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1.
Nucleic Acids Res ; 50(14): 8154-8167, 2022 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-35848927

RESUMO

RNase P is a ribonucleoprotein (RNP) that catalyzes removal of the 5' leader from precursor tRNAs in all domains of life. A recent cryo-EM study of Methanocaldococcus jannaschii (Mja) RNase P produced a model at 4.6-Å resolution in a dimeric configuration, with each holoenzyme monomer containing one RNase P RNA (RPR) and one copy each of five RNase P proteins (RPPs; POP5, RPP30, RPP21, RPP29, L7Ae). Here, we used native mass spectrometry (MS), mass photometry (MP), and biochemical experiments that (i) validate the oligomeric state of the Mja RNase P holoenzyme in vitro, (ii) find a different stoichiometry for each holoenzyme monomer with up to two copies of L7Ae, and (iii) assess whether both L7Ae copies are necessary for optimal cleavage activity. By mutating all kink-turns in the RPR, we made the discovery that abolishing the canonical L7Ae-RPR interactions was not detrimental for RNase P assembly and function due to the redundancy provided by protein-protein interactions between L7Ae and other RPPs. Our results provide new insights into the architecture and evolution of RNase P, and highlight the utility of native MS and MP in integrated structural biology approaches that seek to augment the information obtained from low/medium-resolution cryo-EM models.


Assuntos
Proteínas Arqueais , RNA Catalítico , Proteínas Arqueais/metabolismo , Methanocaldococcus/genética , RNA Catalítico/metabolismo , RNA de Transferência/metabolismo , Ribonuclease P/metabolismo , Relação Estrutura-Atividade
2.
Microbiol Spectr ; 10(1): e0209321, 2022 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-35107346

RESUMO

Methanocaldococcus sp. strain FS406-22, a hyperthermophilic methanogen, fixes nitrogen with a minimal set of known nif genes. Only four structural nif genes, nifH, nifD, nifK, and nifE, are present in a cluster, and a nifB homolog is present elsewhere in the genome. nifN, essential for the final synthesis of the iron-molybdenum cofactor of nitrogenase in well-characterized diazotrophs, is absent from FS406-22. In addition, FS406-22 encodes four novel hypothetical proteins, and a ferredoxin, in the nif cluster. Here, we develop a set of genetic tools for FS406-22 and test the functionality of genes in the nif cluster by making markerless in-frame deletion mutations. Deletion of the gene for one hypothetical protein, designated Hp4, delayed the initiation of diazotrophic growth and decreased the growth rate, an effect we confirmed by genetic complementation. NifE also appeared to play a role in diazotrophic growth, and the encoding of Hp4 and NifE in a single operon suggested they may work together in some way in the synthesis of the nitrogenase cofactor. No role could be discerned for any of the other hypothetical proteins, nor for the ferredoxin, despite the presence of these genes in a variety of related organisms. Possible pathways and evolutionary scenarios for the synthesis of the nitrogenase cofactor in an organism that lacks nifN are discussed. IMPORTANCE Methanocaldococcus has been considered a model genus, but genetic tools have not been forthcoming until recently. Here, we develop and illustrate the utility of positive selection with either of two selective agents (simvastatin and neomycin), negative selection, generation of markerless in-frame deletion mutations, and genetic complementation. These genetic tools should be useful for a variety of related species. We address the question of the minimal set of nif genes, which has implications for how nitrogen fixation evolved.


Assuntos
Proteínas de Bactérias/genética , Methanocaldococcus/genética , Fixação de Nitrogênio/genética , Nitrogenase/genética , Genes Bacterianos/genética , Methanocaldococcus/enzimologia , Methanocaldococcus/metabolismo , Nitrogenase/metabolismo , Óperon , Regiões Promotoras Genéticas , Deleção de Sequência
3.
Nat Commun ; 13(1): 710, 2022 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-35132062

RESUMO

Archaea use a molecular machine, called the archaellum, to swim. The archaellum consists of an ATP-powered intracellular motor that drives the rotation of an extracellular filament composed of multiple copies of proteins named archaellins. In many species, several archaellin homologs are encoded in the same operon; however, previous structural studies indicated that archaellum filaments mainly consist of only one protein species. Here, we use electron cryo-microscopy to elucidate the structure of the archaellum from Methanocaldococcus villosus at 3.08 Å resolution. The filament is composed of two alternating archaellins, suggesting that the architecture and assembly of archaella is more complex than previously thought. Moreover, we identify structural elements that may contribute to the filament's flexibility.


