Effects of producing high levels of hyperthermophile-specific C25,C25-archaeal membrane lipids in Escherichia coli.

A hyperthermophilic archaeon, Aeropyrum pernix, synthesizes C25,C25-archaeal membrane lipids, or extended archaeal membrane lipids, which contain two C25 isoprenoid chains that are linked to glycerol-1-phosphate via ether bonds and are longer than the usual C20,C20-archaeal membrane lipids. The C25,C25-archaeal membrane lipids are believed to allow the archaeon to survive under harsh conditions, because they are able to form lipid membranes that are impermeable at temperatures approaching the boiling point. The effect that C25,C25-archaeal membrane lipids exert on living cells, however, remains unproven along with an explanation for why the hyperthermophilic archaeon synthesizes these specific lipids instead of the more common C20,C20-archaeal lipids or double-headed tetraether lipids. To shed light on the effects that these hyperthermophile-specific membrane lipids exert on living cells, we have constructed an E. coli strain that produces C25,C25-archaeal membrane lipids. However, a resultant low level of productivity would not allow us to assess the effects of their production in E. coli cells. Herein, we report an enhancement of the productivity of C25,C25-archaeal membrane lipids in engineered E. coli strains via the introduction of metabolic pathways such as an artificial isoprenol utilization pathway where the precursors of isoprenoids are synthesized via a two-step phosphorylation of prenol and isoprenol supplemented to a growth medium. In the strain with the highest titer, a major component of C25,C25-archaeal membrane lipids reached ∼11 % of total lipids of E. coli. It is noteworthy that the high production of the extended archaeal lipids did not significantly affect the growth of the bacterial cells. The permeability of the cell membrane of the strain became slightly lower in the presence of the exogenous membrane lipids with longer hydrocarbon chains, which demonstrated the possibility to enhance bacterial cell membranes by the hyperthermophile-specific lipids, along with the surprising robustness of the E. coli cell membrane.

Self-assembly of Aeropyrum pernix bacilliform virus 1 (APBV1) major capsid protein and its application as building blocks for nanomaterials.

Virus capsid proteins have various applications in diverse fields such as biotechnology, electronics, and medicine. In this study, the major capsid protein of bacilliform clavavitus APBV1, which infects the hyperthermophilic archaeon Aeropyrum pernix, was successfully expressed in Escherichia coli. The gene product was expressed as a histidine-tagged protein in E. coli and purified to homogeneity using single-step nickel affinity chromatography. The purified recombinant protein self-assembled to form bacilliform virus-like particles at room temperature. The particles exhibited tolerance against high concentrations of organic solvents and protein denaturants. In addition, we succeeded in fabricating functional nanoparticles with amine functional groups on the surface of ORF6-81 nanoparticles. These robust protein nanoparticles can potentially be used as a scaffold in nanotechnological applications.

NAD+-dependent RNA terminal 2' and 3' phosphomonoesterase activity of a subset of Tpt1 enzymes.

The enzyme Tpt1 removes the 2'-PO4 at the splice junction generated by fungal tRNA ligase; it does so via a two-step reaction in which (i) the internal RNA 2'-PO4 attacks NAD+ to form an RNA-2'-phospho-ADP-ribosyl intermediate; and (ii) transesterification of the ribose O2″ to the 2'-phosphodiester yields 2'-OH RNA and ADP-ribose-1″,2″-cyclic phosphate products. The role that Tpt1 enzymes play in taxa that have no fungal-type RNA ligase remains obscure. An attractive prospect is that Tpt1 enzymes might catalyze reactions other than internal RNA 2'-PO4 removal, via their unique NAD+-dependent transferase mechanism. This study extends the repertoire of the Tpt1 enzyme family to include the NAD+-dependent conversion of RNA terminal 2' and 3' monophosphate ends to 2'-OH and 3'-OH ends, respectively. The salient finding is that different Tpt1 enzymes vary in their capacity and positional specificity for terminal phosphate removal. Clostridium thermocellum and Aeropyrum pernix Tpt1 proteins are active on 2'-PO4 and 3'-PO4 ends, with a 2.4- to 2.6-fold kinetic preference for the 2'-PO4 The accumulation of a terminal 3'-phospho-ADP-ribosylated RNA intermediate during the 3'-phosphotransferase reaction suggests that the geometry of the 3'-p-ADPR adduct is not optimal for the ensuing transesterification step. Chaetomium thermophilum Tpt1 acts specifically on a terminal 2'-PO4 end and not with a 3'-PO4 In contrast, Runella slithyformis Tpt1 and human Tpt1 are ineffective in removing either a 2'-PO4 or 3'-PO4 end.

