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Episodic ataxias (EAs) are rare neurological conditions affecting the nervous system and typically leading to motor impairment. EA6 is linked to the mutation of a highly conserved proline into an arginine in the glutamate transporter EAAT1. In vitro studies showed that this mutation leads to a reduction in the substrates transport and an increase in the anion conductance. It was hypothesised that the structural basis of these opposed functional effects might be the straightening of transmembrane helix 5, which is kinked in the wild-type protein. In this study, we present the functional and structural implications of the mutation P208R in the archaeal homologue of glutamate transporters Glt Tk . We show that also in Glt Tk the P208R mutation leads to reduced aspartate transport activity and increased anion conductance, however a cryo-EM structure reveals that the kink is preserved. The arginine side chain of the mutant points towards the lipidic environment, where it may engage in interactions with the phospholipids, thereby potentially interfering with the transport cycle and contributing to stabilisation of an anion conducting state., (© 2023. The Author(s).)

Histones, ubiquitous in eukaryotes as DNA-packing proteins, find their evolutionary origins in archaea. Unlike the characterized histone proteins of a number of methanogenic and themophilic archaea, previous research indicated that HpyA, the sole histone encoded in the model halophile Halobacterium salinarum, is not involved in DNA packaging. Instead, it was found to have widespread but subtle effects on gene expression and to maintain wild type cell morphology. However, the precise function of halophilic histone-like proteins remain unclear. Here we use quantitative phenotyping, genetics, and functional genomics to investigate HpyA function. These experiments revealed that HpyA is important for growth and rod-shaped morphology in reduced salinity. HpyA preferentially binds DNA at discrete genomic sites under low salt to regulate expression of ion uptake, particularly iron. HpyA also globally but indirectly activates other ion uptake and nucleotide biosynthesis pathways in a salt-dependent manner. Taken together, these results demonstrate an alternative function for an archaeal histone-like protein as a transcriptional regulator, with its function tuned to the physiological stressors of the hypersaline environment., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

CsaA is known to function as a protein secretion chaperone in bacteria. Homologs of CsaA are also found in archaea while they are absent in eukaryotes. This paper presents the biophysical, biochemical analysis and crystallographic structure determination of CsaA from a thermoacidophilic archaeon Picrophilus torridus (PtCsaA). The PtCsaA appears to prevent the aggregation of heat denatured Bovine Carbonic Anhydrase II (BCAII). Differential denaturation of PtCsaA by guanidine hydrochloride (Gdn-HCl) and urea indicates the stabilization of the protein via salt bridges. Denaturant mediated decrease in 8-Anilinonaphthalene-1-sulfonic acid (ANS) binding and shift in wavelength signifies the partial unfolding of the protein molecule and exposure of hydrophobic patches to solvent on denaturation. The crystal structure of PtCsaA was solved to a resolution of 1.7 Å. The structure of PtCsaA appears to be similar to bacterial CsaA in architecture. Docking of a six amino acid peptide in the substrate binding pocket of PtCsaA suggests conservation in the substrate binding cavity. Residues involved in the formation of the binding cavity and hydrogen bonds responsible for the dimerization of PtCsaA were compared with those observed in the structure of Bacillus subtilis CsaA. The similarities and differences in electrostatic surface potential of the substrate binding cavities in bacterial CsaA and PtCsaA are discussed., (Copyright © 2021. Published by Elsevier B.V.)

The origin of the eukaryotic cell is one of the greatest mysteries in modern biology. Eukaryotic-wide specific biological processes arose in the lost ancestors of eukaryotes. These distinctive features, such as the actin cytoskeleton, define what it is to be a eukaryote. Recent sequencing, characterization, and isolation of Asgard archaea have opened an intriguing window into the pre-eukaryotic cell. Firstly, sequencing of anaerobic sediments identified a group of uncultured organisms, Asgard archaea, which contain genes with homology to eukaryotic signature genes. Secondly, characterization of the products of these genes at the protein level demonstrated that Asgard archaea have related biological processes to eukaryotes. Finally, the isolation of an Asgard archaeon has produced a model organism in which the morphological consequences of the eukaryotic-like processes can be studied. Here, we consider the consequences for the Asgard actin cytoskeleton and for the evolution of a regulated actin system in the archaea-to-eukaryotic transition., Competing Interests: Conflict of interest statement Nothing declared., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Cmr-β is a type III-B CRISPR-Cas complex that, upon target RNA recognition, unleashes a multifaceted immune response against invading genetic elements, including single-stranded DNA (ssDNA) cleavage, cyclic oligoadenylate synthesis, and also a unique UA-specific single-stranded RNA (ssRNA) hydrolysis by the Cmr2 subunit. Here, we present the structure-function relationship of Cmr-β, unveiling how binding of the target RNA regulates the Cmr2 activities. Cryoelectron microscopy (cryo-EM) analysis revealed the unique subunit architecture of Cmr-β and captured the complex in different conformational stages of the immune response, including the non-cognate and cognate target-RNA-bound complexes. The binding of the target RNA induces a conformational change of Cmr2, which together with the complementation between the 5' tag in the CRISPR RNAs (crRNA) and the 3' antitag of the target RNA activate different configurations in a unique loop of the Cmr3 subunit, which acts as an allosteric sensor signaling the self- versus non-self-recognition. These findings highlight the diverse defense strategies of type III complexes., Competing Interests: Declaration of Interests G.M. and S.S. are co-founders and board members of Twelve Bio., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Cells require a sensory system and a motility structure to achieve directed movement. Bacteria and archaea possess rotating filamentous motility structures that work in concert with the sensory chemotaxis system. This allows microorganisms to move along chemical gradients. The central response regulator protein CheY can bind to the motor of the motility structure, the flagellum in bacteria, and the archaellum in archaea. Both motility structures have a fundamentally different protein composition and structural organization. Yet, both systems receive input from the chemotaxis system. So far, it was unknown how the signal is transferred from the archaeal CheY to the archaellum motor to initiate motor switching. We applied a fluorescent microscopy approach in the model euryarchaeon Haloferax volcanii and shed light on the sequence order in which signals are transferred from the chemotaxis system to the archaellum. Our findings indicate that the euryarchaeal-specific ArlCDE are part of the archaellum motor and that they directly receive input from the chemotaxis system via the adaptor protein CheF. Hence, ArlCDE are an important feature of the archaellum of euryarchaea, are essential for signal transduction during chemotaxis and represent the archaeal switch complex., (© 2020 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Sulfolobus acidocaldarius is the closest experimentally tractable archaeal relative of eukaryotes and, despite lacking obvious cyclin-dependent kinase and cyclin homologs, has an ordered eukaryote-like cell cycle with distinct phases of DNA replication and division. Here, in exploring the mechanism of cell division in S. acidocaldarius , we identify a role for the archaeal proteasome in regulating the transition from the end of one cell cycle to the beginning of the next. Further, we identify the archaeal ESCRT-III homolog, CdvB, as a key target of the proteasome and show that its degradation triggers division by allowing constriction of the CdvB1:CdvB2 ESCRT-III division ring. These findings offer a minimal mechanism for ESCRT-III-mediated membrane remodeling and point to a conserved role for the proteasome in eukaryotic and archaeal cell cycle control., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Thermostability plays an important role in the application of L-asparaginase in the pharmaceutical and food industries. Understanding the key residues and structures that influence thermostability in L-asparaginase is necessary to obtain suitable L-asparaginase candidates. In this study, special residues and structures that altered thermostability in thermophilic L-asparaginase and non-thermophilic L-asparaginase II were identified. Interchanging these special residues and structures of L-asparaginases from the four strains, that is, Pyrococcus yayanosii CH1 (PYA), Thermococcus gammatolerans (TGA), Bacillus subtilis (BSA II), and Escherichia coli (ECA II), revealed the 51st and 298th residues of PYA (corresponding to 57th, 305th residues of ECA II) as the key residues responsible for thermal stability of thermophilic L-asparaginase and non-thermophilic L-asparaginase II. Moreover, the C terminal tightness, loop rigidity, and low surface charge around activity sites were of great significance to the thermostability of L-asparaginase. This study therefore revealed the crucial amino acid residues and structures responsible for the difference in thermostability of the thermophilic and non-thermophilic L-asparaginase and provides a reference for engineering thermostability in L-asparaginase II.