Assuntos
Flagelos/química , Methanocaldococcus/química , Proteínas Arqueais/química , Sítios de Ligação , Microscopia Crioeletrônica , Flagelos/fisiologia , Flagelina/química , Glicosilação , Metais/química , Methanocaldococcus/fisiologia , Modelos Moleculares , Multimerização Proteica , Subunidades Proteicas
4.
Chembiochem ; 23(1): e202100437, 2022 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-34606675

RESUMO

Methylation and demethylation of DNA, RNA and proteins constitutes a major regulatory mechanism in epigenetic processes. Investigations would benefit from the ability to install photo-cleavable groups at methyltransferase target sites that block interactions with reader proteins until removed by non-damaging light in the visible spectrum. Engineered methionine adenosyltransferases (MATs) have been exploited in cascade reactions with methyltransferases (MTases) to modify biomolecules with non-natural groups, including first evidence for accepting photo-cleavable groups. We show that an engineered MAT from Methanocaldococcus jannaschii (PC-MjMAT) is 308-fold more efficient at converting ortho-nitrobenzyl-(ONB)-homocysteine than the wildtype enzyme. PC-MjMAT is active over a broad range of temperatures and compatible with MTases from mesophilic organisms. We solved the crystal structures of wildtype and PC-MjMAT in complex with AdoONB and a red-shifted derivative thereof. These structures reveal that aromatic stacking interactions within the ligands are key to accommodating the photocaging groups in PC-MjMAT. The enlargement of the binding pocket eliminates steric clashes to enable AdoMet analogue binding. Importantly, PC-MjMAT exhibits remarkable activity on methionine analogues with red-shifted ONB-derivatives enabling photo-deprotection of modified DNA by visible light.


Assuntos
DNA/química , Luz , Metionina Adenosiltransferase/química , RNA/química , DNA/genética , DNA/metabolismo , Methanocaldococcus/enzimologia , Metionina Adenosiltransferase/genética , Metionina Adenosiltransferase/metabolismo , Estrutura Molecular , Processos Fotoquímicos , Engenharia de Proteínas , RNA/genética , RNA/metabolismo
5.
Nat Commun ; 12(1): 6982, 2021 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-34848707

RESUMO

Phospholipids are the major components of the membrane in all type of cells and organelles. They also are critical for cell metabolism, signal transduction, the immune system and other critical cell functions. The biosynthesis of phospholipids is a complex multi-step process with high-energy intermediates. Several enzymes in different metabolic pathways are involved in the initial phospholipid synthesis and its subsequent conversion. While the "Kennedy pathway" is the main pathway in mammalian cells, in bacteria and lower eukaryotes the precursor CDP-DAG is used in the de novo pathway by CDP-DAG alcohol O-phosphatidyl transferases to synthetize the basic lipids. Here we present the high-resolution structures of phosphatidyl serine synthase from Methanocaldococcus jannaschii crystallized in four different states. Detailed structural and functional analysis of the different structures allowed us to identify the substrate binding site and show how CDP-DAG, serine and two essential metal ions are bound and oriented relative to each other. In close proximity to the substrate binding site, two anions were identified that appear to be highly important for the reaction. The structural findings were confirmed by functional activity assays and suggest a model for the catalytic mechanism of CDP-DAG alcohol O-phosphatidyl transferases, which synthetize the phospholipids essential for the cells.


