Archaeal Protein Research Articles

ProSeqAProDB: Prosequence Assisted Protein Database

In early 1990s, several proteins were shown to depend on additional stretches of polypeptide (termed as prosequence/prodomain) for their folding. These regions of the protein were often termed as IMCs (Intra Molecular Chaperones), since they would be cleaved from the mature folded protein eventually. Such proteins were hypothesized to face a kinetic barrier to their folding, which was probably lowered by the prosequences. In last three decades, numerous examples of such proteins have accumulated in literature. Yet, no study has been reported so far attempting to understand the evolutionary differences and similaritess of such proteins. Till date such proteins are continued to be treated as anomalous variants, rather than as representatives of any alternate protein folding strategy. Do such proteins have any distinctive structural facets OR typical biological roles, necessitating an unconventional strategy of protein folding? Do prosequences carry any unique or conserved features that are essential to their function?ProSeqAProDb: ProSequence Assisted Protein Database, (which can be accessed at https://proseqaprodb.mkulab.in) was built as a comprehensive database, to systematically study such proteins along with their pro-sequences. The database currently contains 2140 prosequence assisted proteins (1848 eukaryotic, 255 bacterial, 24 viral and 13 archaeal proteins), from 690 organisms later categorised into 960 families. We envisage that the availability of this curated dataset will enable the researchers worldwide to further their investigation in the origin, importance and evolution of such proteins, leading to better understanding of the protein folding process as a whole.

Halorarius litoreus gen. nov., sp. nov., Halorarius halobius sp. nov., Haloglomus halophilum sp. nov., Haloglomus salinum sp. nov., and Natronomonas marina sp. nov., extremely halophilic archaea isolated from tidal flat and marine solar salt

Five novel halophilic archaeal strains, named BND22T, ZY10T, ZY41T, ZY58T, and ZY43T, were isolated from the coastal saline sediment of the intertidal zone located in Qingdao and the natural sea salt produced from Huanghua marine solar saltern, PRChina. These five strains demonstrated the typical morphology and growth characteristics of haloarchaea. The comparison of 16S rRNA gene revealed that strain BDN22T was associated with Salinirubellus salinus ZS-35-S2T (95.2% similarity), strain ZY10T was related to Halosegnis rubeus F17-44T (95.1% similarity), both strains ZY41T and ZY58T were closely related to Haloglomus irregulare F16-60T (98.1% and 98.2% similarities, respectively), and strain ZY43T was close to Natronomonas salina YPL13T (98.0% similarity). In addition to the 16S rRNA gene, the rpoB′ gene is undoubtedly another important molecular marker for the identification of halophilic archaea, and concatenated-conserved-protein phylogeny was widely used in archaeal classification in recent years. Phylogenetic and phylogenomic analyses based on rpoB′ genes and 122 concatenated archaeal protein genes showed that these haloarchaea construct three different clades and gathered together with the current members of Haloarculaceae. Strains BND22T and ZY10T formed two distinct clades separated from Salinirubellus salinus ZS-35-S2T and Halosegnis members, strains ZY41T and ZY58T clustered with Haloglomus irregulare F16-60T, and strain ZY43T gathered with the current members of Natronomonas. The ANI, DDH, and AAI values of these five strains against phylogenetic neighbours were no more than 91%, 45%, and 92%, respectively, far below the cut-off values for species delineation, supporting their placements in new taxa. Based on the phenotypic, chemotaxonomic, phylogenetic, and phylogenomic properties, these five strains represent five novel taxa of the family Haloarculaceae, Halorarius litoreus gen. nov., sp. nov. (type strain BND22T = CGMCC 1.18780T = JCM 34966T), Halorarius halobius sp. nov. (type strain ZY10T = CGMCC 1.17475T = JCM 34319T), Haloglomus halophilum sp. nov. (type strain ZY41T = CGMCC 1.17030T = JCM 34161T), Haloglomus salinum sp. nov. (type strain ZY58T = CGMCC 1.17216T = JCM 34163T), and Natronomonas marina sp. nov. (type strain ZY43T = CGMCC 1.17202T = JCM 34162T). This is the first report of description of a novel haloarchaeon isolated from a marine intertidal zone.

Stereoisomer-specific reprogramming of a bacterial flagellin sialyltransferase.