How DNA affects the hyperthermophilic protein Ape10b2 for oligomerization: an investigation using multiple short molecular dynamics simulations.

Alba2 is a hyperthermophilic DNA-binding protein, and DNA plays a crucial role in the Alba2 oligomerization process. It is a pity that there is limited research in terms of how DNA affects the conformational change of Alba2 in oligomerization. Herein, we complement the crystal structure of the Ape10b2 (belongs to Alba2)-dsDNA complex (PDB ID: 3U6Y) and employ multiple short molecular dynamics (MSMD) simulations to illuminate the influence of DNA on Ape10b2 at four temperatures (300, 343, 363, and 373 K). Our results indicate that DNA could cause the conformational changes of two important regions (loop1 and loop5), which may be beneficial for protein oligomerization. The results of hydrogen bond analysis show that the increasing number of hydrogen bonds between two monomers of Ape10b2 may also be a favorable factor for oligomerization. In addition, Ape10b2 can stabilize DNA by electrostatic interactions with an increase in temperature, and five residues (Arg40, Arg42, Asn43, Asn45, and Arg46) play a stabilizing role during protein binding to DNA. Our findings could help in understanding the favorable factors leading to protein oligomerization, which contributes to enzyme engineering research from an industrial perspective.

Total Synthesis and Structure Confirmation of trans-Anhydromevalonate-5-phosphate, a Key Biosynthetic Intermediate of the Archaeal Mevalonate Pathway.

The mevalonate pathway is an upstream terpenoid biosynthetic route of terpenoids for providing the two five-carbon units, dimethylallyl diphosphate, and isopentenyl diphosphate. Recently, trans-anhydromevalonate-5-phosphate (tAHMP) was isolated as a new biosynthetic intermediate of the archaeal mevalonate pathway. In this study, we would like to report the first synthesis of tAHMP and its enzymatic transformation using one of the key enzymes, mevalonate-5-phosphate dehydratase from a hyperthermophilic archaeon, Aeropyrum pernix. Starting from methyl tetrolate, a Cu-catalyzed allylation provided an E-trisubstituted olefin in a stereoselective manner. The resulting E-olefin was transformed to tAHMP by cleavage of the olefin and phosphorylation. The structure of the synthetic tAHMP was unambiguously determined by NOESY analysis.

Modified mevalonate pathway of the archaeon Aeropyrum pernix proceeds via trans-anhydromevalonate 5-phosphate.

The modified mevalonate pathway is believed to be the upstream biosynthetic route for isoprenoids in general archaea. The partially identified pathway has been proposed to explain a mystery surrounding the lack of phosphomevalonate kinase and diphosphomevalonate decarboxylase by the discovery of a conserved enzyme, isopentenyl phosphate kinase. Phosphomevalonate decarboxylase was considered to be the missing link that would fill the vacancy in the pathway between mevalonate 5-phosphate and isopentenyl phosphate. This enzyme was recently discovered from haloarchaea and certain Chroloflexi bacteria, but their enzymes are close homologs of diphosphomevalonate decarboxylase, which are absent in most archaea. In this study, we used comparative genomic analysis to find two enzymes from a hyperthermophilic archaeon, Aeropyrum pernix, that can replace phosphomevalonate decarboxylase. One enzyme, which has been annotated as putative aconitase, catalyzes the dehydration of mevalonate 5-phosphate to form a previously unknown intermediate, trans-anhydromevalonate 5-phosphate. Then, another enzyme belonging to the UbiD-decarboxylase family, which likely requires a UbiX-like partner, converts the intermediate into isopentenyl phosphate. Their activities were confirmed by in vitro assay with recombinant enzymes and were also detected in cell-free extract from A. pernix These data distinguish the modified mevalonate pathway of A. pernix and likely, of the majority of archaea from all known mevalonate pathways, such as the eukaryote-type classical pathway, the haloarchaea-type modified pathway, and another modified pathway recently discovered from Thermoplasma acidophilum.

ICTV Virus Taxonomy Profile: Spiraviridae.