Assuntos
CDPdiacilglicerol-Serina O-Fosfatidiltransferase/química , CDPdiacilglicerol-Serina O-Fosfatidiltransferase/metabolismo , Methanocaldococcus/enzimologia , Sítios de Ligação , CDPdiacilglicerol-Serina O-Fosfatidiltransferase/genética , Cristalografia por Raios X , Cistina Difosfato , Escherichia coli , Lipídeos de Membrana/química , Fosfatidilserinas , Fosfolipídeos , Fosfotransferases , Transferases
6.
Chem Commun (Camb) ; 57(45): 5511-5513, 2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-33988635

RESUMO

We report a filamentous chaperone-based protein hydrogel capable of stabilizing enzymes against thermal inactivation. The hydrogel backbone consists of a thermostable chaperone protein, the gamma-prefoldin (γPFD) from Methanocaldococcus jannaschii, which self-assembles into a fibrous structure. Specific coiled-coil interactions engineered into the wildtype γPFD trigger the formation of a cross-linked network of protein filaments. The structure of the filamentous chaperone is preserved through the designed coiled-coil interactions. The resulting hydrogel enables entrapped enzymes to retain greater activity after exposure to high temperatures, presumably by virtue of the inherent chaperone activity of the γPFD.


Assuntos
Hidrogéis/química , Methanocaldococcus/química , Chaperonas Moleculares/química , Reagentes de Ligações Cruzadas/química , Temperatura Alta , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Estabilidade Proteica
7.
Biophys J ; 120(11): 2276-2286, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33812848

RESUMO

MJ0366 from Methanocaldococcus jannaschii is the smallest topologically knotted protein known to date. 92 residues in length, MJ0366 ties a trefoil (31) knot by threading its C-terminal helix through a buttonhole formed by the remainder of the secondary structure elements. By generating a library of point mutations at positions pertinent to the knot formation, we systematically evaluated the contributions of individual residues to the folding stability and kinetics of MJ0366. The experimental Φ-values were used as restraints to computationally generate an ensemble of conformations that correspond to the transition state of MJ0366, which revealed several nonnative contacts. The importance of these nonnative contacts in stabilizing the transition state of MJ0366 was confirmed by a second round of mutagenesis, which also established the pivotal role of F15 in stapling the network of hydrophobic interactions around the threading C-terminal helix. Our converging experimental and computational results show that, despite the small size, the transition state of MJ0366 is formed at a very late stage of the folding reaction coordinate, following a polarized pathway. Eventually, the formation of extensive native contacts, as well as a number of nonnative ones, leads to the threading of the C-terminal helix that defines the topological knot.


Assuntos
Dobramento de Proteína , Proteínas , Cinética , Methanocaldococcus , Conformação Proteica , Proteínas/genética , Termodinâmica
8.
J Struct Biol ; 213(2): 107735, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33831508

RESUMO

Protein structures are directly linked to biological functions. However, there is a gap of knowledge between the decoded genome and the structure. To bridge the gap, we focused on the secondary structure (SS). From a comprehensive analysis of predicted SS of proteins in different types of organisms, we have arrived at the following findings: The proportions of SS in genomes were different among phylogenic domains. The distributions of strand lengths indicated structural limitations in all of the species. Different from bacteria and archaea, eukaryotes have an abundance of α-helical and random coil proteins. Interestingly, there was a relationship between SS and post-translational modifications. By calculating hydrophobicity moments of helices and strands, highly amphipathic fragments of SS were found, which might be related to the biological functions. In conclusion, comprehensive predictions of SS will provide valuable perspectives to understand the entire protein structures in genomes and will help one to discover or design functional proteins.


Assuntos
Estrutura Secundária de Proteína , Proteínas/química , Proteínas/metabolismo , Aminoácidos/química , Proteínas Arqueais/química , Proteínas Arqueais/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Bases de Dados de Proteínas , Interações Hidrofóbicas e Hidrofílicas , Proteínas Intrinsicamente Desordenadas/química , Methanocaldococcus/genética , Sinais de Exportação Nuclear , Sinais de Localização Nuclear , Fosforilação , Conformação Proteica em alfa-Hélice , Processamento de Proteína Pós-Traducional , Proteínas/classificação , Proteínas/genética , Pyrobaculum/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Especificidade da Espécie
9.
Nat Chem Biol ; 17(5): 585-592, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33707784

