ArcS from Thermococcus kodakarensis transfers L-lysine to preQ0 nucleoside derivatives as minimum substrate RNAs.

Archaeosine (G+) is an archaea-specific tRNA modification synthesized via multiple steps. In the first step, archaeosine tRNA guanine transglucosylase (ArcTGT) exchanges the G15 base in tRNA with 7-cyano-7-deazaguanine (preQ0). In Euryarchaea, preQ015 in tRNA is further modified by archaeosine synthase (ArcS). Thermococcus kodakarensis ArcS catalyzes a lysine-transfer reaction to produce preQ0-lysine (preQ0-Lys) as an intermediate. The resulting preQ0-Lys15 in tRNA is converted to G+15 by a radical S-adenosyl-L-methionine enzyme for archaeosine formation (RaSEA), which forms a complex with ArcS. Here, we focus on the substrate tRNA recognition mechanism of ArcS. Kinetic parameters of ArcS for lysine and tRNA-preQ0 were determined using a purified enzyme. RNA fragments containing preQ0 were prepared from Saccharomyces cerevisiae tRNAPhe-preQ015. ArcS transferred 14C-labeled lysine to RNA fragments. Furthermore, ArcS transferred lysine to preQ0 nucleoside and preQ0 nucleoside 5'-monophosphate. Thus, the L-shaped structure and the sequence of tRNA are not essential for the lysine-transfer reaction by ArcS. However, the presence of D-arm structure accelerates the lysine-transfer reaction. Because ArcTGT from thermophilic archaea recognizes the common D-arm structure, we expected the combination of T. kodakarensis ArcTGT and ArcS and RaSEA complex would result in the formation of preQ0-Lys15 in all tRNAs. This hypothesis was confirmed using 46 T. kodakarensis tRNA transcripts and three Haloferax volcanii tRNA transcripts. In addition, ArcTGT did not exchange the preQ0-Lys15 in tRNA with guanine or preQ0 base, showing that formation of tRNA-preQ0-Lys by ArcS plays a role in preventing the reverse reaction in G+ biosynthesis.

Structural and functional insights into Archaeoglobus fulgidus m2G10 tRNA methyltransferase Trm11 and its Trm112 activator.

tRNAs play a central role during the translation process and are heavily post-transcriptionally modified to ensure optimal and faithful mRNA decoding. These epitranscriptomics marks are added by largely conserved proteins and defects in the function of some of these enzymes are responsible for neurodevelopmental disorders and cancers. Here, we focus on the Trm11 enzyme, which forms N2-methylguanosine (m2G) at position 10 of several tRNAs in both archaea and eukaryotes. While eukaryotic Trm11 enzyme is only active as a complex with Trm112, an allosteric activator of methyltransferases modifying factors (RNAs and proteins) involved in mRNA translation, former studies have shown that some archaeal Trm11 proteins are active on their own. As these studies were performed on Trm11 enzymes originating from archaeal organisms lacking TRM112 gene, we have characterized Trm11 (AfTrm11) from the Archaeoglobus fulgidus archaeon, which genome encodes for a Trm112 protein (AfTrm112). We show that AfTrm11 interacts directly with AfTrm112 similarly to eukaryotic enzymes and that although AfTrm11 is active as a single protein, its enzymatic activity is strongly enhanced by AfTrm112. We finally describe the first crystal structures of the AfTrm11-Trm112 complex and of Trm11, alone or bound to the methyltransferase inhibitor sinefungin.

Archaeal fibrillarin-Nop5 heterodimer 2'-O-methylates RNA independently of the C/D guide RNP particle.

Archaeal fibrillarin (aFib) is a well-characterized S-adenosyl methionine (SAM)-dependent RNA 2'-O-methyltransferase that is known to act in a large C/D ribonucleoprotein (RNP) complex together with Nop5 and L7Ae proteins and a box C/D guide RNA. In the reaction, the guide RNA serves to direct the methylation reaction to a specific site in tRNA or rRNA by sequence complementarity. Here we show that a Pyrococcus abyssi aFib-Nop5 heterodimer can alone perform SAM-dependent 2'-O-methylation of 16S and 23S ribosomal RNAs in vitro independently of L7Ae and C/D guide RNAs. Using tritium-labeling, mass spectrometry, and reverse transcription analysis, we identified three in vitro 2'-O-methylated positions in the 16S rRNA of P. abyssi, positions lying outside of previously reported pyrococcal C/D RNP methylation sites. This newly discovered stand-alone activity of aFib-Nop5 may provide an example of an ancestral activity retained in enzymes that were recruited to larger complexes during evolution.