The family Spiraviridae includes viruses that replicate in hyperthermophilic archaea from the genus Aeropyrum. The non-enveloped, hollow, cylindrical virions are formed from a coiling fibre that consists of two intertwining halves of a single circular nucleoprotein filament. A short appendage protrudes from each end of the cylindrical virion. The genome is circular, positive-sense, single-stranded DNA of 24 893 nucleotides. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) report on the family Spiraviridae, which is available at ictv.global/report/spiraviridae.

Insights into the Maturation of Pernisine, a Subtilisin-Like Protease from the Hyperthermophilic Archaeon Aeropyrum pernix.

Pernisine is a subtilisin-like protease that was originally identified in the hyperthermophilic archaeon Aeropyrum pernix, which lives in extreme marine environments. Pernisine shows exceptional stability and activity due to the high-temperature conditions experienced by A. pernix Pernisine is of interest for industrial purposes, as it is one of the few proteases that has demonstrated prion-degrading activity. Like other extracellular subtilisins, pernisine is synthesized in its inactive pro-form (pro-pernisine), which needs to undergo maturation to become proteolytically active. The maturation processes of mesophilic subtilisins have been investigated in detail; however, less is known about the maturation of their thermophilic homologs, such as pernisine. Here, we show that the structure of pro-pernisine is disordered in the absence of Ca2+ ions. In contrast to the mesophilic subtilisins, pro-pernisine requires Ca2+ ions to adopt the conformation suitable for its subsequent maturation. In addition to several Ca2+-binding sites that have been conserved from the thermostable Tk-subtilisin, pernisine has an additional insertion sequence with a Ca2+-binding motif. We demonstrate the importance of this insertion for efficient folding and stabilization of pernisine during its maturation. Moreover, analysis of the pernisine propeptide explains the high-temperature requirement for pro-pernisine maturation. Of note, the propeptide inhibits the pernisine catalytic domain more potently at high temperatures. After dissociation, the propeptide is destabilized at high temperatures only, which leads to its degradation and finally to pernisine activation. Our data provide new insights into and understanding of the thermostable subtilisin autoactivation mechanism.IMPORTANCE Enzymes from thermophilic organisms are of particular importance for use in industrial applications, due to their exceptional stability and activity. Pernisine, from the hyperthermophilic archaeon Aeropyrum pernix, is a proteolytic enzyme that can degrade infective prion proteins and thus has a potential use for disinfection of prion-contaminated surfaces. Like other subtilisin-like proteases, pernisine needs to mature through an autocatalytic process to become an active protease. In the present study, we address the maturation of pernisine and show that the process is regulated specifically at high temperatures by the propeptide. Furthermore, we demonstrate the importance of a unique Ca2+-binding insertion for stabilization of mature pernisine. Our results provide a novel understanding of thermostable subtilisin autoactivation, which might advance the development of these enzymes for commercial use.

Periplasmic production of pernisine in Escherichia coli and determinants for its high thermostability.

Pernisine is a subtilisin-like serine proteinase secreted by the hyperthermophilic archaeon Aeropyrum pernix. The significant properties of this proteinase are remarkable stability and ability to degrade the infectious prion proteins. Here we show the production of pernisine in the periplasm of Escherichia coli. This strategy prevented the aggregation of pernisine in the cytoplasm and increased the purity of the isolated pernisine. The thermostability of this recombinant pernisine was significantly increased compared with previous studies. In addition, several truncated pernisine variants were constructed and expressed in E. coli to identify the minimally active domain. The catalytic domain of pernisine consists of the αẞα structurally similar core flanked by the N-terminal and C-terminal outer regions. The deletion of the C-terminal α helix did not affect the pernisine activity at 90 °C. However, the complete deletion of the C-terminal outer region resulted in loss of proteolytic activity. The pernisine variant, in which the N-terminal outer region was deleted, had a reduced activity at 90 °C. These results underline the importance of the Ca2+ binding sites predicted in these outer regions for stability and activity of pernisine. KEY POINTS: • Aggregation of produced pernisine was prevented by translocation into periplasm. • Thermostability of mature pernisine was increased. • The outer regions of the catalytic core are required for pernisine thermostability.

NAD+-dependent synthesis of a 5'-phospho-ADP-ribosylated RNA/DNA cap by RNA 2'-phosphotransferase Tpt1.