It is unknown how the archaellum-the rotary propeller used by Archaea for motility-works. To further understand the molecular mechanism by which the hexameric ATPase motor protein FlaI drives rotation of the membrane-embedded archaellar motor, we determined motor torque by imposition of various loads on Halobacterium salinarum archaella. Markers of different sizes were attached to single archaella, and their trajectories were quantified using three-dimensional tracking and high-speed recording. We show that rotation slows as the viscous drag of markers increases, but torque remains constant at 160 pN·nm independent of rotation speed. Notably, the estimated work done in a single rotation is twice the expected energy that would come from hydrolysis of six ATP molecules in the hexamer, indicating that more ATP molecules are required for one rotation of archaellum. To reconcile the apparent contradiction, we suggest a new and general model for the mechanism of ATP-driven rotary motors., Competing Interests: Competing interestsThe authors declare no competing interests.

Main Conclusion: Cotton GaTOP6B is involved in cellular endoreduplication and a positive response to drought stress via promoting plant leaf and root growth. Drought is deemed as one of adverse conditions that could cause substantial reductions in crop yields worldwide. Since cotton exhibits a moderate-tolerant phenotype under water-deficit conditions, the plant could therefore be used to characterize potential new genes regulating drought tolerance in crop plants. In this work, GaTOP6B, encoding DNA topoisomerase VI subunit B, was identified in Asian cotton (Gossypium arboreum). Virus-induced gene silencing (VIGS) and overexpression (OE) were used to investigate the biological function of GaTOP6B in G. arboreum and Arabidopsis thaliana under drought stress. The GaTOP6B-silencing plants showed a reduced ploidy level, and displayed a compromised tolerance phenotype including lowered relative water content (RWC), decreased proline content and antioxidative enzyme activity, and an increased malondialdehyde (MDA) content under drought stress. GaTOP6B-overexpressing Arabidopsis lines, however, had increased ploidy levels, and were more tolerant to drought treatment, associated with improved RWC maintenance, higher proline accumulation, and reduced stomatal aperture under drought stress. Transcriptome analysis showed that genes involved in the processes like cell cycle, transcription and signal transduction, were substantially up-regulated in GaTOP6B-overexpressing Arabidopsis, promoting plant growth and development. More specifically, under drought stress, the genes involved in the biosynthesis of secondary metabolites such as phenylpropanoid, starch and sucrose were selectively enhanced to improve tolerance in plants. Taken together, the results demonstrated that GaTOP6B could coordinately regulate plant leaf and root growth via cellular endoreduplication, and positively respond to drought stress. Thus, GaTOP6B could be a competent candidate gene for improvement of drought tolerance in crop species.

Members of the ancient family of Argonaute (Ago) proteins are present in all domains of life. The common feature of Ago proteins is the ability to bind small nucleic acid guides and use them for sequence-specific recognition-and sometimes cleavage-of complementary targets. While eukaryotic Ago (eAgo) proteins are key players in RNA interference and related pathways, the properties and functions of these proteins in archaeal and bacterial species have just started to emerge. We undertook comprehensive exploration of prokaryotic Ago (pAgo) proteins in sequenced genomes and revealed their striking diversity in comparison with eAgos. Many pAgos contain divergent variants of the conserved domains involved in interactions with nucleic acids, while having extra domains that are absent in eAgos, suggesting that they might have unusual specificities in the nucleic acid recognition and cleavage. Many pAgos are associated with putative nucleases, helicases, and DNA binding proteins in the same gene or operon, suggesting that they are involved in target processing. The great variability of pAgos revealed by our analysis opens new ways for exploration of their functions in host cells and for their use as potential tools in genome editing. IMPORTANCE The eukaryotic Ago proteins and the RNA interference pathways they are involved in are widely used as a powerful tool in research and as potential therapeutics. In contrast, the properties and functions of prokaryotic Ago (pAgo) proteins have remained poorly understood. Understanding the diversity and functions of pAgos holds a huge potential for discovery of new cellular pathways and novel tools for genome manipulations. Only few pAgos have been characterized by structural or biochemical approaches, while previous genomic studies discovered about 300 proteins in archaeal and eubacterial genomes. Since that time the number of bacterial strains with sequenced genomes has greatly expanded, and many previously sequenced genomes have been revised. We undertook comprehensive analysis of pAgo proteins in sequenced genomes and almost tripled the number of known genes of this family. Our research thus forms a foundation for further experimental characterization of pAgo functions that will be important for understanding of the basic biology of these proteins and their adoption as a potential tool for genome engineering in the future., (Copyright © 2018 Ryazansky et al.)