RESUMO

YcaO enzymes catalyze several post-translational modifications on peptide substrates, including thioamidation, which substitutes an amide oxygen with sulfur. Most predicted thioamide-forming YcaO enzymes are encoded adjacent to TfuA, which when present, is required for thioamidation. While activation of the peptide amide backbone is well established for YcaO enzymes, the function of TfuA has remained enigmatic. Here we characterize the TfuA protein involved in methyl-coenzyme M reductase thioamidation and demonstrate that TfuA catalyzes the hydrolysis of thiocarboxylated ThiS (ThiS-COSH), a proteinaceous sulfur donor, and enhances the affinity of YcaO toward the thioamidation substrate. We also report a crystal structure of a TfuA, which displays a new protein fold. Our structural and mutational analyses of TfuA have uncovered conserved binding interfaces with YcaO and ThiS in addition to revealing a hydrolase-like active site featuring a Ser-Lys catalytic pair.


Assuntos
Proteínas Arqueais/química , Euryarchaeota/enzimologia , Methanobacteriaceae/enzimologia , Methanocaldococcus/enzimologia , Oxirredutases/química , Tioamidas/química , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Euryarchaeota/genética , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Histidina/química , Histidina/genética , Histidina/metabolismo , Cinética , Lectina de Ligação a Manose/química , Lectina de Ligação a Manose/genética , Lectina de Ligação a Manose/metabolismo , Methanobacteriaceae/genética , Methanocaldococcus/genética , Modelos Moleculares , Mutação , Oligopeptídeos/química , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por Substrato , Tioamidas/metabolismo
10.
Proc Natl Acad Sci U S A ; 118(11)2021 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-33688044

RESUMO

Sequence-specific protein ligations are widely used to produce customized proteins "on demand." Such chimeric, immobilized, fluorophore-conjugated or segmentally labeled proteins are generated using a range of chemical, (split) intein, split domain, or enzymatic methods. Where short ligation motifs and good chemoselectivity are required, ligase enzymes are often chosen, although they have a number of disadvantages, for example poor catalytic efficiency, low substrate specificity, and side reactions. Here, we describe a sequence-specific protein ligase with more favorable characteristics. This ligase, Connectase, is a monomeric homolog of 20S proteasome subunits in methanogenic archaea. In pulldown experiments with Methanosarcina mazei cell extract, we identify a physiological substrate in methyltransferase A (MtrA), a key enzyme of archaeal methanogenesis. Using microscale thermophoresis and X-ray crystallography, we show that only a short sequence of about 20 residues derived from MtrA and containing a highly conserved KDPGA motif is required for this high-affinity interaction. Finally, in quantitative activity assays, we demonstrate that this recognition tag can be repurposed to allow the ligation of two unrelated proteins. Connectase catalyzes such ligations at substantially higher rates, with higher yields, but without detectable side reactions when compared with a reference enzyme. It thus presents an attractive tool for the development of new methods, for example in the preparation of selectively labeled proteins for NMR, the covalent and geometrically defined attachment of proteins on surfaces for cryo-electron microscopy, or the generation of multispecific antibodies.


Assuntos
Proteínas Arqueais/metabolismo , Ligases/metabolismo , Methanocaldococcus/enzimologia , Methanosarcina/enzimologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Cristalografia por Raios X , Complexo de Endopeptidases do Proteassoma/química , Conformação Proteica , Especificidade por Substrato
11.
Commun Biol ; 4(1): 289, 2021 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-33674723

RESUMO

Bioprocesses converting carbon dioxide with molecular hydrogen to methane (CH4) are currently being developed to enable a transition to a renewable energy production system. In this study, we present a comprehensive physiological and biotechnological examination of 80 methanogenic archaea (methanogens) quantifying growth and CH4 production kinetics at hyperbaric pressures up to 50 bar with regard to media, macro-, and micro-nutrient supply, specific genomic features, and cell envelope architecture. Our analysis aimed to systematically prioritize high-pressure and high-performance methanogens. We found that the hyperthermophilic methanococci Methanotorris igneus and Methanocaldococcoccus jannaschii are high-pressure CH4 cell factories. Furthermore, our analysis revealed that high-performance methanogens are covered with an S-layer, and that they harbour the amino acid motif Tyrα444 Glyα445 Tyrα446 in the alpha subunit of the methyl-coenzyme M reductase. Thus, high-pressure biological CH4 production in pure culture could provide a purposeful route for the transition to a carbon-neutral bioenergy sector.