RNA 2'-phosphotransferase Tpt1 converts an internal RNA 2'-monophosphate to a 2'-OH via a two-step NAD+-dependent mechanism in which: (i) the 2'-phosphate attacks the C1″ of NAD+ to expel nicotinamide and form a 2'-phospho-ADP-ribosylated RNA intermediate; and (ii) the ADP-ribose O2″ attacks the phosphate of the RNA 2'-phospho-ADPR intermediate to expel the RNA 2'-OH and generate ADP-ribose 1″-2″ cyclic phosphate. Tpt1 is an essential component of the fungal tRNA splicing pathway that generates a unique 2'-PO4, 3'-5' phosphodiester splice junction during tRNA ligation. The wide distribution of Tpt1 enzymes in taxa that have no fungal-type RNA ligase raises the prospect that Tpt1 might catalyze reactions other than RNA 2'-phosphate removal. A survey of Tpt1 enzymes from diverse sources reveals that whereas all of the Tpt1 enzymes are capable of NAD+-dependent conversion of an internal RNA 2'-PO4 to a 2'-OH (the canonical Tpt1 reaction), a subset of Tpt1 enzymes also catalyzed NAD+-dependent ADP-ribosylation of an RNA or DNA 5'-monophosphate terminus. Aeropyrum pernix Tpt1 (ApeTpt1) is particularly adept in this respect. One-step synthesis of a 5'-phospho-ADP-ribosylated cap structure by ApeTpt1 (with no subsequent 5'-phosphotransferase step) extends the repertoire of the Tpt1 enzyme family and the catalogue of ADP-ribosylation reactions involving nucleic acid acceptors.

A Shared Mechanism for the Folding of Voltage-Gated K+ Channels.

In this study, we probe the folding of KvAP, a voltage-gated K+ (Kv) channel. The KvAP channel, though of archaebacterial origin, is structurally and functionally similar to eukaryotic Kv channels. An advantage of the KvAP channel is that it can be folded in vitro from an extensively unfolded state and the folding can be controlled by temperature. We utilize these properties of the KvAP channel to separately study the membrane insertion and the tetramerization stages during folding. We use two quantitative assays: a Cys PEGylation assay to monitor membrane insertion and a cross-linking assay to monitor tetramerization. We show that during folding the KvAP polypeptide is rapidly inserted into the lipid bilayer with a "native-like" topology. We identify a segment at the C-terminus that is important for multimerization of the KvAP channel. We show that this C-terminal domain forms a dimer, which raises the possibility that the tetramerization of the KvAP channel proceeds through a dimer of dimers pathway. Our studies show that the in vitro folding of the KvAP channel mirrors aspects of the cellular assembly pathway for voltage-gated K+ channels and therefore suggest that evolutionarily distinct Kv channels share a common folding pathway. The pathway for the folding and assembly of a Kv channel is of central importance as defects in this pathway have been implicated in the etiology of several disease states. Our studies indicate that the KvAP channel provides an experimentally tractable system for elucidating the folding mechanism of Kv channels.

ICTV Virus Taxonomy Profile: Clavaviridae.

The family Clavaviridae includes viruses that replicate in hyperthermophilic archaea from the genus Aeropyrum. The non-enveloped rigid virions are rod-shaped, with dimensions of about 143×16 nm, and have terminal cap structures, one of which is pointed and carries short fibres, while the other is rounded. The virion displays helical symmetry and is constructed from a single major α-helical protein, which is heavily glycosylated, and several minor capsid proteins. The 5278 bp, circular, double-stranded DNA genome of Aeropyrum pernix bacilliform virus 1 is packed inside the virion as a left-handed superhelix. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Clavaviridae, which is available at www.ictv.global/report/clavaviridae.

Extracellular production of the engineered thermostable protease pernisine from Aeropyrum pernix K1 in Streptomyces rimosus.