Glycosylation of surface structures diversifies cells chemically and physically. Nucleotide-activated sialic acids commonly serve as glycosyl donors, particularly pseudaminic acid (Pse) and its stereoisomer legionaminic acid (Leg), which decorate eubacterial and archaeal surface layers or protein appendages. FlmG, a recently identified protein sialyltransferase, O-glycosylates flagellins, the subunits of the flagellar filament. We show that flagellin glycosylation and motility in Caulobacter crescentus and Brevundimonas subvibrioides is conferred by functionally insulated Pse and Leg biosynthesis pathways, respectively, and by specialized FlmG orthologs. We established a genetic glyco-profiling platform for the classification of Pse or Leg biosynthesis pathways, discovered a signature determinant of eubacterial and archaeal Leg biosynthesis, and validated it by reconstitution experiments in a heterologous host. Finally, by rewiring FlmG glycosylation using chimeras, we defined two modular determinants that govern flagellin glycosyltransferase specificity: a glycosyltransferase domain that either donates Leg or Pse and a specialized flagellin-binding domain that identifies the acceptor.

An archaeal Cas3 protein facilitates rapid recovery from DNA damage.

CRISPR-Cas systems provide heritable acquired immunity against viruses to archaea and bacteria. Cas3 is a CRISPR-associated protein that is common to all Type I systems, possesses both nuclease and helicase activities, and is responsible for degradation of invading DNA. Involvement of Cas3 in DNA repair had been suggested in the past, but then set aside when the role of CRISPR-Cas as an adaptive immune system was realized. Here we show that in the model archaeon Haloferax volcanii a cas3 deletion mutant exhibits increased resistance to DNA damaging agents compared with the wild-type strain, but its ability to recover quickly from such damage is reduced. Analysis of cas3 point mutants revealed that the helicase domain of the protein is responsible for the DNA damage sensitivity phenotype. Epistasis analysis indicated that cas3 operates with mre11 and rad50 in restraining the homologous recombination pathway of DNA repair. Mutants deleted for Cas3 or deficient in its helicase activity showed higher rates of homologous recombination, as measured in pop-in assays using non-replicating plasmids. These results demonstrate that Cas proteins act in DNA repair, in addition to their role in defense against selfish elements and are an integral part of the cellular response to DNA damage.

Fusion of a bacterial cadherin-like domain and green fluorescent protein as a specific probe to study biofilm matrix formation in Rhizobium spp.

Rhizobium adhering proteins or 'Raps' are secreted proteins identified in a very restricted group of rhizobial strains, specifically those belonging to R. leguminosarum and R. etli. The distinctive feature of members of the Rap family is the presence of one or two cadherin-like domains or CHDLs that are also present in numerous extracellular bacterial and archaeal proteins and were proposed to confer carbohydrate binding ability. We have previously made an in-depth characterization of RapA2, a calcium-binding lectin, composed by two CHDLs, involved in biofilm matrix remodelling in R. leguminosarum bv. viciae 3841. In this study, CHDLs derived from RapA2 were analysed in detail, finding significant structural and functional differences despite their considerable sequence similarity. Only the carboxy-terminal CHDL retained properties similar to those displayed by RapA2. Our findings were used to obtain a novel fluorescent probe to study biofilm matrix development by confocal laser scanning microscopy, and also to shed some light on the role of the ubiquitous CHDL domains in bacterial secreted proteins.

A subclass of archaeal U8-tRNA sulfurases requires a [4Fe–4S] cluster for catalysis

Sulfuration of uridine 8, in bacterial and archaeal tRNAs, is catalyzed by enzymes formerly known as ThiI, but renamed here TtuI. Two different classes of TtuI proteins, which possess a PP-loop-containing pyrophosphatase domain that includes a conserved cysteine important for catalysis, have been identified. The first class, as exemplified by the prototypic Escherichia coli enzyme, possesses an additional C-terminal rhodanese domain harboring a second cysteine, which serves to form a catalytic persulfide. Among the second class of TtuI proteins that do not possess the rhodanese domain, some archaeal proteins display a conserved CXXC+C motif. We report here spectroscopic and enzymatic studies showing that TtuI from Methanococcus maripaludis and Pyrococcus furiosus can assemble a [4Fe-4S] cluster that is essential for tRNA sulfuration activity. Moreover, structural modeling studies, together with previously reported mutagenesis experiments of M. maripaludis TtuI, indicate that the [4Fe-4S] cluster is coordinated by the three cysteines of the CXXC+C motif. Altogether, our results raise a novel mechanism for U8-tRNA sulfuration, in which the cluster is proposed to catalyze the transfer of sulfur atoms to the activated tRNA substrate.