Archaea have many unique physiological features of which the lipid composition of their cellular membranes is the most striking. Archaeal ether-linked isoprenoidal membranes can occur as bilayers or monolayers, possess diverse polar head groups, and a multiplicity of ring structures in the isoprenoidal cores. These lipid structures are proposed to provide protection from the extreme temperature, pH, salinity, and nutrient-starved conditions that many archaea inhabit. However, many questions remain regarding the synthesis and physiological role of some of the more complex archaeal lipids. In this study, we identify a radical S -adenosylmethionine (SAM) protein in Sulfolobus acidocaldarius required for the synthesis of a unique cyclopentyl head group, known as calditol. Calditol-linked glycerol dibiphytanyl glycerol tetraethers (GDGTs) are membrane spanning lipids in which calditol is ether bonded to the glycerol backbone and whose production is restricted to a subset of thermoacidophilic archaea of the Sulfolobales order within the Crenarchaeota phylum. Several studies have focused on the enzymatic mechanism for the synthesis of the calditol moiety, but to date no protein that catalyzes this reaction has been discovered. Phylogenetic analyses of this putative calditol synthase (Cds) reveal the genetic potential for calditol-GDGT synthesis in phyla other than the Crenarchaeota, including the Korarchaeota and Marsarchaeota. In addition, we identify Cds homologs in metagenomes predominantly from acidic ecosystems. Finally, we demonstrate that deletion of calditol synthesis renders S. acidocaldarius sensitive to extremely low pH, indicating that calditol plays a critical role in protecting archaeal cells from acidic stress., Competing Interests: The authors declare no conflict of interest.

In archaeal translation initiation, a preinitiation complex (PIC) made up of aIF1, aIF1A, the ternary complex (TC, e/aIF2-GTP-Met-tRNAiMet) and mRNA bound to the small ribosomal subunit is responsible for start codon selection. Many archaeal mRNAs contain a Shine-Dalgarno (SD) sequence allowing the PIC to be prepositioned in the vicinity of the start codon. Nevertheless, cryo-EM studies have suggested local scanning to definitely establish base pairing of the start codon with the tRNA anticodon. Here, using fluorescence anisotropy, we show that aIF1 and mRNA have synergistic binding to the Pyrococcus abyssi 30S. Stability of 30S:mRNA:aIF1 strongly depends on the SD sequence. Further, toeprinting experiments show that aIF1-containing PICs display a dynamic conformation with the tRNA not firmly accommodated in the P site. AIF1-induced destabilization of the PIC is favorable for proofreading erroneous initiation complexes. After aIF1 departure, the stability of the PIC increases reflecting initiator tRNA fully base-paired to the start codon. Altogether, our data support the idea that some of the main events governing start codon selection in eukaryotes and archaea occur within a common structural and functional core. However, idiosyncratic features in loop 1 sequence involved in 30S:mRNA binding suggest adjustments of e/aIF1 functioning in the two domains.

Protein synthesis is a complex and highly coordinated process requiring many different protein factors as well as various types of nucleic acids. All translation machinery components require multiple maturation events to be functional. These include post-transcriptional and post-translational modification steps and methylations are the most frequent among these events. In eukaryotes, Trm112, a small protein (COG2835) conserved in all three domains of life, interacts and activates four methyltransferases (Bud23, Trm9, Trm11 and Mtq2) that target different components of the translation machinery (rRNA, tRNAs, release factors). To clarify the function of Trm112 in archaea, we have characterized functionally and structurally its interaction network using Haloferax volcanii as model system. This led us to unravel that methyltransferases are also privileged Trm112 partners in archaea and that this Trm112 network is much more complex than anticipated from eukaryotic studies. Interestingly, among the identified enzymes, some are functionally orthologous to eukaryotic Trm112 partners, emphasizing again the similarity between eukaryotic and archaeal translation machineries. Other partners display some similarities with bacterial methyltransferases, suggesting that Trm112 is a general partner for methyltransferases in all living organisms.

Deregulation of cellular proteostasis due to the failure of the ubiquitin proteasome system to dispose of misfolded aggregation-prone proteins is a hallmark of various neurodegenerative diseases in humans. Microorganisms have evolved to survive massive protein misfolding and aggregation triggered by heat shock using their protein-unfolding ATPases (unfoldases) from the Hsp100 family. Because the Hsp100 chaperones are absent in homoeothermic mammals, we hypothesized that the vulnerability of mammalian neurons to misfolded proteins could be mitigated by expressing a xenogeneic unfoldase. To test this idea, we expressed proteasome-activating nucleotidase (PAN), a protein-unfolding ATPase from thermophilic Archaea , which is homologous to the 19S eukaryotic proteasome and similar to the Hsp100 family chaperones in rod photoreceptors of mice. We found that PAN had no obvious effect in healthy rods; however, it effectively counteracted protein-misfolding retinopathy in Gγ 1 knock-out mice. We conclude that archaeal PAN can rescue a protein-misfolding neurodegenerative disease, likely by recognizing misfolded mammalian proteins. SIGNIFICANCE STATEMENT This study demonstrates successful therapeutic application of an archaeal molecular chaperone in an animal model of neurodegenerative disease. Introducing the archaeal protein-unfolding ATPase proteasome-activating nucleotidase (PAN) into the retinal photoreceptors of mice protected these neurons from the cytotoxic effect of misfolded proteins. We propose that xenogeneic protein-unfolding chaperones could be equally effective against other types of neurodegenerative diseases of protein-misfolding etiology., (Copyright © 2018 the authors 0270-6474/18/387248-07$15.00/0.)

While bacteria and eukaryotes show distinct mechanisms of DNA damage response (DDR) regulation, investigation of ultraviolet (UV)-responsive expression in a few archaea did not yield any conclusive evidence for an archaeal DDR regulatory network. Nevertheless, expression of Orc1-2, an ortholog of the archaeal origin recognition complex 1/cell division control protein 6 (Orc1/Cdc6) superfamily proteins was strongly activated in Sulfolobus solfataricus and Sulfolobus acidocaldarius upon UV irradiation. Here, a series of experiments were conducted to investigate the possible functions of Orc1-2 in DNA damage repair in Sulfolobus islandicus. Study of DDR in Δorc1-2 revealed that Orc1-2 deficiency abolishes DNA damage-induced differential expression of a large number of genes and the mutant showed hypersensitivity to DNA damage treatment. Reporter gene and DNase I footprinting assays demonstrated that Orc1-2 interacts with a conserved hexanucleotide motif present in several DDR gene promoters and regulates their expression. Manipulation of orc1-2 expression by promoter substitution in this archaeon revealed that a high level of orc1-2 expression is essential but not sufficient to trigger DDR. Together, these results have placed Orc1-2 in the heart of the archaeal DDR regulation, and the resulting Orc1-2-centered regulatory circuit represents the first DDR network identified in Archaea, the third domain of life.

