RESUMEN
The ß-barrel assembly machinery (BAM) inserts outer membrane ß-barrel proteins (OMPs) in the outer membrane of Gram-negative bacteria. In Enterobacteriacea, BAM also mediates export of the stress sensor lipoprotein RcsF to the cell surface by assembling RcsF-OMP complexes. Here, we report the crystal structure of the key BAM component BamA in complex with RcsF. BamA adopts an inward-open conformation, with the lateral gate to the membrane closed. RcsF is lodged deep within the lumen of the BamA barrel, binding regions proposed to undergo outward and lateral opening during OMP insertion. On the basis of our structural and biochemical data, we propose a push-and-pull model for RcsF export following conformational cycling of BamA, and provide a mechanistic explanation for how RcsF uses its interaction with BamA to detect envelope stress. Our data also suggest that the flux of incoming OMP substrates is involved in the control of BAM activity.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Cristalografía por Rayos X , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Modelos Moleculares , Conformación ProteicaRESUMEN
N6-threonyl-carbamoyl adenosine (t6A) is a universal tRNA modification found at position 37, next to the anticodon, in almost all tRNAs decoding ANN codons (where N = A, U, G, or C). t6A stabilizes the codon-anticodon interaction and hence promotes translation fidelity. The first step of the biosynthesis of t6A, the production of threonyl-carbamoyl adenylate (TC-AMP), is catalyzed by the Sua5/TsaC family of enzymes. While TsaC is a single domain protein, Sua5 enzymes are composed of the TsaC-like domain, a linker and an extra domain called SUA5 of unknown function. In the present study, we report structure-function analysis of Pyrococcus abyssi Sua5 (Pa-Sua5). Crystallographic data revealed binding sites for bicarbonate substrate and pyrophosphate product. The linker of Pa-Sua5 forms a loop structure that folds into the active site gorge and closes it. Using structure-guided mutational analysis, we established that the conserved sequence motifs in the linker and the domain-domain interface are essential for the function of Pa-Sua5. We propose that the linker participates actively in the biosynthesis of TC-AMP by binding to ATP/PPi and by stabilizing the N-carboxy-l-threonine intermediate. Hence, TsaC orthologs which lack such a linker and SUA5 domain use a different mechanism for TC-AMP synthesis.
Asunto(s)
Adenosina/análogos & derivados , Proteínas Arqueales/química , Pyrococcus abyssi/enzimología , ARN de Transferencia/metabolismo , Adenosina/biosíntesis , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Secuencias de Aminoácidos , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Modelos Moleculares , Mutación , Conformación Proteica , Dominios Proteicos , ARN de Transferencia/química , Relación Estructura-ActividadRESUMEN
Protein synthesis is a complex and highly coordinated process requiring many different protein factors as well as various types of nucleic acids. All translation machinery components require multiple maturation events to be functional. These include post-transcriptional and post-translational modification steps and methylations are the most frequent among these events. In eukaryotes, Trm112, a small protein (COG2835) conserved in all three domains of life, interacts and activates four methyltransferases (Bud23, Trm9, Trm11 and Mtq2) that target different components of the translation machinery (rRNA, tRNAs, release factors). To clarify the function of Trm112 in archaea, we have characterized functionally and structurally its interaction network using Haloferax volcanii as model system. This led us to unravel that methyltransferases are also privileged Trm112 partners in archaea and that this Trm112 network is much more complex than anticipated from eukaryotic studies. Interestingly, among the identified enzymes, some are functionally orthologous to eukaryotic Trm112 partners, emphasizing again the similarity between eukaryotic and archaeal translation machineries. Other partners display some similarities with bacterial methyltransferases, suggesting that Trm112 is a general partner for methyltransferases in all living organisms.