Biomarker metaproteomics for relative taxa abundances across soil organisms

Soil organisms are often classified using methods targeting individual groups of taxa (e.g., bacteria, fungi and invertebrates), which hampers our ability to directly compare the relative abundance of different groups across environmental gradients. We posit that the use of protein biomarkers could help to provide a more real representation of the cross-kingdom soil microbial populations. Here, we tested if the abundant proteins ATP synthase F(0) complex (ATPS), elongation factors (EF), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), GroEL, pyruvate dehydrogenase (PyrDH), RNA polymerase beta chain (RNAP), and translation initiation factor 2 (TIF) could be used to describe the taxonomic composition of microbial communities. As positive control, we used a mock community with different relative abundances of algae, archaea, bacteria, and viruses. We tested this approach on a previously published soil metaproteomes from which we randomly selected samples from forests, grasslands, and shrublands (each n = 10). Unfortunately, the biomarker approach is not feasible for viruses as these organisms do not share single genes. All biomarkers showed decent accuracy to determine the relative abundances of archaea, bacteria, and eukaryota in the mock community. However, false positive hits dominated on phylum level probably due to sequence homology. Archaeal proteins were only detected in the soil samples when EF was used as biomarker at an abundance of 0.7%. Bacteria dominated the EF-metaproteome and were most abundant in shrublands (64.4%) while eukaryotes were more abundant in forests (25.6%). In compliance with previously published results, the correlation analysis revealed the impact of mean annual temperature and pH on both bacteria and eukaryota. Our approach not only shows the potential to use biomarker metaproteomics to unveil the relative taxa abundances across soil organisms but also the need to create mock communities comprising members of all soil taxa.

Halorussus vallis sp. nov., Halorussus aquaticus sp. nov., Halorussus gelatinilyticus sp. nov., Halorussus limi sp. nov., Halorussus salilacus sp. nov., Halorussus salinisoli sp. nov.: six extremely halophilic archaea isolated from solar saltern, salt lake and saline soil.

Six novel halophilic archaeal strains of XZYJT10T, XZYJ18T, XZYJT40T, XZYJT49T, YCN54T and LT46T were isolated from a solar saltern in Tibet, a salt lake in Shanxi, and a saline soil in Xinjiang, China. Sequence similarities of 16S rRNA and rpoB' genes among strains XZYJT10T, XZYJ18T, XZYJT40T, XZYJT49T, YCN54T, LT46T and current members of Halorussus were 90.6-97.8% and 87.8-96.4%, respectively. The average nucleotide identity and in silico DNA-DNA hybridization values among these six strains and current Halorussus members were in the range of 76.5-87.5% and 21.0-33.8%, respectively. These values were all below the species boundary threshold values. The phylogenomic tree based on 122 conserved archaeal protein marker genes revealed that the six novel strains formed individual distinct branches and clustered tightly with Halorussus members. Several phenotypic characteristics distinguished the six strains from current Halorussus members. Polar lipid analysis showed that the six novel strains contained phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate and two to three glycolipids. Phenotypic, chemotaxonomic and phylogenetic properties showed that the six strains represented six novel species within the genus Halorussus, for which the names Halorussus vallis sp. nov., Halorussus aquaticus sp. nov., Halorussus gelatinilyticus sp. nov., Halorussus limi sp. nov., Halorussus salilacus sp. nov., and Halorussus salinisoli sp. nov. are proposed.

Asgard ESCRT-III and VPS4 reveal conserved chromatin binding properties of the ESCRT machinery.