The cellular replicative DNA polymerases cannot initiate DNA synthesis without a priming 3' OH. During DNA replication, this is supplied in the context of a short RNA primer molecule synthesized by DNA primase. The primase of archaea and eukaryotes, despite having varying subunit compositions, share sequence and structural homology. Intriguingly, archaeal primase has been demonstrated to possess the ability to synthesize DNA de novo, a property shared with the eukaryotic PrimPol enzymes. The dual RNA and DNA synthetic capabilities of the archaeal DNA primase have led to the proposal that there may be a sequential hand-off between these synthetic modes of primase. In the current work, we dissect the functional interplay between DNA and RNA synthetic modes of primase. In addition, we determine the key determinants that govern primer length definition by the archaeal primase. Our results indicate a primer measuring system that functions akin to a caliper., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Molecular chaperones promote the correct folding of proteins in aggregation-prone cellular environments by stabilizing nascent polypeptide chains and providing appropriate folding conditions. Prefoldins (PFDs) are molecular chaperones found in archaea and eukaryotes, generally characterized by a unique jellyfish-like hexameric structure consisting of a rigid beta-barrel backbone with protruding flexible coiled-coils. Unlike eukaryotic PFDs that mainly interact with cytoskeletal components, archaeal PFDs can stabilize a wide range of substrates; such versatility reflects PFD's role as a key element in archaeal chaperone systems, which often lack general nascent-chain binding chaperone components such as Hsp70. While archaeal PFDs mainly exist as hexameric complexes, their structural diversity ranges from tetramers to filamentous oligomers. PFDs bind and stabilize nonnative proteins using varying numbers of coiled-coils, and subsequently transfer the substrate to a group II chaperonin (CPN) for refolding. The distinct structure and specific function of archaeal PFDs have been exploited for a broad range of applications in biotechnology; furthermore, a filament-forming variant of PFD has been used to fabricate nanoscale architectures of defined shapes, demonstrating archaeal PFDs' potential applicability in nanotechnology.

During sexual reproduction haploid gametes are generated out of diploid mother cells. This ploidy reduction is accomplished during meiosis and, in most species, relies on the occurrence of homologous recombination that is triggered by the induction of a large number of DNA double strand breaks (DSBs). The mechanism by which such DSBs are generated without provoking massive DNA breakdown in gamete mother cells is still poorly understood. However, the recent characterisation, in plants and in mammals, of a new component of the meiotic DSB forming machinery, defining a meiotic-specific TOPOVIB-Like protein family, has established a clear connection between the meiotic DSB activity and topoisomerases, enzymes that modify the DNA topology by introducing transient DSBs., (© 2017 médecine/sciences – Inserm.)

Organisms within the archaeal domain of life possess a simplified version of the eukaryotic DNA replication machinery. While some archaea possess a bacterial-like mode of DNA replication with single origins of replication per chromosome, the majority of species characterized to date possess chromosomes with multiple replication origins. Genetic, structural, and biochemical studies have revealed the nature of archaeal origin specification. Recent work has begun to shed light on the mechanisms of replication initiation in these organisms.

Acrylic acid and propionic acid are important chemicals requiring affordable, renewable production solutions. Here, we metabolically engineered Escherichia coli with genes encoding components of the 3-hydroxypropionate/4-hydroxybutyrate cycle from Metallosphaera sedula for conversion of glucose to acrylic and propionic acids. To construct an acrylic acid-producing pathway in E. coli, heterologous expression of malonyl-CoA reductase (MCR), malonate semialdehyde reductase (MSR), 3-hydroxypropionyl-CoA synthetase (3HPCS), and 3-hydroxypropionyl-CoA dehydratase (3HPCD) from M. sedula was accompanied by overexpression of succinyl-CoA synthetase (SCS) from E. coli. The engineered strain produced 13.28 ± 0.12 mg/L of acrylic acid. To construct a propionic acid-producing pathway, the same five genes were expressed, with the addition of M. sedula acryloyl-CoA reductase (ACR). The engineered strain produced 1430 ± 30 mg/L of propionic acid. This approach can be expanded to synthesize many important organic chemicals, creating new opportunities for the production of chemicals by carbon dioxide fixation.

Voltage-dependent potassium (Kv) channels allow for the selective permeability of potassium ions in a membrane potential dependent manner, playing crucial roles in neurotransmission and muscle contraction. Kv channel is a tetramer, in which each subunit possesses a voltage-sensing domain (VSD) and a pore domain (PD). Although several lines of evidence indicated that membrane depolarization is sensed as the movement of helix S4 of the VSD, the detailed voltage-sensing mechanism remained elusive, due to the difficulty of structural analyses at resting potential. In this study, we conducted a comprehensive disulfide locking analysis of the VSD using 36 double Cys mutants, in order to identify the proximal residue pairs of the VSD in the presence or absence of a membrane potential. An intramolecular SS-bond was formed between 6 Cys pairs under both polarized and depolarized environment, and one pair only under depolarized environment. The multiple conformations captured by the SS-bond can be divided by two states, up and down, where S4 lies on the extracellular and intracellular sides of the membrane, respectively, with axial rotation of 180°. The transition between these two states is caused by the S4 translocation of 12 Å, enabling allosteric regulation of the gating at the PD.

Eukaryotic and archaeal translation initiation complexes have a common structural core comprising e/aIF1, e/aIF1A, the ternary complex (TC, e/aIF2-GTP-Met-tRNA i Met ) and mRNA bound to the small ribosomal subunit. e/aIF2 plays a crucial role in this process but how this factor controls start codon selection remains unclear. Here, we present cryo-EM structures of the full archaeal 30S initiation complex showing two conformational states of the TC. In the first state, the TC is bound to the ribosome in a relaxed conformation with the tRNA oriented out of the P site. In the second state, the tRNA is accommodated within the peptidyl (P) site and the TC becomes constrained. This constraint is compensated by codon/anticodon base pairing, whereas in the absence of a start codon, aIF2 contributes to swing out the tRNA. This spring force concept highlights a mechanism of codon/anticodon probing by the initiator tRNA directly assisted by aIF2.

Water and protein dynamics on a nanometer scale were measured by quasi-elastic neutron scattering in the piezophile archaeon Thermococcus barophilus and the closely related pressure-sensitive Thermococcus kodakarensis, at 0.1 and 40 MPa. We show that cells of the pressure sensitive organism exhibit higher intrinsic stability. Both the hydration water dynamics and the fast protein and lipid dynamics are reduced under pressure. In contrast, the proteome of T. barophilus is more pressure sensitive than that of T. kodakarensis. The diffusion coefficient of hydration water is reduced, while the fast protein and lipid dynamics are slightly enhanced with increasing pressure. These findings show that the coupling between hydration water and cellular constituents might not be simply a master-slave relationship. We propose that the high flexibility of the T. barophilus proteome associated with reduced hydration water may be the keys to the molecular adaptation of the cells to high hydrostatic pressure.