RNA-Dependent Cysteine Biosynthesis in Bacteria and Archaea.

The diversity of the genetic code systems used by microbes on earth is yet to be elucidated. It is known that certain methanogenic archaea employ an alternative system for cysteine (Cys) biosynthesis and encoding; tRNACys is first acylated with phosphoserine (Sep) by O-phosphoseryl-tRNA synthetase (SepRS) and then converted to Cys-tRNACys by Sep-tRNA:Cys-tRNA synthase (SepCysS). In this study, we searched all genomic and metagenomic protein sequence data in the Integrated Microbial Genomes (IMG) system and at the NCBI to reveal new clades of SepRS and SepCysS proteins belonging to diverse archaea in the four major groups (DPANN, Euryarchaeota, TACK, and Asgard) and two groups of bacteria ("Candidatus Parcubacteria" and Chloroflexi). Bacterial SepRS and SepCysS charged bacterial tRNACys species with cysteine in vitro Homologs of SepCysE, a scaffold protein facilitating SepRS⋅SepCysS complex assembly in Euryarchaeota class I methanogens, are found in a few groups of TACK and Asgard archaea, whereas the C-terminally truncated homologs exist fused or genetically coupled with diverse SepCysS species. Investigation of the selenocysteine (Sec)- and pyrrolysine (Pyl)-utilizing traits in SepRS-utilizing archaea and bacteria revealed that the archaea carrying full-length SepCysE employ Sec and that SepRS is often found in Pyl-utilizing archaea and Chloroflexi bacteria. We discuss possible contributions of the SepRS-SepCysS system for sulfur assimilation, methanogenesis, and other metabolic processes requiring large amounts of iron-sulfur enzymes or Pyl-containing enzymes.IMPORTANCE Comprehensive analyses of all genomic and metagenomic protein sequence data in public databases revealed the distribution and evolution of an alternative cysteine-encoding system in diverse archaea and bacteria. The finding that the SepRS-SepCysS-SepCysE- and the selenocysteine-encoding systems are shared by the Euryarchaeota class I methanogens, the Crenarchaeota AK8/W8A-19 group, and an Asgard archaeon suggests that ancient archaea may have used both systems. In contrast, bacteria may have obtained the SepRS-SepCysS system from archaea. The SepRS-SepCysS system sometimes coexists with a pyrrolysine-encoding system in both archaea and bacteria. Our results provide additional bioinformatic evidence for the contribution of the SepRS-SepCysS system for sulfur assimilation and diverse metabolisms which require vast amounts of iron-sulfur enzymes and proteins. Among these biological activities, methanogenesis, methylamine metabolism, and organohalide respiration may have local and global effects on earth. Taken together, uncultured bacteria and archaea provide an expanded record of the evolution of the genetic code.

Crystal structure of the archaeosine synthase QueF-like-Insights into amidino transfer and tRNA recognition by the tunnel fold.

The tunneling-fold (T-fold) structural superfamily has emerged as a versatile protein scaffold of diverse catalytic activities. This is especially evident in the pathways to the 7-deazaguanosine modified nucleosides of tRNA queuosine and archaeosine. Four members of the T-fold superfamily have been confirmed in these pathways and here we report the crystal structure of a fifth enzyme; the recently discovered amidinotransferase QueF-Like (QueF-L), responsible for the final step in the biosynthesis of archaeosine in the D-loop of tRNA in a subset of Crenarchaeota. QueF-L catalyzes the conversion of the nitrile group of the 7-cyano-7-deazaguanine (preQ0 ) base of preQ0 -modified tRNA to a formamidino group. The structure, determined in the presence of preQ0 , reveals a symmetric T-fold homodecamer of two head-to-head facing pentameric subunits, with 10 active sites at the inter-monomer interfaces. Bound preQ0 forms a stable covalent thioimide bond with a conserved active site cysteine similar to the intermediate previously observed in the nitrile reductase QueF. Despite distinct catalytic functions, phylogenetic distributions, and only 19% sequence identity, the two enzymes share a common preQ0 binding pocket, and likely a common mechanism of thioimide formation. However, due to tight twisting of its decamer, QueF-L lacks the NADPH binding site present in QueF. A large positively charged molecular surface and a docking model suggest simultaneous binding of multiple tRNA molecules and structure-specific recognition of the D-loop by a surface groove. The structure sheds light on the mechanism of nitrile amidation, and the evolution of diverse chemistries in a common fold. Proteins 2016; 85:103-116. © 2016 Wiley Periodicals, Inc.