The archaeal Asgard superphylum currently stands as the most promising prokaryotic candidate, from which eukaryotic cells emerged. This unique superphylum encodes for eukaryotic signature proteins (ESP) that could shed light on the origin of eukaryotes, but the properties and function of these proteins is largely unresolved. Here, we set to understand the function of an Asgard archaeal protein family, namely the ESCRT machinery, that is conserved across all domains of life and executes basic cellular eukaryotic functions, including membrane constriction during cell division. We find that ESCRT proteins encoded in Loki archaea, express in mammalian and yeast cells, and that the Loki ESCRT-III protein, CHMP4-7, resides in the eukaryotic nucleus in both organisms. Moreover, Loki ESCRT-III proteins associated with chromatin, recruited their AAA-ATPase VPS4 counterpart to organize in discrete foci in the mammalian nucleus, and directly bind DNA. The human ESCRT-III protein, CHMP1B, exhibited similar nuclear properties and recruited both human and Asgard VPS4s to nuclear foci, indicating interspecies interactions. Mutation analysis revealed a role for the N terminal region of ESCRT-III in mediating these phenotypes in both human and Asgard ESCRTs. These findings suggest that ESCRT proteins hold chromatin binding properties that were highly preserved through the billion years of evolution separating Asgard archaea and humans. The conserved chromatin binding properties of the ESCRT membrane remodeling machinery, reported here, may have important implications for the origin of eukaryogenesis.

Structural characterisation of a MAPR-related archaeal cytochrome b5M protein.

We recently reported that the membrane-associated progesterone receptor (MAPR) protein family (mammalian members: PGRMC1, PGRMC2, NEUFC and NENF) originated from a new class of prokaryotic cytochrome b5 (cytb5 ) domain proteins, called cytb5M (MAPR-like). Relative to classical cytb5 proteins, MAPR and ctyb5M proteins shared unique sequence elements and a distinct heme-binding orientation at an approximately 90° rotation relative to classical cytb5 , as demonstrated in the archetypal crystal structure of a cytb5M protein (PDB accession number 6NZX). Here, we present the crystal structure of an archaeal cytb5M domain (Methanococcoides burtonii WP_011499504.1, PDB:6VZ6). It exhibits similar heme binding to the 6NZX cytb5M , supporting the deduction that MAPR-like heme orientation was inherited from the prokaryotic ancestor of the original eukaryotic MAPR gene.

Isolation and Characterization of Cell Envelope Fragments Comprising Archaeal S-Layer Proteins.

The outermost component of cell envelopes of most bacteria and almost all archaea comprise a protein lattice, which is termed Surface (S-)layer. The S-layer lattice constitutes a highly porous structure with regularly arranged pores in the nm-range. Some archaea thrive in extreme milieus, thus producing highly stable S-layer protein lattices that aid in protecting the organisms. In the present study, fragments of the cell envelope from the hyperthermophilic acidophilic archaeon Saccharolobus solfataricus P2 (SSO) have been isolated by two different methods and characterized. The organization of the fragments and the molecular sieving properties have been elucidated by transmission electron microscopy and by determining the retention efficiency of proteins varying in size, respectively. The porosity of the archaeal S-layer fragments was determined to be 45%. S-layer fragments of SSO showed a retention efficiency of up to 100% for proteins having a molecular mass of ≥ 66 kDa. Moreover, the extraction costs for SSO fragments have been reduced by more than 80% compared to conventional methods, which makes the use of these archaeal S-layer material economically attractive.

Purification and Characterization of a Stable, Membrane-Associated Peptidoglycan Responsive Adenylate Cyclase LRR Domain from Human Commensal Candida albicans.

The evolutionarily conserved leucine rich repeat (LRR) protein domain is a unique structural motif found in many viral, bacterial, archaeal, and eukaryotic proteins. The LRR domain serves many roles, including being a signaling domain and a pathogen recognition receptor. In the human innate immune system, it serves an essential role by recognizing fragments of bacterial cell walls. Interestingly, the human fungal pathogen Candida albicans also uses an LRR domain-containing protein, Cyrp1, to sense bacterial cell wall fragments. However, the dynamics of signaling and detection of bacterial peptidoglycan fragments by the LRR of Cyr1p remains poorly characterized. Here we develop optimal recombinant expression workflows and provide characterization of the entire region of the LRR domain of Cyr1p as a peripheral membrane protein. Using a newly designed peptidoglycan enrichment bead assay, we demonstrate that this domain can bind bacterial peptidoglycan fragments under native conditions. The new membrane-associated Cyr1p-LRR construct sets the stage for the development of antifungal agents via high-throughput campaigns to inhibit cell wall-Cyr1p interactions.