The motor of the membrane-anchored archaeal motility structure, the archaellum, contains FlaX, FlaI and FlaH. FlaX forms a 30 nm ring structure that acts as a scaffold protein and was shown to interact with the bifunctional ATPase FlaI and FlaH. However, the structure and function of FlaH has been enigmatic. Here we present structural and functional analyses of isolated FlaH and archaellum motor subcomplexes. The FlaH crystal structure reveals a RecA/Rad51 family fold with an ATP bound on a conserved and exposed surface, which presumably forms an oligomerization interface. FlaH does not hydrolyze ATP in vitro, but ATP binding to FlaH is essential for its interaction with FlaI and for archaellum assembly. FlaH interacts with the C-terminus of FlaX, which was earlier shown to be essential for FlaX ring formation and to mediate interaction with FlaI. Electron microscopy reveals that FlaH assembles as a second ring inside the FlaX ring in vitro. Collectively these data reveal central structural mechanisms for FlaH interactions in mediating archaellar assembly: FlaH binding within the FlaX ring and nucleotide-regulated FlaH binding to FlaI form the archaellar basal body core., Competing Interests: The authors declare no conflict of interests., (© 2015 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

The COMBREX database (COMBREX-DB; combrex.bu.edu) is an online repository of information related to (i) experimentally determined protein function, (ii) predicted protein function, (iii) relationships among proteins of unknown function and various types of experimental data, including molecular function, protein structure, and associated phenotypes. The database was created as part of the novel COMBREX (COMputational BRidges to EXperiments) effort aimed at accelerating the rate of gene function validation. It currently holds information on ∼ 3.3 million known and predicted proteins from over 1000 completely sequenced bacterial and archaeal genomes. The database also contains a prototype recommendation system for helping users identify those proteins whose experimental determination of function would be most informative for predicting function for other proteins within protein families. The emphasis on documenting experimental evidence for function predictions, and the prioritization of uncharacterized proteins for experimental testing distinguish COMBREX from other publicly available microbial genomics resources. This article describes updates to COMBREX-DB since an initial description in the 2011 NAR Database Issue., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The Cmr complex is the multisubunit effector complex of the type III-B clustered regularly interspaced short palindromic repeats (CRISPR)-Cas immune system. The Cmr complex recognizes a target RNA through base pairing with the integral CRISPR RNA (crRNA) and cleaves the target at multiple regularly spaced locations within the complementary region. To understand the molecular basis of the function of this complex, we have assembled information from electron microscopic and X-ray crystallographic structural studies and mutagenesis of a complete Pyrococcus furiosus Cmr complex. Our findings reveal that four helically packed Cmr4 subunits, which make up the backbone of the Cmr complex, act as a platform to support crRNA binding and target RNA cleavage. Interestingly, we found a hook-like structural feature associated with Cmr4 that is likely the site of target RNA binding and cleavage. Our results also elucidate analogies in the mechanisms of crRNA and target molecule binding by the distinct Cmr type III-A and Cascade type I-E complexes., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

The Cmr complex is an RNA-guided endonuclease that cleaves foreign RNA targets as part of the CRISPR prokaryotic defense system. We investigated the molecular architecture of the P. furiosus Cmr complex using an integrative structural biology approach. We determined crystal structures of P. furiosus Cmr1, Cmr2, Cmr4, and Cmr6 and combined them with known structural information to interpret the cryo-EM map of the complex. To support structure determination, we obtained residue-specific interaction data using protein crosslinking and mass spectrometry. The resulting pseudoatomic model reveals how the superhelical backbone of the complex is defined by the polymerizing principles of Cmr4 and Cmr5 and how it is capped at the extremities by proteins of similar folds. The inner surface of the superhelix exposes conserved residues of Cmr4 that we show are required for target-cleavage activity. The structural and biochemical data thus identify Cmr4 as the conserved endoribonuclease of the Cmr complex., (Copyright © 2014 Elsevier Inc. All rights reserved.)

In all organisms of the three living domains (Bacteria, Archaea, Eucarya) chromosome-associated proteins play a key role in genome functional organization. They not only compact and shape the genome structure, but also regulate its dynamics, which is essential to allow complex genome functions. Elucidation of chromatin composition and regulation is a critical issue in biology, because of the intimate connection of chromatin with all the essential information processes (transcription, replication, recombination, and repair). Chromatin proteins include architectural proteins and DNA topoisomerases, which regulate genome structure and remodelling at two hierarchical levels. This review is focussed on architectural proteins and topoisomerases from hyperthermophilic Archaea. In these organisms, which live at high environmental temperature (>80 °C <113 °C), chromatin proteins and modulation of the DNA secondary structure are concerned with the problem of DNA stabilization against heat denaturation while maintaining its metabolic activity.

With the aim of unraveling their potential involvement in the regulation of nitrogen metabolism in Methanosarcina mazei strain Gö1, we characterized five genes that are differentially transcribed in response to changing nitrogen availability and encoding putative transcriptional regulators. Study of the respective mutant strains under nitrogen-limited conditions revealed a growth delay for M. mazei MM0444::pac and MM1708::pac, and strongly reduced diazotrophic growth for MM0872::pac, whereas the absence of MM2441 or MM2525 did not affect growth behaviour. Transcriptome analyses further demonstrated that only MM1708 - encoding a CxxCG zinc finger protein - plays a regulatory role in nitrogen metabolism, most likely by specifically enhancing transcription of the N2 fixation (nif) operon under nitrogen-limited conditions. In agreement with this, a palindromic binding motif was predicted in silico in the nifH promoter region, nine nucleotides upstream of the BRE box, and confirmed to bind purified maltose-binding protein-MM1708 by electromobility shift assays. As MM1708 itself is under the control of the global nitrogen repressor NrpR, this adds a secondary level to the transcriptional regulation of the nif genes, and is most likely crucial for maximal nif induction under nitrogen-limited conditions. This is in accordance with the finding that protein expression of NifH is highly reduced in the absence of MM1708 under nitrogen-limited conditions. On the basis of our findings, we hypothesize that, in M. mazei, nitrogen fixation is controlled by a hierarchical network of two transcriptional regulators, the global nitrogen repressor NrpR, and the newly identified activator NrpA (MM1708), thereby providing tight control of N2 fixation., (© 2014 FEBS.)

Available structures of HAMP domains suggest rotation as one potential mechanism in intraprotein signal transduction. It has been proposed that in poly-HAMP modules the signal sign is inverted with each additional HAMP. We examined signal transduction through the HAMP tandem domain from the phototaxis transducer of the halophilic archaeon Natronomonas pharaonis in membrane-bound chimeras consisting of the Escherichia coli chemotaxis receptor for serine, Tsr, as an input and the mycobacterial adenylyl cyclase Rv3645 as an output domain, i.e. the basic chimera was 'Tsr-NpHAMP tandem-Rv3645 cyclase'. Neither of the NpHAMP units alone nor the NpHAMP tandem transduced a serine signal. After five targeted point mutations in the first α-helix of NpHAMP1 , the non-functional NpHAMP modules combined into a functional HAMP tandem. 1 mm serine significantly inhibited cyclase activity (-35%; IC50  = 30 μm) in disagreement with the structure-based predictions. Surprisingly, replacement of NpAS11 in the tandem by the respective AS1 from HAMPT sr resulted in signal inversion, i.e. serine activated cyclase (+129%; EC50  = 10 μm). Examination of 48 mutants of AS11 in the HAMP tandem including two residues of a putative N-terminal control cable identified five residues in NpAS11 which probably define different ground states of the output domain and thus affect the sign of signal output. The data question the predicted HAMP rotation as the predominant mechanism of intraprotein signal transduction and point to as yet unrecognized conformational motions of HAMP domains in intraprotein signaling., (© 2014 FEBS.)