Mechanistic Studies of the Radical S-Adenosyl-L-methionine Enzyme 4-Demethylwyosine Synthase Reveal the Site of Hydrogen Atom Abstraction.

TYW1 catalyzes the formation of 4-demethylwyosine via the condensation of N-methylguanosine (m¹G) with carbons 2 and 3 of pyruvate. In this study, labeled transfer ribonucleic acid (tRNA) and pyruvate were utilized to determine the site of hydrogen atom abstraction and regiochemistry of the pyruvate addition. tRNA containing a ²H-labeled m¹G methyl group was used to identify the methyl group of m¹G as the site of hydrogen atom abstraction by 5'-deoxyadenosyl radical. [2-¹³C1-3,3,3-²H3]Pyruvate was used to demonstrate retention of all the pyruvate protons, indicating that C2 of pyruvate forms the bridging carbon of the imidazoline ring and C3 the methyl.

Heptameric targeting ligands against EGFR and HER2 with high stability and avidity.

Multivalency of targeting ligands provides significantly increased binding strength towards their molecular targets. Here, we report the development of a novel heptameric targeting system, with general applications, constructed by fusing a target-binding domain with the heptamerization domain of the Archaeal RNA binding protein Sm1 through a flexible hinge peptide. The previously reported affibody molecules against EGFR and HER2, Z(EGFR) and Z(HER2), were used as target binding moieties. The fusion molecules were highly expressed in E. coli as soluble proteins and efficiently self-assembled into multimeric targeting ligands with the heptamer as the predominant form. We demonstrated that the heptameric molecules were resistant to protease-mediated digestion or heat- and SDS-induced denaturation. Surface plasmon resonance (SPR) analysis showed that both heptameric Z(EGFR) and Z(HER2) ligands have a significantly enhanced binding strength to their target receptors with a nearly 100 to 1000 fold increase relative to the monomeric ligands. Cellular binding assays showed that heptameric ligands maintained their target-binding specificities similar to the monomeric forms towards their respective receptor. The non-toxic property of each heptameric ligand was demonstrated by the cell proliferation assay. In general,, the heptamerization strategy we describe here could be applied to the facile and efficient engineering of other protein domain- or short peptide-based affinity molecules to acquire significantly improved target-binding strengths with potential applications in the targeted delivery of various imaging or therapeutic agents..

A genetic investigation of the KEOPS complex in halophilic Archaea.

KEOPS is an important cellular complex conserved in Eukarya, with some subunits conserved in Archaea and Bacteria. This complex was recently found to play an essential role in formation of the tRNA modification threonylcarbamoyladenosine (t(6)A), and was previously associated with telomere length maintenance and transcription. KEOPS subunits are conserved in Archaea, especially in the Euryarchaea, where they had been studied in vitro. Here we attempted to delete the genes encoding the four conserved subunits of the KEOPS complex in the euryarchaeote Haloferax volcanii and study their phenotypes in vivo. The fused kae1-bud32 gene was shown to be essential as was cgi121, which is dispensable in yeast. In contrast, pcc1 (encoding the putative dimerizing unit of KEOPS) was not essential in H. volcanii. Deletion of pcc1 led to pleiotropic phenotypes, including decreased growth rate, reduced levels of t(6)A modification, and elevated levels of intra-cellular glycation products.

Biogenesis of 2-agmatinylcytidine catalyzed by the dual protein and RNA kinase TiaS.