Assuntos
Microbiologia Industrial , Metano/metabolismo , Methanocaldococcaceae/metabolismo , Methanocaldococcus/metabolismo , Motivos de Aminoácidos , Ensaios de Triagem em Larga Escala , Cinética , Glicoproteínas de Membrana/metabolismo , Methanocaldococcaceae/crescimento & desenvolvimento , Methanocaldococcus/crescimento & desenvolvimento , Oxirredutases/metabolismo , Pressão , Energia Renovável
12.
Nucleic Acids Res ; 49(2): 1075-1093, 2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33398350

RESUMO

Defects in the posttranscriptional modifications of mitochondrial tRNAs have been linked to human diseases, but their pathophysiology remains elusive. In this report, we investigated the molecular mechanism underlying a deafness-associated tRNAIle 4295A>G mutation affecting a highly conserved adenosine at position 37, 3' adjacent to the tRNA's anticodon. Primer extension and methylation activity assays revealed that the m.4295A>G mutation introduced a tRNA methyltransferase 5 (TRMT5)-catalyzed m1G37 modification of tRNAIle. Molecular dynamics simulations suggested that the m.4295A>G mutation affected tRNAIle structure and function, supported by increased melting temperature, conformational changes and instability of mutated tRNA. An in vitro processing experiment revealed that the m.4295A>G mutation reduced the 5' end processing efficiency of tRNAIle precursors, catalyzed by RNase P. We demonstrated that cybrid cell lines carrying the m.4295A>G mutation exhibited significant alterations in aminoacylation and steady-state levels of tRNAIle. The aberrant tRNA metabolism resulted in the impairment of mitochondrial translation, respiratory deficiency, decreasing membrane potentials and ATP production, increasing production of reactive oxygen species and promoting autophagy. These demonstrated the pleiotropic effects of m.4295A>G mutation on tRNAIle and mitochondrial functions. Our findings highlighted the essential role of deficient posttranscriptional modifications in the structure and function of tRNA and their pathogenic consequence of deafness.


Assuntos
Perda Auditiva Neurossensorial/genética , Mutação Puntual , RNA de Transferência de Isoleucina/genética , Trifosfato de Adenosina/biossíntese , Adulto , Proteínas Arqueais/metabolismo , Autofagia , Sequência de Bases , Linhagem Celular , DNA Mitocondrial/genética , Feminino , Pleiotropia Genética , Perda Auditiva Neurossensorial/etnologia , Humanos , Isoleucina/metabolismo , Masculino , Herança Materna , Potencial da Membrana Mitocondrial , Methanocaldococcus/enzimologia , Metilação , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Fosforilação Oxidativa , Linhagem , Biossíntese de Proteínas , Processamento Pós-Transcricional do RNA , Proteínas Recombinantes/metabolismo , Aminoacilação de RNA de Transferência , Adulto Jovem , tRNA Metiltransferases/metabolismo
13.
Environ Microbiol ; 23(7): 3614-3626, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33022088

RESUMO

Deep-sea hydrothermal vents are inhabited by complex communities of microbes and their viruses. Despite the importance of viruses in controlling the diversity, adaptation and evolution of their microbial hosts, to date, only eight bacterial and two archaeal viruses isolated from abyssal ecosystems have been described. Thus, our efforts focused on gaining new insights into viruses associated with deep-sea autotrophic archaea. Here, we provide the first evidence of an infection of hyperthermophilic methanogenic archaea by a head-tailed virus, Methanocaldococcus fervens tailed virus 1 (MFTV1). MFTV1 has an isometric head of 50 nm in diameter and a 150 nm-long non-contractile tail. Virions are released continuously without causing a sudden drop in host growth. MFTV1 infects Methanocaldococcus species and is the first hyperthermophilic head-tailed virus described thus far. The viral genome is a double-stranded linear DNA of 31 kb. Interestingly, our results suggest potential strategies adopted by the plasmid pMEFER01, carried by M. fervens, to spread horizontally in hyperthermophilic methanogens. The data presented here open a new window of understanding on how the abyssal mobilome interacts with hyperthermophilic marine archaea.