Discovery of archaeal fusexins homologous to eukaryotic HAP2/GCS1 gamete fusion proteins

Sexual reproduction consists of genome reduction by meiosis and subsequent gamete fusion. The presence of genes homologous to eukaryotic meiotic genes in archaea and bacteria suggests that DNA repair mechanisms evolved towards meiotic recombination. However, fusogenic proteins resembling those found in gamete fusion in eukaryotes have so far not been found in prokaryotes. Here, we identify archaeal proteins that are homologs of fusexins, a superfamily of fusogens that mediate eukaryotic gamete and somatic cell fusion, as well as virus entry. The crystal structure of a trimeric archaeal fusexin (Fusexin1 or Fsx1) reveals an archetypical fusexin architecture with unique features such as a six-helix bundle and an additional globular domain. Ectopically expressed Fusexin1 can fuse mammalian cells, and this process involves the additional globular domain and a conserved fusion loop. Furthermore, archaeal fusexin genes are found within integrated mobile elements, suggesting potential roles in cell-cell fusion and gene exchange in archaea, as well as different scenarios for the evolutionary history of fusexins.

Conserved and lineage-specific hypothetical proteins may have played a central role in the rise and diversification of major archaeal groups

BackgroundArchaea play fundamental roles in the environment, for example by methane production and consumption, ammonia oxidation, protein degradation, carbon compound turnover, and sulfur compound transformations. Recent genomic analyses have profoundly reshaped our understanding of the distribution and functionalities of Archaea and their roles in eukaryotic evolution.ResultsHere, 1179 representative genomes were selected from 3197 archaeal genomes. The representative genomes clustered based on the content of 10,866 newly defined archaeal protein families (that will serve as a community resource) recapitulates archaeal phylogeny. We identified the co-occurring proteins that distinguish the major lineages. Those with metabolic roles were consistent with experimental data. However, two families specific to Asgard were determined to be new eukaryotic signature proteins. Overall, the blocks of lineage-specific families are dominated by proteins that lack functional predictions.ConclusionsGiven that these hypothetical proteins are near ubiquitous within major archaeal groups, we propose that they were important in the origin of most of the major archaeal lineages. Interestingly, although there were clearly phylum-specific co-occurring proteins, no such blocks of protein families were shared across superphyla, suggesting a burst-like origin of new lineages early in archaeal evolution.

A Lipoate-Protein Ligase Is Required for De Novo Lipoyl-Protein Biosynthesis in the Hyperthermophilic Archaeon Thermococcus kodakarensis.

Lipoic acid is an organosulfur cofactor essential for several key enzyme complexes in oxidative and one-carbon metabolism. It is covalently bound to the lipoyl domain of the E2 subunit in some 2-oxoacid dehydrogenase complexes and the H-protein in the glycine cleavage system. Lipoate-protein ligase (Lpl) is involved in the salvage of exogenous lipoate and attaches free lipoate to the E2 subunit or the H-protein in an ATP-dependent manner. In the hyperthermophilic archaeon Thermococcus kodakarensis, TK1234 and TK1908 are predicted to encode the N- and C-terminal regions of Lpl, respectively. TK1908 and TK1234 recombinant proteins form a heterodimer and together displayed significant ligase activity toward octanoate in addition to lipoate when a chemically synthesized octapeptide was used as the acceptor. The proteins also displayed activity toward other fatty acids, indicating broad fatty acid specificity. On the other hand, lipoyl synthase from T. kodakarensis only recognized octanoyl-peptide as a substrate. Examination of individual proteins indicated that the TK1908 protein alone was able to catalyze the ligase reaction although with a much lower activity. Gene disruption of TK1908 led to lipoate/serine auxotrophy, whereas TK1234 gene deletion did not. Acyl carrier protein homologs are not found on the archaeal genomes, and the TK1908/TK1234 protein complex did not utilize octanoyl-CoA, raising the possibility that the substrate of the ligase reaction is octanoic acid itself. Although Lpl has been considered as an enzyme involved in lipoate salvage, the results imply that in T. kodakarensis, the TK1908 and TK1234 proteins function in de novo lipoyl-protein biosynthesis. IMPORTANCE Based on previous studies in bacteria and eukaryotes, lipoate-protein ligases (Lpls) have been considered to be involved exclusively in lipoate salvage. The genetic analyses in this study on the lipoate-protein ligase in T. kodakarensis, however, suggest otherwise and that the enzyme is additionally involved in de novo protein lipoylation. We also provide biochemical evidence that the lipoate-protein ligase displays broad substrate specificity and is capable of ligating acyl groups of various chain-lengths to the peptide substrate. We show that this apparent ambiguity in Lpl is resolved by the strict substrate specificity of the lipoyl synthase LipS in this organism, which only recognizes octanoyl-peptide. The results provide relevant physiological insight into archaeal protein lipoylation.