Asunto(s)
Proteínas Arqueales/fisiología , Proteínas Bacterianas/fisiología , Haloferax volcanii/enzimología , Procesamiento Postranscripcional del ARN , ARNt Metiltransferasas/fisiología , Proteínas Bacterianas/genética , Cristalografía por Rayos X , Conjuntos de Datos como Asunto , Activación Enzimática , Células Eucariotas/enzimología , Evolución Molecular , Holoenzimas/fisiología , Inmunoprecipitación , Espectrometría de Masas , Metilación , Modelos Moleculares , Unión Proteica , Conformación Proteica , Mapeo de Interacción de Proteínas , Proteómica , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Especificidad de la Especie , ARNt Metiltransferasas/deficiencia , ARNt Metiltransferasas/genéticaRESUMEN
Most of the factors involved in translation (tRNA, rRNA and proteins) are subject to post-transcriptional and post-translational modifications, which participate in the fine-tuning and tight control of ribosome and protein synthesis processes. In eukaryotes, Trm112 acts as an obligate activating platform for at least four methyltransferases (MTase) involved in the modification of 18S rRNA (Bud23), tRNA (Trm9 and Trm11) and translation termination factor eRF1 (Mtq2). Trm112 is then at a nexus between ribosome synthesis and function. Here, we present a structure-function analysis of the Trm9-Trm112 complex, which is involved in the 5-methoxycarbonylmethyluridine (mcm(5)U) modification of the tRNA anticodon wobble position and hence promotes translational fidelity. We also compare the known crystal structures of various Trm112-MTase complexes, highlighting the structural plasticity allowing Trm112 to interact through a very similar mode with its MTase partners, although those share less than 20% sequence identity.
Asunto(s)
Proteínas de Saccharomyces cerevisiae/química , ARNt Metiltransferasas/química , Biocatálisis , Dominio Catalítico , Cristalografía por Rayos X , Modelos Moleculares , Proteínas de Saccharomyces cerevisiae/metabolismo , Yarrowia/enzimología , ARNt Metiltransferasas/metabolismoRESUMEN
The eukaryotic small ribosomal subunit carries only four ribosomal (r) RNA methylated bases, all close to important functional sites. N(7)-methylguanosine (m(7)G) introduced at position 1575 on 18S rRNA by Bud23-Trm112 is at a ridge forming a steric block between P- and E-site tRNAs. Here we report atomic resolution structures of Bud23-Trm112 in the apo and S-adenosyl-L-methionine (SAM)-bound forms. Bud23 and Trm112 interact through formation of a ß-zipper involving main-chain atoms, burying an important hydrophobic surface and stabilizing the complex. The structures revealed that the coactivator Trm112 undergoes an induced fit to accommodate its methyltransferase (MTase) partner. We report important structural similarity between the active sites of Bud23 and Coffea canephora xanthosine MTase, leading us to propose and validate experimentally a model for G1575 coordination. We identify Bud23 residues important for Bud23-Trm112 complex formation and recruitment to pre-ribosomes. We report that though Bud23-Trm112 binds precursor ribosomes at an early nucleolar stage, m(7)G methylation occurs at a late step of small subunit biogenesis, implying specifically delayed catalytic activation. Finally, we show that Bud23-Trm112 interacts directly with the box C/D snoRNA U3-associated DEAH RNA helicase Dhr1 supposedly involved in central pseudoknot formation; this suggests that Bud23-Trm112 might also contribute to controlling formation of this irreversible and dramatic structural reorganization essential to overall folding of small subunit rRNA. Our study contributes important new elements to our understanding of key molecular aspects of human ribosomopathy syndromes associated with WBSCR22 (human Bud23) malfunction.