Assuntos
Vírus de Archaea , Vírus , Archaea/genética , Vírus de Archaea/genética , Ecossistema , Methanocaldococcus
14.
Chembiochem ; 22(8): 1379-1384, 2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33350556

RESUMO

Site-specific incorporation of unnatural amino acids (uAAs) bearing a bioorthogonal group has enabled the attachment - typically at a single site or at a few sites per protein - of chemical groups at precise locations for protein and biomaterial labeling, conjugation, and functionalization. Herein, we report the evolution of chromosomal Methanocaldococcus jannaschii tyrosyl-tRNA synthetase (aaRS) for the alkyne-bearing uAA, 4-propargyloxy-l-phenylalanine (pPR), with ∼30-fold increased production of green fluorescent protein containing three instances of pPR compared with a previously described M. jannaschii-derived aaRS for pPR, when expressed from a single chromosomal copy. We show that when expressed from multicopy plasmids, the evolved aaRSs enable the production - using a genomically recoded Escherichia coli and the non-recoded BL21 E. coli strain - of elastin-like polypeptides (ELPs) containing multiple pPR residues in high yields. We further show that the multisite incorporation of pPR in ELPs facilitates the rapid, robust, and nontoxic fluorescent labeling of these proteins in bacteria. The evolved variants described in this work can be used to produce a variety of protein and biomaterial conjugates and to create efficient minimal tags for protein labeling.


Assuntos
Aminoácidos/metabolismo , Aminoacil-tRNA Sintetases/metabolismo , Materiais Biocompatíveis/metabolismo , Methanocaldococcus/metabolismo , Aminoácidos/química , Aminoacil-tRNA Sintetases/química , Materiais Biocompatíveis/química , Methanocaldococcus/enzimologia , Estrutura Molecular , Fenilalanina/química , Fenilalanina/metabolismo
15.
Nat Commun ; 11(1): 6233, 2020 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-33277478

RESUMO

The KEOPS complex, which is conserved across archaea and eukaryotes, is composed of four core subunits; Pcc1, Kae1, Bud32 and Cgi121. KEOPS is crucial for the fitness of all organisms examined. In humans, pathogenic mutations in KEOPS genes lead to Galloway-Mowat syndrome, an autosomal-recessive disease causing childhood lethality. Kae1 catalyzes the universal and essential tRNA modification N6-threonylcarbamoyl adenosine, but the precise roles of all other KEOPS subunits remain an enigma. Here we show using structure-guided studies that Cgi121 recruits tRNA to KEOPS by binding to its 3' CCA tail. A composite model of KEOPS bound to tRNA reveals that all KEOPS subunits form an extended tRNA-binding surface that we have validated in vitro and in vivo to mediate the interaction with the tRNA substrate and its modification. These findings provide a framework for understanding the inner workings of KEOPS and delineate why all KEOPS subunits are essential.


Assuntos
Proteínas Arqueais/química , Methanocaldococcus/metabolismo , Complexos Multiproteicos/química , RNA de Transferência/química , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Cristalografia por Raios X , Methanocaldococcus/genética , Modelos Moleculares , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Conformação de Ácido Nucleico , Ligação Proteica , Domínios Proteicos , RNA de Transferência/genética , RNA de Transferência/metabolismo , RNA de Transferência de Lisina/química , RNA de Transferência de Lisina/genética , RNA de Transferência de Lisina/metabolismo
16.
Cell Rep ; 33(3): 108294, 2020 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-33086053

RESUMO

The UbiA superfamily of intramembrane prenyltransferases catalyzes an isoprenyl transfer reaction in the biosynthesis of lipophilic compounds involved in cellular physiological processes. Digeranylgeranylglyceryl phosphate (DGGGP) synthase (DGGGPase) generates unique membrane core lipids for the formation of the ether bond between the glycerol moiety and the alkyl chains in archaea and has been confirmed to be a member of the UbiA superfamily. Here, the crystal structure is reported to exhibit nine transmembrane helices along with a large lateral opening covered by a cytosolic cap domain and a unique substrate-binding central cavity. Notably, the lipid-bound states of this enzyme demonstrate that the putative substrate-binding pocket is occupied by the lipidic molecules used for crystallization, indicating the binding mode of hydrophobic substrates. Collectively, these structural and functional studies provide not only an understanding of lipid biosynthesis by substrate-specific lipid-modifying enzymes but also insights into the mechanisms of lipid membrane remodeling and adaptation.