Archaea live under different environmental conditions, such as high salinity, extreme pHs and cold or hot temperatures. How energy is conserved under such harsh environmental conditions is a major question in cellular bioenergetics of archaea. The key enzymes in energy conservation are the archaeal A1AO ATP synthases, a class of ATP synthases distinct from the F1FO ATP synthase ATP synthase found in bacteria, mitochondria and chloroplasts and the V1VO ATPases of eukaryotes. A1AO ATP synthases have distinct structural features such as a collar-like structure, an extended central stalk, and two peripheral stalks possibly stabilizing the A1AO ATP synthase during rotation in ATP synthesis/hydrolysis at high temperatures as well as to provide the storage of transient elastic energy during ion-pumping and ATP synthesis/-hydrolysis. High resolution structures of individual subunits and subcomplexes have been obtained in recent years that shed new light on the function and mechanism of this unique class of ATP synthases. An outstanding feature of archaeal A1AO ATP synthases is their diversity in size of rotor subunits and the coupling ion used for ATP synthesis with H(+), Na(+) or even H(+) and Na(+) using enzymes. The evolution of the H(+) binding site to a Na(+) binding site and its implications for the energy metabolism and physiology of the cell are discussed., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Many leguminous plants have a unique ability to reset and alter the fate of differentiated root cortical cells to form new organs of nitrogen-fixing root nodules during legume-Rhizobium symbiosis. Recent genetic studies on the role of cytokinin signaling reveal that activation of cytokinin signaling is crucial to the nodule organogenesis process. However, the genetic mechanism underlying the initiation of nodule organogenesis is poorly understood due to the low number of genes that have been identified. Here, we have identified a novel nodulation-deficient mutant named vagrant infection thread 1 (vag1) after suppressor mutant screening of spontaneous nodule formation 2, a cytokinin receptor gain-of-function mutant in Lotus japonicus. The VAG1 gene encodes a protein that is putatively orthologous to Arabidopsis ROOT HAIRLESS 1/HYPOCOTYL 7, a component of the plant DNA topoisomerase VI that is involved in the control of endoreduplication. Nodule phenotype of the vag1 mutant shows that VAG1 is required for the ploidy-dependent cell growth of rhizobial-infected cells. Furthermore, VAG1 mediates the onset of endoreduplication in cortical cells during early nodule development, which may be essential for the initiation of cortical cell proliferation that leads to nodule primordium formation. In addition, cortical infection is severely impaired in the vag1 mutants, whereas the epidermal infection threads formation is normal. This suggests that the VAG1-mediated endoreduplication of cortical cells may be required for the guidance of symbiotic bacteria to host meristematic cells., (© 2014. Published by The Company of Biologists Ltd.)

The 20S core particle proteasome is a molecular machine playing an important role in cellular function by degrading protein substrates that no longer are required or that have become damaged. Regulation of proteasome activity occurs, in part, through a gating mechanism controlling the sizes of pores at the top and bottom ends of the symmetric proteasome barrel and restricting access to catalytic sites sequestered in the lumen of the structure. Although atomic resolution models of both open and closed states of the proteasome have been elucidated, the mechanism by which gates exchange between these states remains to be understood. Here, this is investigated by using magnetization transfer NMR spectroscopy focusing on the 20S proteasome core particle from Thermoplasma acidophilum. We show from viscosity-dependent proteasome gating kinetics that frictional forces originating from random solvent motions are critical for driving the gating process. Notably, a small effective hydrodynamic radius (EHR; <4Å) is obtained, providing a picture in which gate exchange proceeds through many steps involving only very small segment sizes. A small EHR further suggests that the kinetics of gate interconversion will not be affected appreciably by large viscogens, such as macromolecules found in the cell, so long as they are inert. Indeed, measurements in cell lysate reveal that the gate interconversion rate decreases only slightly, demonstrating that controlled studies in vitro provide an excellent starting point for understanding regulation of 20S core particle function in complex, biologically relevant environments.

Background: Although many hyperthermophilic endoglucanases have been reported from archaea and bacteria, a complete survey and classification of all sequences in these species from disparate evolutionary groups, and the relationship between their molecular structures and functions are lacking. The completion of several high-quality gene or genome sequencing projects provided us with the unique opportunity to make a complete assessment and thorough comparative analysis of the hyperthermophilic endoglucanases encoded in archaea and bacteria., Results: Structure alignment of the 19 hyperthermophilic endoglucanases from archaea and bacteria which grow above 80°C revealed that Gly30, Pro63, Pro83, Trp115, Glu131, Met133, Trp135, Trp175, Gly227 and Glu229 are conserved amino acid residues. In addition, the average percentage composition of residues cysteine and histidine of 19 endoglucanases is only 0.28 and 0.74 while it is high in thermophilic or mesophilic one. It can be inferred from the nodes that there is a close relationship among the 19 protein from hyperthermophilic bacteria and archaea based on phylogenetic analysis. Among these conserved amino acid residues, as far as Cel12B concerned, two Glu residues might be the catalytic nucleophile and proton donor, Gly30, Pro63, Pro83 and Gly227 residues might be necessary to the thermostability of protein, and Trp115, Met133, Trp135, Trp175 residues is related to the binding of substrate. Site-directed mutagenesis results reveal that Pro63 and Pro83 contribute to the thermostability of Cel12B and Met133 is confirmed to have role in enhancing the binding of substrate., Conclusions: The conserved acids have been shown great importance to maintain the structure, thermostability, as well as the similarity of the enzymatic properties of those proteins. We have made clear the function of these conserved amino acid residues in Cel12B protein, which is helpful in analyzing other undetailed molecular structure and transforming them with site directed mutagenesis, as well as providing the theoretical basis for degrading cellulose from woody and herbaceous plants.

