarly defined by rapidly exchanging electrostatic interactions mediated by disordered regions.
Asunto(s)
Núcleo Celular/metabolismo , Histonas/metabolismo , Carioferinas/metabolismo , Transporte Activo de Núcleo Celular , Animales , Sitios de Unión , Núcleo Celular/genética , Núcleo Celular/ultraestructura , Microscopía por Crioelectrón , Humanos , Carioferinas/genética , Carioferinas/ultraestructura , Modelos Moleculares , Complejos Multiproteicos , Mutación , Proteínas de Complejo Poro Nuclear/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Electricidad Estática , Relación Estructura-Actividad , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis , beta Carioferinas/genética , beta Carioferinas/metabolismo , Proteína de Unión al GTP ran/metabolismoRESUMEN
Heterochromatin is a distinctive chromatin structure that is essential for chromosome segregation, genome stability and regulation of gene expression. H3K9 methylation (H3K9me), a hallmark of heterochromatin, is deposited by the Su(var)3-9 family of proteins; however, the mechanism by which H3K9 methyltransferases bind and methylate the nucleosome is poorly understood. In this work we determined the interaction of Clr4, the fission yeast H3K9 methyltransferase, with nucleosomes using nuclear magnetic resonance, biochemical and genetic assays. Our study shows that the Clr4 chromodomain binds the H3K9me3 tail and that both, the chromodomain and the disordered region connecting the chromodomain and the SET domain, bind the nucleosome core. We show that interaction of the disordered region with the nucleosome core is independent of H3K9me and contributes to H3K9me in vitro and in vivo. Moreover, we show that those interactions with the nucleosome core are contributing to de novo deposition of H3K9me and to establishment of heterochromatin.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , ADN de Hongos/metabolismo , Heterocromatina/metabolismo , Código de Histonas , N-Metiltransferasa de Histona-Lisina/metabolismo , Metiltransferasas/metabolismo , Nucleosomas/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Eliminación de Gen , Heterocromatina/ultraestructura , N-Metiltransferasa de Histona-Lisina/química , Metiltransferasas/química , Metiltransferasas/genética , Modelos Moleculares , Mutagénesis Insercional , Mutación Missense , Conformación de Ácido Nucleico , Unión Proteica , Conformación Proteica , Dominios Proteicos , Mapeo de Interacción de Proteínas , ARN de Hongos/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Schizosaccharomyces/crecimiento & desarrollo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genéticaRESUMEN
The linear chromosome of Streptomyces coelicolor contains two paralogous ssb genes, ssbA and ssbB. Following mutational analysis, we concluded that ssbA is essential, whereas ssbB plays a key role in chromosome segregation during sporulation. In the ssbB mutant, â¼30% of spores lacked DNA. The two ssb genes were expressed differently; in minimal medium, gene expression was prolonged for both genes and significantly upregulated for ssbB. The ssbA gene is transcribed as part of a polycistronic mRNA from two initiation sites, 163 bp and 75 bp upstream of the rpsF translational start codon. The ssbB gene is transcribed as a monocistronic mRNA, from an unusual promoter region, 73 bp upstream of the AUG codon. Distinctive DNA-binding affinities of single-stranded DNA-binding proteins monitored by tryptophan fluorescent quenching and electrophoretic mobility shift were observed. The crystal structure of SsbB at 1.7 Å resolution revealed a common OB-fold, lack of the clamp-like structure conserved in SsbA and previously unpublished S-S bridges between the A/B and C/D subunits. This is the first report of the determination of paralogous single-stranded DNA-binding protein structures from the same organism. Phylogenetic analysis revealed frequent duplication of ssb genes in Actinobacteria, whereas their strong retention suggests that they are involved in important cellular functions.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/fisiología , Segregación Cromosómica , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/fisiología , Streptomyces coelicolor/genética , Actinobacteria/clasificación , Actinobacteria/genética , Proteínas Bacterianas/genética , Secuencia de Bases , ADN de Cadena Simple , Proteínas de Unión al ADN/genética , Modelos Moleculares , Datos de Secuencia Molecular , Filogenia , Regiones Promotoras Genéticas , Unión Proteica , Esporas Bacterianas/genética , Streptomyces coelicolor/metabolismo , Streptomyces coelicolor/fisiología , Relación Estructura-Actividad , Sitio de Iniciación de la TranscripciónRESUMEN
Aminoacyl-tRNA synthetases (aaRSs) are ancient and evolutionary conserved enzymes catalyzing the formation of aminoacyl-tRNAs, that are used as substrates for ribosomal protein biosynthesis. In addition to full length aaRS genes, genomes of many organisms are sprinkled with truncated genes encoding single-domain aaRS-like proteins, which often have relinquished their canonical role in genetic code translation. We have identified the genes for putative seryl-tRNA synthetase homologs widespread in bacterial genomes and characterized three of them biochemically and structurally. The proteins encoded are homologous to the catalytic domain of highly diverged, atypical seryl-tRNA synthetases (aSerRSs) found only in methanogenic archaea and are deprived of the tRNA-binding domain. Remarkably, in comparison to SerRSs, aSerRS homologs display different and relaxed amino acid specificity. aSerRS homologs lack canonical tRNA aminoacylating activity and instead transfer activated amino acid to phosphopantetheine prosthetic group of putative carrier proteins, whose genes were identified in the genomic surroundings of aSerRS homologs. Detailed kinetic analysis confirmed that aSerRS homologs aminoacylate these carrier proteins efficiently and specifically. Accordingly, aSerRS homologs were renamed amino acid:[carrier protein] ligases (AMP forming). The enzymatic activity of aSerRS homologs is reminiscent of adenylation domains in nonribosomal peptide synthesis, and thus they represent an intriguing link between programmable ribosomal protein biosynthesis and template-independent nonribosomal peptide synthesis.