Essentiality of core hydrophobicity to the structure and function of archaeal chromatin protein Cren7

Studies on the structure–function relationship of protein greatly help to understand not only the principles of protein folding but also the rationales of protein engineering. Crenarchaeal chromatin protein Cren7 provides an excellent research model for this issue. The small protein adopts a ‘β-barrel’ fold, formed by the double-stranded antiparallel β-sheet 1 tightly packing with the triple-stranded antiparallel β-sheet 2. The simple structure of Cren7 is stabilized by the hydrophobic core between the β-sheets, consisting of the side chains of V8, V10, L20, V25, F41 and F50. In the present work, mutation analyses by alanine substitution of each of the residues in the hydrophobic core were performed. Circular dichroism spectra and nuclear magnetic resonance analyses showed that mutation of F41 led to a significant misfolding of Cren7 through disruption of the β-sheets. Meanwhile, the mutant F41A showed a reduced thermostatility (Tm of 53.2 °C), as compared with the wild-type Cren7 (Tm > 80 °C). Biolayer interferometry and nick-closure assays showed the largely unchanged activities in DNA binding and supercoiling of F41A, indicating the DNA interface of Cren7 was generally retained in F41A. However, F41A was unable to mediate DNA bridging, probably due to the impairment in forming oligomers/polymers on DNA. Atomic force microscopic images of the F41A-DNA complexes also revealed that F41A nearly completely lost the ability to compact DNA into highly condensed structures. Our results not only reveal the critical role of F41 in protein folding of Cren7 but also provide new insights into the structure-function relationships of thermostable proteins.

Minimal Yet Powerful: The Role of Archaeal Small Heat Shock Proteins in Maintaining Protein Homeostasis.

Small heat shock proteins (sHsp) are a ubiquitous group of ATP-independent chaperones found in all three domains of life. Although sHsps in bacteria and eukaryotes have been studied extensively, little information was available on their archaeal homologs until recently. Interestingly, archaeal heat shock machinery is strikingly simplified, offering a minimal repertoire of heat shock proteins to mitigate heat stress. sHsps play a crucial role in preventing protein aggregation and holding unfolded protein substrates in a folding-competent form. Besides protein aggregation protection, archaeal sHsps have been shown recently to stabilize membranes and contribute to transferring captured substrate proteins to chaperonin for refolding. Furthermore, recent studies on archaeal sHsps have shown that environment-induced oligomeric plasticity plays a crucial role in maintaining their functional form. Despite being prokaryotes, the archaeal heat shock protein repository shares several features with its highly sophisticated eukaryotic counterpart. The minimal nature of the archaeal heat shock protein repository offers ample scope to explore the function and regulation of heat shock protein(s) to shed light on their evolution. Moreover, similar structural dynamics of archaeal and human sHsps have made the former an excellent system to study different chaperonopathies since archaeal sHsps are more stable under in vitro experiments.

Biochemical characterization of a new 7,8‐dihydro‐6‐hydroxymethylpterin pyrophosphokinase (HPPK) from Methanocaldococcus jannaschii