BACKGROUND: The thermostable serine protease pernisine originates from the hyperthermophilic Archaeaon Aeropyrum pernix and has valuable industrial applications. Due to its properties, A. pernix cannot be cultivated in standard industrial fermentation facilities. Furthermore, pernisine is a demanding target for heterologous expression in mesophilic heterologous hosts due to the relatively complex processing step involved in its activation. RESULTS: We achieved production of active extracellular pernisine in a Streptomyces rimosus host through heterologous expression of the codon-optimised gene by applying step-by-step protein engineering approaches. To ensure secretion of fully active enzyme, the srT signal sequence from the S. rimosus protease was fused to pernisine. To promote correct processing and folding of pernisine, the srT functional cleavage site motif was fused directly to the core pernisine sequence, this way omitting the proregion. Comparative biochemical analysis of the wild-type and recombinant pernisine confirmed that the enzyme produced by S. rimosus retained all of the desired properties of native pernisine. Importantly, the recombinant pernisine also degraded cellular and infectious bovine prion proteins, which is one of the particular applications of this protease. CONCLUSION: Functional pernisine that retains all of the advantageous properties of the native enzyme from the thermophilic host was successfully produced in a S. rimosus heterologous host. Importantly, we achieved extracellular production of active pernisine, which significantly simplifies further downstream procedures and also omits the need for any pre-processing step for its activation. We demonstrate that S. rimosus can be used as an attractive host for industrial production of recombinant proteins that originate from thermophilic organisms.

Biosynthetic machinery for C25,C25-diether archaeal lipids from the hyperthermophilic archaeon Aeropyrum pernix.

Archaea that thrive in harsh environments usually produce membrane lipids with specific structures such as bipolar tetraether lipids. Only a few genera of archaea, which are hyperthermophiles or halophiles, are known to utilize diether lipids with extended, C25 isoprenoid hydrocarbon chains. In the present study, we identify two prenyltransferases and a prenyl reductase responsible for the biosynthesis of C25,C25-diether lipids in the hyperthermophilic archaeon Aeropyrum pernix. These enzymes are more specific to C25 isoprenoid chains than to C20 chains, which are used for the biosynthesis of ordinary C20,C20-diether archaeal lipids. The recombinant expression of these enzymes with two known archaeal enzymes allows the production of C25,C25-diether archaeal lipids in the cells of Escherichia coli.

A L-proline/O2 biofuel cell using L-proline dehydrogenase (LPDH) from Aeropyrum pernix.

To utilize amino acids from food waste as an energy source, L-proline/O2 biofuel cell was constructed using a stable enzyme from hyperthermophilic archaeon for long-term operation. On the anode, the electrocatalytic oxidation of L-proline by L-proline dehydrogenase from Aeropyrum pernix was observed in the presence of ferrocenecarboxylic acid as mediator. On the cathode, electrocatalytic oxygen reduction was detected. Ketjenblack modification of carbon cloth substrate increased the current density due to increased laccase loading and enhanced electron transfer reaction. The biofuel cell using these electrodes achieved a current density of 6.00 µA/cm2. We successfully constructed the first biofuel cell that generates power from L-proline.

Temperature dependent mistranslation in a hyperthermophile adapts proteins to lower temperatures.

All organisms universally encode, synthesize and utilize proteins that function optimally within a subset of growth conditions. While healthy cells are thought to maintain high translational fidelity within their natural habitats, natural environments can easily fluctuate outside the optimal functional range of genetically encoded proteins. The hyperthermophilic archaeon Aeropyrum pernix (A. pernix) can grow throughout temperature variations ranging from 70 to 100°C, although the specific factors facilitating such adaptability are unknown. Here, we show that A. pernix undergoes constitutive leucine to methionine mistranslation at low growth temperatures. Low-temperature mistranslation is facilitated by the misacylation of tRNA(Leu) with methionine by the methionyl-tRNA synthetase (MetRS). At low growth temperatures, the A. pernix MetRS undergoes a temperature dependent shift in tRNA charging fidelity, allowing the enzyme to conditionally charge tRNA(Leu) with methionine. We demonstrate enhanced low-temperature activity for A. pernix citrate synthase that is synthesized during leucine to methionine mistranslation at low-temperature growth compared to its high-fidelity counterpart synthesized at high-temperature. Our results show that conditional leucine to methionine mistranslation can make protein adjustments capable of improving the low-temperature activity of hyperthermophilic proteins, likely by facilitating the increasing flexibility required for greater protein function at lower physiological temperatures.

Antioxidative Activity of Methanolic and Water Extracts from the Hyperthermophilic Archaeon Aeropyrum pernix K1.