Assuntos
Proteínas Arqueais/metabolismo , Glicerofosfatos/biossíntese , Methanocaldococcus/enzimologia , Archaea/enzimologia , Proteínas Arqueais/biossíntese , Proteínas Arqueais/genética , Glicerofosfatos/metabolismo , Lipídeos de Membrana , Methanocaldococcus/metabolismo , Estrutura Secundária de Proteína
17.
J Mol Biol ; 432(24): 166692, 2020 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-33122004

RESUMO

PLP-dependent enzymes catalyze a plethora of chemical reactions affecting diverse physiological functions. Here we report the structural determinants of the reaction mechanism in a Group II PLP-dependent decarboxylase by assigning two early intermediates. The in-crystallo complexes of the PLP bound form, and the Dunathan and quinonoid intermediates, allowed direct observation of the active site interactions. The structures reveal that a subtle rearrangement of a conserved Arg residue in concert with a water-mediated interaction with the carboxylate of the Dunathan intermediate, appears to directly stabilize the alignment and facilitate the release of CO2 to yield the quinonoid. Modeling indicates that the conformational change of a dynamic catalytic loop to a closed form controls a conserved network of hydrogen bond interactions between catalytic residues to protonate the quinonoid. Our results provide a structural framework to elucidate mechanistic roles of residues that govern reaction specificity and catalysis in PLP-dependent decarboxylation.


Assuntos
Catálise , Conformação Proteica , Fosfato de Piridoxal/análogos & derivados , Tirosina Descarboxilase/ultraestrutura , Aspartato Aminotransferases/química , Aspartato Aminotransferases/genética , Dióxido de Carbono/metabolismo , Domínio Catalítico/genética , Cristalografia por Raios X , Ligação de Hidrogênio , Methanocaldococcus/enzimologia , Fosfato de Piridoxal/química , Fosfato de Piridoxal/genética , Tirosina Descarboxilase/química , Tirosina Descarboxilase/genética , Água/química
18.
Nat Chem Biol ; 16(12): 1434-1439, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32929278

RESUMO

Compared with green fluorescent protein-based biosensors, red fluorescent protein (RFP)-based biosensors are inherently advantageous because of reduced phototoxicity, decreased autofluorescence and enhanced tissue penetration. However, existing RFP-based biosensors often suffer from small dynamic ranges, mislocalization and undesired photoconversion. In addition, the choice of available RFP-based biosensors is limited, and development of each biosensor requires substantial effort. Herein, we describe a general and convenient method, which introduces a genetically encoded noncanonical amino acid, 3-aminotyrosine, to the chromophores of green fluorescent protein-like proteins and biosensors for spontaneous and efficient green-to-red conversion. We demonstrated that this method could be used to quickly expand the repertoire of RFP-based biosensors. With little optimization, the 3-aminotyrosine-modified biosensors preserved the molecular brightness, dynamic range and responsiveness of their green fluorescent predecessors. We further applied spectrally resolved biosensors for multiplexed imaging of metabolic dynamics in pancreatic ß-cells.


Assuntos
Técnicas Biossensoriais , Proteínas de Fluorescência Verde/análise , Proteínas Luminescentes/análise , Imagem Óptica/métodos , Engenharia de Proteínas/métodos , Tirosina/análogos & derivados , Animais , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Linhagem Celular , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Transferência Ressonante de Energia de Fluorescência , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Glucose/farmacologia , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Humanos , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/genética , Methanocaldococcus/química , Methanocaldococcus/enzimologia , Camundongos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tirosina/genética , Tirosina/metabolismo , Tirosina-tRNA Ligase/genética , Tirosina-tRNA Ligase/metabolismo
19.
Mol Cell Proteomics ; 19(12): 1997-2015, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32883800