The archaeal AUA-codon specific tRNA(Ile) contains 2-agmatinylcytidine (agm(2)C or agmatidine) at the anticodon wobble position (position 34). The formation of this essential modification is catalyzed by tRNA(Ile)-agm(2)C synthetase (TiaS) using agmatine and ATP as substrates. TiaS has a previously unknown catalytic domain, which we have named the Thr18-Cyt34 kinase domain (TCKD). Biochemical analyses of Archaeoglobus fulgidus TiaS and its mutants revealed that the TCKD first hydrolyzes ATP into AMP and pyrophosphate, then phosphorylates the C2 position of C34 with the γ-phosphate. Next, the amino group of agmatine attacks this position to release the phosphate and form agm(2)C. Notably, the TCKD also autophosphorylates the Thr18 of TiaS, which may be involved in agm(2)C formation. Thus, the unique kinase domain of TiaS catalyzes dual phosphorylation of protein and RNA substrates.

Structural basis of tRNA agmatinylation essential for AUA codon decoding.

The cytidine at the first position of the anticodon (C34) in the AUA codon-specific archaeal tRNA(Ile2) is modified to 2-agmatinylcytidine (agm(2)C or agmatidine), an agmatine-conjugated cytidine derivative, which is crucial for the precise decoding of the genetic code. Agm(2)C is synthesized by tRNA(Ile)-agm(2)C synthetase (TiaS) in an ATP-dependent manner. Here we present the crystal structures of the Archaeoglobus fulgidus TiaS-tRNA(Ile2) complexed with ATP, or with AMPCPP and agmatine, revealing a previously unknown kinase module required for activating C34 by phosphorylation, and showing the molecular mechanism by which TiaS discriminates between tRNA(Ile2) and tRNA(Met). In the TiaS-tRNA(Ile2)-ATP complex, C34 is trapped within a pocket far away from the ATP-binding site. In the agmatine-containing crystals, C34 is located near the AMPCPP γ-phosphate in the kinase module, demonstrating that agmatine is essential for placing C34 in the active site. These observations also provide the structural dynamics for agm(2)C formation.

The archaeal exosome.

The archaeal exosome is a protein complex with structural similarities to the eukaryotic exosome and bacterial PNPase. Its catalytic core is formed by alternating Rrp41 and Rrp42 polypeptides, arranged in a hexameric ring. A flexible RNA binding cap composed of the evolutionarily conserved proteins Rrp4 and/or Csl4 is bound at the top of the ring and seems to be involved in recruitment of specific substrates and their unwinding. Additionally, the protein complex contains an archaea-specific subunit annotated as DnaG, the function of which is still unknown. The archaeal exosome degrades RNA phosphorolytically in 3' to 5' direction. In a reverse reaction, it synthesizes heteropolymeric RNA tails using nucleoside diphosphates. The functional similarity between the archaeal exosome and PNPase shows that important processes of RNA degradation and posttranscriptional modification in Archaea are similar to the processes in Bacteria and organelles.

The archaeal exosome.

The archaeal exosome is aprotein complex with structural similarities to the eukaryotic exosome and bacterial PNPase. Its catalytic core is formed by alternating Rrp41 and Rrp42 polypeptides, arranged in a hexameric ring. A flexible RNA binding cap composed of the evolutionarily conserved proteins Rrp4 and/or Cs14 is bound at the top of the ring and seems to be involved in recruitment of specific substrates and their unwinding. Additionally, the protein complex contains an archaea-specific subunit annotated as DnaG, the function of which is still unknown. The archaeal exosome degrades RNA phosphorolytically in 3' to 5' direction. In a reverse reaction, it synthesizes heteropolymeric RNA tails using nucleoside diphosphates. The functional similarity between the archaeal exosome and PNPase shows that important processes of RNA degradation and posttranscriptional modification in Archaea are similar to the processes in Bacteria and organelles.

Structural basis of AdoMet-dependent aminocarboxypropyl transfer reaction catalyzed by tRNA-wybutosine synthesizing enzyme, TYW2.