Methanogenic archaea are strict anaerobes that produce methane as a byproduct of their essential energy metabolism known as methanogenesis. Tetrahydromethanopterin (H4MPT), a specialized cofactor similar to tetrahydrofolate (H4F), is a C1carrier required for the methanogenesis pathway. The biosynthesis of H4MPT utilizes a similar series of reactions used in H4F biosynthesis, but they are catalyzed by nonorthologous enzymes. Here, we report the recombinant expression, purification, and enzymatic properties of MptE (MJ1634), a novel 7,8‐dihydro‐6‐hydroxymethylpterin pyrophosphokinase (HPPK) identified to catalyze the final step in the biosynthesis of the pterin portion in H4MPT in Methancaldococcus jannaschii by catalyzing the transfer of a pyrophosphate from ATP to the substrate dihydro‐6‐hydroxymethylpterin (H2HMP). This novel HPPK shows a distinctive sequence from the bacterial counterpart; instead, it belongs to the superfamily of thiamine pyrophosphokinase (TPK) catalytic domain. The gene encoding MptE from M. jannaschii was overexpressed in Escherichia coli, and the his‐tagged recombinant enzyme was purified by metal‐affinity chromatography. After purification, initial enzyme assays were performed and analyzed via HPLC, which revealed a similar catalytic efficiency to bacterial HPPKs. Site‐directed mutagenesis studies coupled with structural analyses revealed that Asp106 and Asp171 are essential for Mg2+ coordination, His129 and Phe169 play important roles in H2HMP binding, and Lys217 is important for transition state stabilization. These residues were selected because they are strictly conserved in the archaeal protein family COG1634 or thiamine pyrophosphokinase (TPK), whose active site resembles those in the COG1634 family. Currently, kinetic studies are underway for additional variants to further identify key residues of structural and catalytic importance.

Evolutionary diversification of the autophagy-related ubiquitin-like conjugation systems

ABSTRACT Two autophagy-related (ATG) ubiquitin-like conjugation systems, the ATG12 and ATG8 systems, play important roles in macroautophagy. While multiple duplications and losses of the ATG conjugation system proteins are found in different lineages, the extent to which the underlying systems diversified across eukaryotes is not fully understood. Here, in order to understand the evolution of the ATG conjugation systems, we constructed a transcriptome database consisting of 94 eukaryotic species covering major eukaryotic clades and systematically identified ATG conjugation system components. Both ATG10 and the C-terminal glycine of ATG12 are essential for the canonical ubiquitin-like conjugation of ATG12 and ATG5. However, loss of ATG10 or the C-terminal glycine of ATG12 occurred at least 16 times in a wide range of lineages, suggesting that possible covalent-to-non-covalent transition is not limited to the species that we previously reported such as Alveolata and some yeast species. Some species have only the ATG8 system (with conjugation enzymes) or only ATG8 (without conjugation enzymes). More than 10 species have ATG8 homologs without the conserved C-terminal glycine, and Tetrahymena has an ATG8 homolog with a predicted transmembrane domain, which may be able to anchor to the membrane independent of the ATG conjugation systems. We discuss the possibility that the ancestor of the ATG12 and ATG8 systems is more similar to ATG8. Overall, our study offers a whole picture of the evolution and diversity of the ATG conjugation systems among eukaryotes, and provides evidence that functional diversifications of the systems are more common than previously thought. Abbreviations: APEAR: ATG8–PE association region; ATG: autophagy-related; LIR: LC3-interacting region; NEDD8: neural precursor cell expressed, developmentally down-regulated gene 8; PE: phosphatidylethanolamine; SAMP: small archaeal modifier protein; SAR: Stramenopiles, Alveolata, and Rhizaria; SMC: structural maintenance of chromosomes; SUMO: small ubiquitin like modifier; TACK: Thaumarchaeota, Aigarchaeota, Crenarchaeota, and Korarchaeota; UBA: ubiquitin like modifier activating enzyme; UFM: ubiquitin fold modifier; URM: ubiquitin related modifier.

Interplay between Alba and Cren7 Regulates Chromatin Compaction in Sulfolobus solfataricus.

Chromatin compaction and regulation are essential processes for the normal function of all organisms, yet knowledge on how archaeal chromosomes are packed into higher-order structures inside the cell remains elusive. In this study, we investigated the role of archaeal architectural proteins Alba and Cren7 in chromatin folding and dynamics. Atomic force microscopy revealed that Sulfolobus solfataricus chromatin is composed of 28 nm fibers and 60 nm globular structures. In vitro reconstitution showed that Alba can mediate the formation of folded DNA structures in a concentration-dependent manner. Notably, it was demonstrated that Alba on its own can form higher-order structures with DNA. Meanwhile, Cren7 was observed to affect the formation of Alba-mediated higher-order chromatin structures. Overall, the results suggest an interplay between Alba and Cren7 in regulating chromatin compaction in archaea.