The hyperthermophilic archaeon Aeropyrum pernix has adapted to optimal growth under high temperatures in saline environments and under oxidizing conditions. In the present study, we focused on the antioxidative activity of proteins from A. pernix K1. Following high temperature methanol and water extractions of the protein from the biomass of A. pernix K1, the total sulphydryl groups and radical scavenging activities were investigated. The total protein in the methanolic extract was 36% lower and showed 10% fewer sulphydryl groups than that from the water extract. However, the radical scavenging activity of the water extract was four-fold greater than for the methanolic extract. The proteins of both of these extracts were separated by two-dimensional electrophoresis, and selected proteins were identified using mass spectrometry. The majority of these identified proteins were intracellular proteins, such as those involved in oxidative stress responses and osmotic stress responses, and proteins with hydrolase and dehydrogenase activities. These proteins are also common to most organisms, and included putative uncharacterized proteins.

Heme A synthase in bacteria depends on one pair of cysteinyls for activity.

Heme A is a prosthetic group unique for cytochrome a-type respiratory oxidases in mammals, plants and many microorganisms. The poorly understood integral membrane protein heme A synthase catalyzes the synthesis of heme A from heme O. In bacteria, but not in mitochondria, this enzyme contains one or two pairs of cysteine residues that are present in predicted hydrophilic polypeptide loops on the extracytoplasmic side of the membrane. We used heme A synthase from the eubacterium Bacillus subtilis and the hyperthermophilic archeon Aeropyrum pernix to investigate the functional role of these cysteine residues. Results with B. subtilis amino acid substituted proteins indicated the pair of cysteine residues in the loop connecting transmembrane segments I and II as being essential for catalysis but not required for binding of the enzyme substrate, heme O. Experiments with isolated A. pernix and B. subtilis heme A synthase demonstrated that a disulfide bond can form between the cysteine residues in the same loop and also between loops showing close proximity of the two loops in the folded enzyme protein. Based on the findings, we propose a classification scheme for the four discrete types of heme A synthase found so far in different organisms and propose that essential cysteinyls mediate transfer of reducing equivalents required for the oxygen-dependent catalysis of heme A synthesis from heme O.

Aeropyrum pernix membrane topology of protein VKOR promotes protein disulfide bond formation in two subcellular compartments.

Disulfide bonds confer stability and activity to proteins. Bioinformatic approaches allow predictions of which organisms make protein disulfide bonds and in which subcellular compartments disulfide bond formation takes place. Such an analysis, along with biochemical and protein structural data, suggests that many of the extremophile Crenarachaea make protein disulfide bonds in both the cytoplasm and the cell envelope. We have sought to determine the oxidative folding pathways in the sequenced genomes of the Crenarchaea, by seeking homologues of the enzymes known to be involved in disulfide bond formation in bacteria. Some Crenarchaea have two homologues of the cytoplasmic membrane protein VKOR, a protein required in many bacteria for the oxidation of bacterial DsbAs. We show that the two VKORs of Aeropyrum pernix assume opposite orientations in the cytoplasmic membrane, when expressed in E. coli. One has its active cysteines oriented toward the E. coli periplasm (ApVKORo) and the other toward the cytoplasm (ApVKORi). Furthermore, the ApVKORo promotes disulfide bond formation in the E. coli cell envelope, while the ApVKORi promotes disulfide bond formation in the E. coli cytoplasm via a co-expressed archaeal protein ApPDO. Amongst the VKORs from different archaeal species, the pairs of VKORs in each species are much more closely related to each other than to the VKORs of the other species. The results suggest two independent occurrences of the evolution of the two topologically inverted VKORs in archaea. Our results suggest a mechanistic basis for the formation of disulfide bonds in the cytoplasm of Crenarchaea.

Crystal structure of translation initiation factor 5B from the crenarchaeon Aeropyrum pernix.

Initiation factor 5B (IF5B) is a universally conserved translational GTPase that catalyzes ribosomal subunit joining. In eukaryotes, IF5B directly interacts via a groove in its domain IV with initiation factor 1A (IF1A), another universally conserved initiation factor, to accomplish efficient subunit joining. Here, we have determined the first structure of a crenarchaeal IF5B, which revealed that the archaea-specific region of IF5B (helix α15) binds and occludes the groove of domain IV. Therefore, archaeal IF5B cannot access IF1A in the same manner as eukaryotic IF5B. This fact suggests that different relationships between IF5B and IF1A exist in archaea and eukaryotes.