Asunto(s)
Aminoacil-ARNt Sintetasas/metabolismo , Proteínas Portadoras/metabolismo , Biosíntesis de Péptidos Independientes de Ácidos Nucleicos , Ribosomas/metabolismo , Acilación , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/metabolismo , Alanina/metabolismo , Secuencia de Aminoácidos , Aminoacil-ARNt Sintetasas/química , Aminoacil-ARNt Sintetasas/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biocatálisis , Bradyrhizobium/genética , Bradyrhizobium/metabolismo , Proteínas Portadoras/genética , Dominio Catalítico , Cristalografía por Rayos X , Cinética , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Serina-ARNt Ligasa/química , Serina-ARNt Ligasa/genética , Serina-ARNt Ligasa/metabolismo , Aminoacilación de ARN de TransferenciaRESUMEN
In actively translating 80S ribosomes the ribosomal protein eS7 of the 40S subunit is monoubiquitinated by the E3 ligase Not4 and deubiquitinated by Otu2 upon ribosomal subunit recycling. Despite its importance for translation efficiency the exact role and structural basis for this translational reset is poorly understood. Here, structural analysis by cryo-electron microscopy of native and reconstituted Otu2-bound ribosomal complexes reveals that Otu2 engages 40S subunits mainly between ribosome recycling and initiation stages. Otu2 binds to several sites on the intersubunit surface of the 40S that are not occupied by any other 40S-binding factors. This binding mode explains the discrimination against 80S ribosomes via the largely helical N-terminal domain of Otu2 as well as the specificity for mono-ubiquitinated eS7 on 40S. Collectively, this study reveals mechanistic insights into the Otu2-driven deubiquitination steps for translational reset during ribosome recycling/(re)initiation.
Asunto(s)
Proteínas Ribosómicas , Ribosomas , Microscopía por Crioelectrón , Biosíntesis de Proteínas , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Ribosomas/metabolismoRESUMEN
Several hydrolases of the SGNH superfamily, including the lipase SrLip from Streptomyces rimosus (Q93MW7), the acyl-CoA thioesterase I TesA from Pseudomonas aeruginosa (Q9HZY8) and the two lipolytic enzymes EstA (from P. aeruginosa, O33407) and EstP (from Pseudomonas putida, Q88QS0), were examined for promiscuity. These enzymes were tested against four chemically different classes of a total of 34 substrates known to be hydrolysed by esterases, thioesterases, lipases, phospholipases, Tweenases and proteases. Furthermore, they were also analysed with respect to their amino acid sequences and structural homology, and their phylogenetic relationship was determined. The Pseudomonas esterases EstA and EstP each have an N-terminal domain with catalytic activity together with a C-terminal autotransporter domain, and so the hybrid enzymes EstA(N)-EstP(C) and EstP(N)-EstA(C) were constructed by swapping the corresponding N- and C-terminal domains, and their hydrolytic activities were compared. Interestingly, substrate specificity and kinetic measurements indicated a significant influence of the autotransporter domains on the catalytic activities of these enzymes in solution. TesA, EstA and EstP were shown to function as esterases with different affinities and catalytic efficacies towards p-nitrophenyl butyrate. Of all the enzymes tested, only SrLip revealed lipase, phospholipase, esterase, thioesterase and Tweenase activities.