S-adenosylmethionine (AdoMet) is a methyl donor used by a wide variety of methyltransferases, and it is also used as the source of an alpha-amino-alpha-carboxypropyl ("acp") group by several enzymes. tRNA-yW synthesizing enzyme-2 (TYW2) is involved in the biogenesis of a hypermodified nucleotide, wybutosine (yW), and it catalyzes the transfer of the "acp" group from AdoMet to the C7 position of the imG-14 base, a yW precursor. This modified nucleoside yW is exclusively located at position 37 of eukaryotic tRNA(Phe), and it ensures the anticodon-codon pairing on the ribosomal decoding site. Although this "acp" group has a significant role in preventing decoding frame shifts, the mechanism of the "acp" group transfer by TYW2 remains unresolved. Here we report the crystal structures and functional analyses of two archaeal homologs of TYW2 from Pyrococcus horikoshii and Methanococcus jannaschii. The in vitro mass spectrometric and radioisotope-labeling analyses confirmed that these archaeal TYW2 homologues have the same activity as yeast TYW2. The crystal structures verified that the archaeal TYW2 contains a canonical class-I methyltransferase (MTase) fold. However, their AdoMet-bound structures revealed distinctive AdoMet-binding modes, in which the "acp" group, instead of the methyl group, of AdoMet is directed to the substrate binding pocket. Our findings, which were confirmed by extensive mutagenesis studies, explain why TYW2 transfers the "acp" group, and not the methyl group, from AdoMet to the nucleobase.

Analysis of a critical interaction within the archaeal box C/D small ribonucleoprotein complex.

In archaea and eukarya, box C/D ribonucleoprotein (RNP) complexes are responsible for 2'-O-methylation of tRNAs and rRNAs. The archaeal box C/D small RNP complex requires a small RNA component (sRNA) possessing Watson-Crick complementarity to the target RNA along with three proteins: L7Ae, Nop5p, and fibrillarin. Transfer of a methyl group from S-adenosylmethionine to the target RNA is performed by fibrillarin, which by itself has no affinity for the sRNA-target duplex. Instead, it is targeted to the site of methylation through association with Nop5p, which in turn binds to the L7Ae-sRNA complex. To understand how Nop5p serves as a bridge between the targeting and catalytic functions of the box C/D small RNP complex, we have employed alanine scanning to evaluate the interaction between the Pyrococcus horikoshii Nop5p domain and an L7Ae box C/D RNA complex. From these data, we were able to construct an isolated RNA-binding domain (Nop-RBD) that folds correctly as demonstrated by x-ray crystallography and binds to the L7Ae box C/D RNA complex with near wild type affinity. These data demonstrate that the Nop-RBD is an autonomously folding and functional module important for protein assembly in a number of complexes centered on the L7Ae-kinkturn RNP.

Anaerobiosis inhibits gas vesicle formation in halophilic Archaea.

The effect of anaerobiosis on the gas vesicle formation was investigated in three Halobacterium salinarum strains, Haloferax mediterranei and in Haloferax volcanii transformants. All these strains significantly reduced gas vesicle formation or lacked these structures under anoxic conditions. When grown by arginine fermentation, Hbt. salinarum PHH4 lacked gas vesicles, whereas Hbt. salinarum PHH1 and NRC-1 contained 5-20 small gas vesicles arranged in two to three aggregates per cell instead of the 30-80 gas vesicles present under oxic conditions. The enlargement presumably stopped due to a depletion of Gvp proteins. Also Hfx. mediterranei and Hfx. volcanii transformants lacked gas vesicles under anoxic growth and yielded a 10-fold reduced gvp transcription. Even the gas vesicle-overproducing DeltaD transformants did not form gas vesicles under anoxic conditions, demonstrating that the repressing protein GvpD was not involved. The presence of large amounts of GvpA implied that the assembly of the gas vesicles was inhibited. When Hbt. salinarum PHH1 and NRC-1 were grown with dimethyl sulphoxide or trimethylamine N-oxid under anoxic conditions the number but not the size of gas vesicles was reduced. This was in contrast to the previously reported overproduction of gas vesicles in NRC-1 that turned out to depend on the citrate-containing medium used for growth.

UPF201 archaeal specific family members reveal structural similarity to RNA-binding proteins but low likelihood for RNA-binding function.