Asunto(s)
Metiltransferasas/metabolismo , ARN Ribosómico 18S/metabolismo , Ribosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , ARNt Metiltransferasas/metabolismo , Catálisis , Metilación , Metiltransferasas/química , Modelos Moleculares , Conformación Proteica , Proteínas de Saccharomyces cerevisiae/química , ARNt Metiltransferasas/químicaRESUMEN
OmpA, a protein commonly found in the outer membrane of Gram-negative bacteria, has served as a paradigm for the study of ß-barrel proteins for several decades. In Escherichia coli, OmpA was previously reported to form complexes with RcsF, a surface-exposed lipoprotein that triggers the Rcs stress response when damage occurs in the outer membrane and the peptidoglycan. How OmpA interacts with RcsF and whether this interaction allows RcsF to reach the surface has remained unclear. Here, we integrated in vivo and in vitro approaches to establish that RcsF interacts with the C-terminal, periplasmic domain of OmpA, not with the N-terminal ß-barrel, thus implying that RcsF does not reach the bacterial surface via OmpA. Our results suggest a novel function for OmpA in the cell envelope: OmpA competes with the inner membrane protein IgaA, the downstream Rcs component, for RcsF binding across the periplasm, thereby regulating the Rcs response.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/genética , Membrana Celular/fisiología , Proteínas de Escherichia coli/genética , Escherichia coli/fisiología , Transducción de Señal , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de Escherichia coli/metabolismoRESUMEN
Post-transcriptional and post-translational modifications are very important for the control and optimal efficiency of messenger RNA (mRNA) translation. Among these, methylation is the most widespread modification, as it is found in all domains of life. These methyl groups can be grafted either on nucleic acids (transfer RNA (tRNA), ribosomal RNA (rRNA), mRNA, etc.) or on protein translation factors. This review focuses on Trm112, a small protein interacting with and activating at least four different eukaryotic methyltransferase (MTase) enzymes modifying factors involved in translation. The Trm112-Trm9 and Trm112-Trm11 complexes modify tRNAs, while the Trm112-Mtq2 complex targets translation termination factor eRF1, which is a tRNA mimic. The last complex formed between Trm112 and Bud23 proteins modifies 18S rRNA and participates in the 40S biogenesis pathway. In this review, we present the functions of these eukaryotic Trm112-MTase complexes, the molecular bases responsible for complex formation and substrate recognition, as well as their implications in human diseases. Moreover, as Trm112 orthologs are found in bacterial and archaeal genomes, the conservation of this Trm112 network beyond eukaryotic organisms is also discussed.
Asunto(s)
Células Eucariotas/enzimología , Metiltransferasas/metabolismo , Biosíntesis de Proteínas , Secuencia de Aminoácidos , Metiltransferasas/química , Modelos Moleculares , Células Procariotas/enzimologíaRESUMEN
Elongator was initially described as an RNA polymerase II-associated factor but has since been associated with a broad range of cellular activities. It has also attracted clinical attention because of its role in certain neurodegenerative diseases. Here we describe the crystal structure of the Saccharomyces cerevisiae subcomplex of Elongator proteins 4, 5 and 6 (Elp456). The subunits each show almost identical RecA folds that form a heterohexameric ring-like structure resembling hexameric RecA-like ATPases. This structural finding is supported by different complementary in vitro and in vivo approaches, including the specific binding of the hexameric Elp456 subcomplex to tRNAs in a manner regulated by ATP. Our results support a role of Elongator in tRNA modification, explain the importance of each of the Elp4, Elp5 and Elp6 subunits for complex integrity and suggest a model for the overall architecture of the holo-Elongator complex.
Asunto(s)
Proteínas de Unión al ARN/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimología , Modelos Moleculares , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , ARN de Transferencia/química , ARN de Transferencia/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidad por SustratoRESUMEN
For high-throughput structural studies of protein complexes of composition inferred from proteomics data, it is crucial that candidate complexes are selected accurately. Herein, we exemplify a procedure that combines a bioinformatics tool for complex selection with in vivo validation, to deliver structural results in a medium-throughout manner. We have selected a set of 20 yeast complexes, which were predicted to be feasible by either an automated bioinformatics algorithm, by manual inspection of primary data, or by literature searches. These complexes were validated with two straightforward and efficient biochemical assays, and heterologous expression technologies of complex components were then used to produce the complexes to assess their feasibility experimentally. Approximately one-half of the selected complexes were useful for structural studies, and we detail one particular success story. Our results underscore the importance of accurate target selection and validation in avoiding transient, unstable, or simply nonexistent complexes from the outset.