RESUMO

AAA+ ATPases constitute a large family of proteins that are involved in a plethora of cellular processes including DNA disassembly, protein degradation and protein complex disassembly. They typically form a hexametric ring-shaped structure with six subunits in a (pseudo) 6-fold symmetry. In a subset of AAA+ ATPases that facilitate protein unfolding and degradation, six subunits cooperate to translocate protein substrates through a central pore in the ring. The number and type of nucleotides in an AAA+ ATPase hexamer is inherently linked to the mechanism that underlies cooperation among subunits and couples ATP hydrolysis with substrate translocation. We conducted a native MS study of a monodispersed form of PAN, an archaeal proteasome AAA+ ATPase, to determine the number of nucleotides bound to each hexamer of the WT protein. We utilized ADP and its analogs (TNP-ADP and mant-ADP), and a nonhydrolyzable ATP analog (AMP-PNP) to study nucleotide site occupancy within the PAN hexamer in ADP- and ATP-binding states, respectively. Throughout all experiments we used a Walker A mutant (PANK217A) that is impaired in nucleotide binding as an internal standard to mitigate the effects of residual solvation on mass measurement accuracy and to serve as a reference protein to control for nonspecific nucleotide binding. This approach led to the unambiguous finding that a WT PAN hexamer carried - from expression host - six tightly bound ADP molecules that could be exchanged for ADP and ATP analogs. Although the Walker A mutant did not bind ADP analogs, it did bind AMP-PNP, albeit at multiple stoichiometries. We observed variable levels of hexamer dissociation and an appearance of multimeric species with the over-charged molecular ion distributions across repeated experiments. We posit that these phenomena originated during ESI process at the final stages of ESI droplet evolution.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Espectrometria de Massas , Nucleotídeos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Multimerização Proteica , Difosfato de Adenosina/metabolismo , Adenilil Imidodifosfato/metabolismo , Proteínas Arqueais/metabolismo , Ligantes , Methanocaldococcus , Proteínas Mutantes/metabolismo , Ligação Proteica , Subunidades Proteicas/metabolismo , Espectrometria de Massas por Ionização por Electrospray
20.
J Struct Biol ; 211(3): 107559, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32653645

RESUMO

Complexes of archaeal ribosomal proteins uL11 and uL10/P0 (the two-domain N-terminal fragment of uL10, uL10NTF/P0NTF) with the adjacent 74 nucleotides of 23S rRNA fragment (23SrRNA(74)) from Methanococcus jannaschii (Mja) were obtained, crystallized and their structures were studied. The comparative structural analysis of the complexes of Mja uL10NTF•23SrRNA(74) and Mja uL10NTF•uL11•23SrRNA(74) shows that the insertion of uL11 in the binary complex does not change the conformation of the 23S rRNA fragment. On the other hand, the interaction with this specific RNA fragment leads to the restructuring of uL11 compared to the structure of this protein in the free state. Besides, although analysis confirmed the mobility of uL10/P0 domain II, disproved the assumption that it may be in contact with rRNA or uL11. In addition, the Mja uL10NTF•uL11•23SrRNA(74) complex was cocrystallized with the antibiotic thiostrepton, and the structure of this complex was solved. The thiostrepton binding site in this archaeal complex was found between the 23S rRNA and the N-terminal domain (NTD) of the Mja uL11 protein, similar to its binding site in the one of bacterial ribosome complex with thiostrepton. Upon binding of thiostrepton, the NTD of uL11 shifts toward rRNA by 7 Å. Such a shift may be the cause of the inhibitory effect of the antibiotic on the recruitment of translation factors to the GTPase-activating region in archaeal ribosomes, similar to its inhibitory effect on protein synthesis in bacterial ribosomes.


Assuntos
Proteínas Arqueais/química , Methanocaldococcus/química , Proteínas Ribossômicas/química , Ribossomos/química , Proteínas Arqueais/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Domínios Proteicos , RNA Ribossômico 23S/química , RNA Ribossômico 23S/metabolismo , Proteínas Ribossômicas/metabolismo , Tioestreptona/metabolismo
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