The 26S proteasome is the main protease in eukaryotes. Proteolysis occurs within the cylindrical 20S proteasome that is constitutively expressed in most tissues. However, three tissue-specific versions of the 20S proteasome have been discovered to date. The immunoproteasome is optimized to process antigens and it directs the differentiation of T helper (Th) cells. The thymoproteasome is selectively expressed in cortical epithelial cells of the thymus where it plays an essential role in the positive selection of T lymphocytes. Finally, the spermatoproteasome is found in the testes where it is required during spermatogenesis. Here, we outline how tissue-specific proteasomes adapt to functional needs in their respective tissues and how their selective inhibition may be used to interfere with autoimmune diseases and cancer., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The complex of sensory rhodopsin II (NpSRII) with its cognate transducer (NpHtrII) mediates negative phototaxis in halobacteria Natronomonas pharaonis. Upon light activation NpSRII triggers, by means of NpHtrII, a signal transduction chain homologous to the two component system in eubacterial chemotaxis. Here we report on the crystal structure of the ground state of the mutant NpSRII-D75N/NpHtrII complex in the space group I212121. Mutations of this aspartic acid in light-driven proton pumps dramatically modify or/and inhibit protein functions. However, in vivo studies show that the similar D75N mutation retains functionality of the NpSRII/NpHtrII complex. The structure provides the molecular basis for the explanation of the unexpected observation that the wild and the mutant complexes display identical physiological response on light excitation., (Copyright © 2013 Elsevier B.V. All rights reserved.)

CRISPR (clustered regularly interspaced short palindromic repeats)-mediated virus defense based on small RNAs is a hallmark of archaea and also found in many bacteria. Archaeal genomes and, in particular, organisms of the extremely thermoacidophilic genus Sulfolobus, carry extensive CRISPR loci each with dozens of sequence signatures (spacers) able to mediate targeting and degradation of complementary invading nucleic acids. The diversity of CRISPR systems and their associated protein complexes indicates an extensive functional breadth and versatility of this adaptive immune system. Sulfolobus solfataricus and S. islandicus represent two of the best characterized genetic model organisms in the archaea not only with respect to the CRISPR system. Here we address and discuss in a broader context particularly recent progress made in understanding spacer recruitment from foreign DNA, production of small RNAs, in vitro activity of CRISPR-associated protein complexes and attack of viruses and plasmids in in vivo test systems.

Superfamily ATPases in type IV pili, type 2 secretion, and archaella (formerly archaeal flagella) employ similar sequences for distinct biological processes. Here, we structurally and functionally characterize prototypical superfamily ATPase FlaI in Sulfolobus acidocaldarius, showing FlaI activities in archaeal swimming-organelle assembly and movement. X-ray scattering data of FlaI in solution and crystal structures with and without nucleotide reveal a hexameric crown assembly with key cross-subunit interactions. Rigid building blocks form between N-terminal domains (points) and neighboring subunit C-terminal domains (crown ring). Upon nucleotide binding, these six cross-subunit blocks move with respect to each other and distinctly from secretion and pilus ATPases. Crown interactions and conformations regulate assembly, motility, and force direction via a basic-clamp switching mechanism driving conformational changes between stable, backbone-interconnected moving blocks. Collective structural and mutational results identify in vivo functional components for assembly and motility, phosphate-triggered rearrangements by ATP hydrolysis, and molecular predictors for distinct ATPase superfamily functions., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The ASCTs (alanine, serine, and cysteine transporters) belong to the solute carrier family 1 (SLC1), which also includes the human glutamate transporters (excitatory amino acid transporters, EAATs) and the prokaryotic aspartate transporter GltPh. Despite the high degree of amino acid sequence identity between family members, ASCTs function quite differently from the EAATs and GltPh. The aim of this study was to mutate ASCT1 to generate a transporter with functional properties of the EAATs and GltPh, to further our understanding of the structural basis for the different transport mechanisms of the SLC1 family. We have identified three key residues involved in determining differences between ASCT1, the EAATs and GltPh. ASCT1 transporters containing the mutations A382T, T459R, and Q386E were expressed in Xenopus laevis oocytes, and their transport and anion channel functions were investigated. A382T and T459R altered the substrate selectivity of ASCT1 to allow the transport of acidic amino acids, particularly l-aspartate. The combination of A382T and T459R within ASCT1 generates a transporter with a similar profile to that of GltPh, with preference for l-aspartate over l-glutamate. Interestingly, the amplitude of the anion conductance activated by the acidic amino acids does not correlate with rates of transport, highlighting the distinction between these two processes. Q386E impaired the ability of ASCT1 to bind acidic amino acids at pH 5.5; however, this was reversed by the additional mutation A382T. We propose that these residues differences in TM7 and TM8 combine to determine differences in substrate selectivity between members of the SLC1 family.

Previously, Lrp-like transcriptional regulator LysM from the hyperthermoacidophilic crenarchaeon Sulfolobus solfataricus was proposed to have a single target, the lysWXJK operon of lysine biosynthesis, and a single effector molecule, l-lysine. Here we identify ∼70 novel binding sites for LysM in the S. solfataricus genome with a LysM-specific nanobody-based chromatin immunoprecipitation assay coupled to microarray hybridization (ChIP-chip) and in silico target site prediction using an energy-based position weight matrix, and validate these findings with in vitro binding. LysM binds to intergenic and coding regions, including promoters of various amino acid biosynthesis and transport genes. We confirm that l-lysine is the most potent effector molecule that reduces, but does not completely abolish, LysM binding, and show that several other amino acids and derivatives, including d-lysine, l-arginine, l-homoarginine, l-glutamine and l-methionine and branched-chain amino acids l-leucine, l-isoleucine and l-valine, significantly affect DNA-binding properties of LysM. Therefore, it appears from this study that LysM is a much more versatile regulator than previously thought, and that it uses a variety of amino acids to sense nutritional quality of the environment and to modulate expression of the metabolic machinery of Sulfolobus accordingly.

The 26S proteasome is thought to be a homogenous complex, consisting of a 20S proteolytic core and a 19S regulatory particle that is required for its activation. Two groups have recently reported the activation of archeal 20S by a p97-related double-ring AAA+ ATPase complex, in a similar fashion to that reported for 19S. Since p97 is found in eukaryotes, the existence of a parallel setting in higher organisms is intriguing. Herein, we present supporting data and hypothesize that in eukaryotes, p97 and CSN form a promiscuous, hence hard-to-detect, "alternative cap", enabling the prompt and precise elimination of particular substrates., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

The conserved protein sequence motifs present in all prokaryotic proteomes, "omnipresent motifs," presumably, correspond to the earliest proteins of the Last Universal Cellular Ancestor, from which all the proteomes have descended. Fifteen proteomes, each representing one of the total 15 diverse phyla of 131 Eubacteria and Archea, from which the omnipresent elements have been originally derived, are exhaustively screened. All those proteins which harbor the omnipresent motifs are identified. Six "omnipresent" protein types are revealed which are located in all 15 proteomes: ABC cassettes, FtsH proteases, translation initiation factors, translation elongation factors, isoleucyl-tRNA synthases, and RNA polymerases β'. In addition to the omnipresent motifs, these proteins also contain other highly conserved motifs, standing for additional modules of the proteins. Remarkably, the identified tentative earliest proteins are responsible for only three basic functions: supply of monomers (ABC transporters and proteases), protein synthesis (initiation and elongation factors, aminoacyl-tRNA synthases), and RNA synthesis (polymerases). No enzymes involved in metabolic activities are present in the list of the earliest proteins derived by this approach. Some of the omnipresent sequence motifs are found, indeed, in the metabolic enzymes (e.g. NTP binding motifs), but these enzymes do not make a sequence matching collection of 15 sequences, i.e. they are not omnipresent. Future analysis of less conserved sequence motifs may reveal at what degree of conservation (stage of evolution) the metabolic enzymes could have entered the scene.