We have determined X-ray crystal structures of four members of an archaeal specific family of proteins of unknown function (UPF0201; Pfam classification: DUF54) to advance our understanding of the genetic repertoire of archaea. Despite low pairwise amino acid sequence identities (10-40%) and the absence of conserved sequence motifs, the three-dimensional structures of these proteins are remarkably similar to one another. Their common polypeptide chain fold, encompassing a five-stranded antiparallel beta-sheet and five alpha-helices, proved to be quite unexpectedly similar to that of the RRM-type RNA-binding domain of the ribosomal L5 protein, which is responsible for binding the 5S- rRNA. Structure-based sequence alignments enabled construction of a phylogenetic tree relating UPF0201 family members to L5 ribosomal proteins and other structurally similar RNA binding proteins, thereby expanding our understanding of the evolutionary purview of the RRM superfamily. Analyses of the surfaces of these newly determined UPF0201 structures suggest that they probably do not function as RNA binding proteins, and that this domain specific family of proteins has acquired a novel function in archaebacteria, which awaits experimental elucidation.

A bifunctional archaeal protein that is a component of 30S ribosomal subunits and interacts with C/D box small RNAs.

We have identified a novel archaeal protein that apparently plays two distinct roles in ribosome metabolism. It is a polypeptide of about 18 kDa (termed Rbp18) that binds free cytosolic C/D box sRNAs in vivo and in vitro and behaves as a structural ribosomal protein, specifically a component of the 30S ribosomal subunit. As Rbp18 is selectively present in Crenarcheota and highly thermophilic Euryarchaeota, we propose that it serves to protect C/D box sRNAs from degradation and perhaps to stabilize thermophilic 30S subunits.

Presence of tRNA-dependent pathways correlates with high cysteine content in methanogenic Archaea.

Archeal proteomes can be clustered into two groups based on their cysteine content. One group of proteomes displays a low cysteine content ( approximately 0.7% of the entire proteome), whereas the second group contains twice as many cysteines as the first ( approximately 1.3%). All cysteine-rich organisms belong to the methanogenic Archaea, which generates special cysteine clusters associated with primitive metabolic reactions. Our findings suggest that cysteine plays an important role in early forms of life.

Crystal structure and mutational study of a unique SpoU family archaeal methylase that forms 2'-O-methylcytidine at position 56 of tRNA.

The conserved cytidine residue at position 56 of tRNA contributes to the maintenance of the L-shaped tertiary structure. aTrm56 catalyzes the 2'-O-methylation of the cytidine residue in archaeal tRNA, using S-adenosyl-L-methionine. Based on the amino acid sequence, aTrm56 is the most distant member of the SpoU family. Here, we determined the crystal structure of Pyrococcus horikoshii aTrm56 complexed with S-adenosyl-L-methionine at 2.48 A resolution. aTrm56 consists of the SPOUT domain, which contains the characteristic deep trefoil knot, and a unique C-terminal beta-hairpin. aTrm56 forms a dimer. The S-adenosyl-L-methionine binding and dimerization of aTrm56 were similar to those of the other SpoU members. A structure-based sequence alignment revealed that aTrm56 conserves only motif II, among the four signature motifs. However, an essential Arg16 residue is located at a novel position within motif I. Biochemical assays showed that aTrm56 prefers the L-shaped tRNA to the lambda form as its substrate.

Structural insights into the first step of RNA-dependent cysteine biosynthesis in archaea.

Cysteine is ligated to tRNA(Cys) by cysteinyl-tRNA synthetase in most organisms. However, in methanogenic archaea lacking cysteinyl-tRNA synthetase, O-phosphoserine is ligated to tRNA(Cys) by O-phosphoseryl-tRNA synthetase (SepRS), and the phosphoseryl-tRNA(Cys) is converted to cysteinyl-tRNA(Cys). In this study, we determined the crystal structure of the SepRS tetramer in complex with tRNA(Cys) and O-phosphoserine at 2.6-A resolution. The catalytic domain of SepRS recognizes the negatively charged side chain of O-phosphoserine at a noncanonical site, using the dipole moment of a conserved alpha-helix. The unique C-terminal domain specifically recognizes the anticodon GCA of tRNA(Cys). On the basis of the structure, we engineered SepRS to recognize tRNA(Cys) mutants with the anticodons UCA and CUA and clarified the anticodon recognition mechanism by crystallography. The mutant SepRS-tRNA pairs may be useful for translational incorporation of O-phosphoserine into proteins in response to the stop codons UGA and UAG.