Asunto(s)
Proteínas Bacterianas/química , Hidrolasas/química , Secuencia de Aminoácidos , Proteínas Bacterianas/clasificación , Proteínas Bacterianas/genética , Butiratos/química , Butiratos/metabolismo , Hidrolasas de Éster Carboxílico/química , Hidrolasas de Éster Carboxílico/genética , Hidrolasas de Éster Carboxílico/metabolismo , Hidrolasas/clasificación , Hidrolasas/metabolismo , Cinética , Lipasa/química , Lipasa/genética , Lipasa/metabolismo , Datos de Secuencia Molecular , Palmitoil-CoA Hidrolasa/química , Palmitoil-CoA Hidrolasa/genética , Palmitoil-CoA Hidrolasa/metabolismo , Filogenia , Estructura Terciaria de Proteína , Alineación de Secuencia , Especificidad por SustratoRESUMEN
H1 linker histones are small basic proteins that have a key role in the formation and maintenance of higher-order chromatin structures. Additionally, many examples have shown that linker histones play an important role in gene regulation, modulated by their various subtypes and posttranslational modifications. Obtaining high amounts of very pure linker histones, especially for efficient antibody production, remains a demanding and challenging procedure. Here we present an easy and fast method to purify human linker histone H1.0 overexpressed in Escherichia coli, as well as its domains: N-terminal/globular domain and C-terminal intrinsically disordered domain. This purification protocol relies on a simple affinity chromatography step followed by cation exchange due to the highly basic properties of histone proteins. Therefore, this protocol can also be applied to other linker histones. Highly pure proteins in amounts sufficient for most biochemical experiments can be obtained. The functional quality of purified H1.0 histone and its domains has been confirmed by pull-down, gel-mobility shift assays and the nuclear import assay.
Asunto(s)
Histonas/aislamiento & purificación , Cromatografía de Afinidad , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Histonas/química , Histonas/genética , Reacción en Cadena de la Polimerasa , Dominios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
Amino acid:[carrier protein] ligases (aa:CP ligases) are recently discovered enzymes that are highly similar to class II aminoacyl-tRNA synthetases (aaRSs). However, while aaRSs aminoacylate tRNA and supply building blocks for ribosomal translation, aa:CP ligases transfer activated amino acids to the phosphopantetheine group of small carrier proteins. We have solved the crystal structure of an aa:CP ligase complexed with the carrier protein (CP). The CP prosthetic group enters the active site from a different direction than tRNA in class II aaRS complexes through an idiosyncratic tunnel. CP binds to aa:CP ligase in a fundamentally different manner compared to tRNA binding by structurally closely related aaRSs. Based on crystallographic analysis, an enzyme of altered CP specificity was designed, and the mechanism of amino acid transfer to the prosthetic group was proposed. The presented study reveals how a conserved class II aaRS catalytic core can adapt to another function through minor structural alterations.
Asunto(s)
Aminoacil-ARNt Sintetasas/química , Bradyrhizobium/química , Proteínas Portadoras/química , Ligasas/química , Modelos Moleculares , Complejos Multiproteicos/química , Conformación Proteica , Aminoacil-ARNt Sintetasas/metabolismo , Aminoacilación , Proteínas Portadoras/metabolismo , Dominio Catalítico/genética , Cristalización , Escherichia coli , Cinética , Ligasas/metabolismo , Estructura Molecular , Complejos Multiproteicos/metabolismo , Unión ProteicaRESUMEN
All seryl-tRNA synthetases (SerRSs) are functional homodimers with a C-terminal active site domain typical for class II aminoacyl-tRNA synthetases and an N-terminal domain involved in tRNA binding. The recently solved three-dimensional structure of Methanosarcina barkeri SerRS revealed the idiosyncratic features of methanogenic-type SerRSs; that is, an active site zinc ion, a unique tRNA binding domain, and an insertion of approximately 30 residues in the catalytic domain, which adopt a helix-turn-helix (HTH) fold. Here, we present biochemical evidence for multiple roles of the HTH motif; it is important for dimerization of the enzyme, contributes to the overall stability, and is critical for the proper positioning of the tRNA binding domain relative to the catalytic domain. The changes in intrinsic fluorescence during denaturation of the wild-type M. barkeri SerRS and of the mutated variant lacking the HTH motif combined with cross-linking and gel analysis of protein subunits during various stages of the unfolding process revealed significantly reduced stability of the mutant dimers. In vitro kinetic analysis of enzymes, mutated in one of the N-terminal helices and the HTH motif, shows impaired tRNA binding and aminoacylation and emphasizes the importance of this domain for the overall architecture of the enzyme. The role of the idiosyncratic HTH motif in dimer stabilization and association between the catalytic and tRNA binding domain has been additionally confirmed by a yeast two-hybrid approach. Furthermore, we provide experimental evidence that tRNA binds across the dimer.