This review highlights the finding that ubiquitin-like (Ubl) proteins of archaea (termed SAMPs) function not only as sulfur carriers but also as protein modifiers. UbaA (an E1 ubiquitin-activating enzyme homolog of archaea) is required for the SAMPs to be covalently attached to proteins. The SAMPs and UbaA are also needed to form sulfur-containing biomolecules (e.g., thiolated tRNA and molybdenum cofactor). These findings provide a new perspective on how Ubl proteins can serve as both sulfur carriers and protein modifiers in the absence of canonical E2 ubiquitin conjugating or E3 ubiquitin ligase enzyme homologs., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Genetic techniques for the Archaea have undergone a rapid expansion in complexity, resulting in increased exploration of the role of Archaea in the environment and detailed analyses of the molecular physiology and information-processing systems in the third domain of life. Complementary gains in describing the ever-increasing diversity of archaeal organisms have allowed these techniques to be leveraged in new and imaginative ways to elucidate shared and unique aspects of archaeal diversity and metabolism. In this review, we introduce the four archaeal clades for which advanced genetic techniques are available--the methanogens, halophiles, Sulfolobales, and Thermococcales--with the aim of providing an overall profile of the advantages and disadvantages of working within each clade, as essentially all of the genetically accessible archaeal organisms require unique culturing techniques that present real challenges. We discuss the full repertoire of techniques possible within these clades while highlighting the recent advances that have been made by taking advantage of the most prominent techniques and approaches.

Wybutosine and its derivatives are found in position 37 of tRNA encoding Phe in eukaryotes and archaea. They are believed to play a key role in the decoding function of the ribosome. The second step in the biosynthesis of wybutosine is catalyzed by TYW1 protein, which is a member of the well established class of metalloenzymes called "Radical-SAM." These enzymes use a [4Fe-4S] cluster, chelated by three cysteines in a CX(3)CX(2)C motif, and S-adenosyl-L-methionine (SAM) to generate a 5'-deoxyadenosyl radical that initiates various chemically challenging reactions. Sequence analysis of TYW1 proteins revealed, in the N-terminal half of the enzyme beside the Radical-SAM cysteine triad, an additional highly conserved cysteine motif. In this study we show by combining analytical and spectroscopic methods including UV-visible absorption, Mössbauer, EPR, and HYSCORE spectroscopies that these additional cysteines are involved in the coordination of a second [4Fe-4S] cluster displaying a free coordination site that interacts with pyruvate, the second substrate of the reaction. The presence of two distinct iron-sulfur clusters on TYW1 is reminiscent of MiaB, another tRNA-modifying metalloenzyme whose active form was shown to bind two iron-sulfur clusters. A possible role for the second [4Fe-4S] cluster in the enzyme activity is discussed.

Background: Recent studies of Haloferax volcanii have begun to elucidate the steps of N-glycosylation in Archaea, where this universal post-translational modification remains poorly described. In Hfx. volcanii, a series of Agl proteins catalyzes the assembly and attachment of a N-linked pentasaccharide to the S-layer glycoprotein. Although roles have been assigned to the majority of Agl proteins, others await description. In the following, the contribution of AglR to N-glycosylation was addressed., Methods: A combination of bioinformatics, gene deletion, mass spectrometry and metabolic radiolabeling served to show a role for AglR in archaeal N-glycosylation at both the dolichol phosphate and reporter glycoprotein levels., Results: The modified behavior of the S-layer glycoprotein isolated from cells lacking AglR points to an involvement of this protein in N-glycosylation. In cells lacking AglR, glycan-charged dolichol phosphate, including mannose-charged dolichol phosphate, accumulates. At the same time, the S-layer glycoprotein does not incorporate mannose, the final subunit of the N-linked pentasaccharide decorating this protein. AglR is a homologue of Wzx proteins, annotated as flippases responsible for delivering lipid-linked O-antigen precursor oligosaccharides across the bacterial plasma membrane during lipopolysaccharide biogenesis., Conclusions: The effects resulting from aglR deletion are consistent with AglR interacting with dolichol phosphate-mannose, possibly acting as a dolichol phosphate-mannose flippase or contributing to such activity., General Significance: Little is known of how lipid-linked oligosaccharides are translocated across membrane during N-glycosylation. The possibility of Hfx. volcanii AglR mediating or contributing to flippase activity could help address this situation., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The complex of sensory rhodopsin II (SRII) and its cognate transducer HtrII (2:2 SRII-HtrII complex) consists of a photoreceptor and its signal transducer, respectively, associated with negative phototaxis in extreme halophiles. In this study to investigate how photoexcitation in SRII affects the structures of the complex, we conducted two series of molecular dynamics simulations of the complex of SRII and truncated HtrII (residues 1-136) of Natronomonas pharaonis linked with a modeled HAMP domain in the lipid bilayer using the two crystal structures of the ground state and the M-intermediate state as the starting structures. The simulation results showed significant enhancements of the structural differences observed between the two crystal structures. Helix F of SRII showed an outward motion, and the C-terminal end of transmembrane domain 2 (TM2) in HtrII rotated by ∼10°. The most significant structural changes were observed in the overall orientations of the two SRII molecules, closed in the ground state and open in the M-state. This change was attributed to substantial differences in the structure of the four-helix bundle of the HtrII dimer causing the apparent rotation of TM2. These simulation results established the structural basis for the various experimental observations explaining the structural differences between the ground state and the M-intermediate state.

Archaea, along with Bacteria and Eukarya, are the three domains of life. In all living cells, chromatin proteins serve a crucial role in maintaining the integrity of the structure and function of the genome. An array of small, abundant and basic DNA-binding proteins, considered candidates for chromatin proteins, has been isolated from the Euryarchaeota and the Crenarchaeota, the two major phyla in Archaea. While most euryarchaea encode proteins resembling eukaryotic histones, crenarchaea appear to synthesize a number of unique DNA-binding proteins likely involved in chromosomal organization. Several of these proteins (e.g., archaeal histones, Sac10b homologs, Sul7d, Cren7, CC1, etc.) have been extensively studied. However, whether they are chromatin proteins and how they function in vivo remain to be fully understood. Future investigation of archaeal chromatin proteins will lead to a better understanding of chromosomal organization and gene expression in Archaea and provide valuable information on the evolution of DNA packaging in cellular life.