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Structure of the Aeropyrum pernix L7Ae multifunctional protein and insight into its extreme thermostability.

ABSTRACT: Archaeal ribosomal protein L7Ae is a multifunctional RNA-binding protein that directs post-transcriptional modification of archaeal RNAs. The L7Ae protein from Aeropyrum pernix (Ap L7Ae), a member of the Crenarchaea, was found to have an extremely high melting temperature (>383?K). The crystal structure of Ap L7Ae has been determined to a resolution of 1.56?Å. The structure of Ap L7Ae was compared with the structures of two homologs: hyperthermophilic Methanocaldococcus jannaschii L7Ae and the mesophilic counterpart mammalian 15.5?kD protein. The primary stabilizing feature in the Ap L7Ae protein appears to be the large number of ion pairs and extensive ion-pair network that connects secondary-structural elements. To our knowledge, Ap L7Ae is among the most thermostable single-domain monomeric proteins presently observed.

SUBMITTER: Bhuiya MW

PROVIDER: S-EPMC3758144 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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Structure of the Aeropyrum pernix L7Ae multifunctional protein and insight into its extreme thermostability.

Bhuiya Mohammad Wadud MW Suryadi Jimmy J Zhou Zholi Z Brown Bernard Andrew BA

Acta crystallographica. Section F, Structural biology and crystallization communications 20130819 Pt 9

Archaeal ribosomal protein L7Ae is a multifunctional RNA-binding protein that directs post-transcriptional modification of archaeal RNAs. The L7Ae protein from Aeropyrum pernix (Ap L7Ae), a member of the Crenarchaea, was found to have an extremely high melting temperature (>383 K). The crystal structure of Ap L7Ae has been determined to a resolution of 1.56 Å. The structure of Ap L7Ae was compared with the structures of two homologs: hyperthermophilic Methanocaldococcus jannaschii L7Ae and the m ...[more]

PMID: 23989144

Similar Datasets

Aeropyrum pernix

Project description:Hyperthermophilic archaeon

| PRJNA35133 | ENA

Crystal structure of archaeal chromatin protein Alba2-double-stranded DNA complex from Aeropyrum pernix K1.

Project description:All thermophilic and hyperthermophilic archaea encode homologs of dimeric Alba (Sac10b) proteins that bind cooperatively at high density to DNA. Here, we report the 2.0 Å resolution crystal structure of an Alba2 (Ape10b2)-dsDNA complex from Aeropyrum pernix K1. A rectangular tube-like structure encompassing duplex DNA reveals the positively charged residues in the monomer-monomer interface of each dimer packing on either side of the bound dsDNA in successive minor grooves. The extended hairpin loop connecting strands β3 and β4 undergoes significant conformational changes upon DNA binding to accommodate the other Alba2 dimer during oligomerization. Mutational analysis of key interacting residues confirmed the specificity of Alba2-dsDNA interactions.

| S-EPMC3322981 | biostudies-literature

Provirus induction in hyperthermophilic archaea: characterization of Aeropyrum pernix spindle-shaped virus 1 and Aeropyrum pernix ovoid virus 1.

Project description:By in silico analysis, we have identified two putative proviruses in the genome of the hyperthermophilic archaeon Aeropyrum pernix, and under special conditions of A. pernix growth, we were able to induce their replication. Both viruses were isolated and characterized. Negatively stained virions of one virus appeared as pleomorphic spindle-shaped particles, 180 to 210 nm by 40 to 55 nm, with tails of heterogeneous lengths in the range of 0 to 300 nm. This virus was named Aeropyrum pernix spindle-shaped virus 1 (APSV1). Negatively stained virions of the other virus appeared as slightly irregular oval particles with one pointed end, while in cryo-electron micrographs, the virions had a regular oval shape and uniform size (70 by 55 nm). The virus was named Aeropyrum pernix ovoid virus 1 (APOV1). Both viruses have circular, double-stranded DNA genomes of 38,049 bp for APSV1 and 13,769 bp for APOV1. Similarities to proteins of other archaeal viruses were limited to the integrase and Dna1-like protein. We propose to classify APOV1 into the family Guttaviridae.

| S-EPMC3187419 | biostudies-literature

Crystal structure of novel dye-linked L-proline dehydrogenase from hyperthermophilic archaeon Aeropyrum pernix.

Project description:Two types of dye-linked L-proline dehydrogenase (PDH1, α4β4-type hetero-octamer, and PDH2, αβγδ-type heterotetramer) have been identified so far in hyperthermophilic archaea. Here, we report the crystal structure of a third type of L-proline dehydrogenase, found in the aerobic hyperthermophilic archaeon Aeropyrum pernix, whose structure (homodimer) is much simpler than those of previously studied L-proline dehydrogenases. The structure was determined at a resolution of 1.92 Å. The asymmetric unit contained one subunit, and a crystallographic 2-fold axis generated the functional dimer. The overall fold of the subunit showed similarity to that of the PDH1 β-subunit, which is responsible for catalyzing L-proline dehydrogenation. However, the situation at the subunit-subunit interface of the A. pernix enzyme was totally different from that in PDH1. The presence of additional surface elements in the A. pernix enzyme contributes to a unique dimer association. Moreover, the C-terminal Leu(428), which is provided by a tail extending from the FAD-binding domain, shielded the active site, and an L-proline molecule was entrapped within the active site cavity. The K(m) value of a Leu(428) deletion mutant for L-proline was about 800 times larger than the K(m) value of the wild-type enzyme, although the k(cat) values did not differ much between the two enzymes. This suggests the C-terminal Leu(428) is not directly involved in catalysis, but it is essential for maintaining a high affinity for the substrate. This is the first description of an LPDH structure with L-proline bound, and it provides new insight into the substrate binding of LPDH.

| S-EPMC3370190 | biostudies-literature

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

Project description: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.

| S-EPMC5845738 | biostudies-literature

Structure and characterization of the 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from Aeropyrum pernix.

Project description:The first enzyme in the shikimic acid biosynthetic pathway, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS), varies significantly in size and complexity in the bacteria and plants that express it. The DAH7PS from the archaebacterium Aeropyrum pernix (DAH7PS(Ap)) is among the smallest and least complex of the DAH7PS enzymes, leading to the hypothesis that DAH7PS(Ap) would not be subject to feedback regulation by shikimic acid pathway products. We overexpressed DAH7PS(Ap) in Escherichia coli, purified it, and characterized its enzymatic activity. We then solved its X-ray crystal structure with a divalent manganese ion and phosphoenolpyruvate bound (PDB ID: 1VS1). DAH7PS(Ap) is a homodimeric metalloenzyme in solution. Its enzymatic activity increases dramatically above 60 °C, with optimum activity at 95 °C. Its pH optimum at 60 °C is 5.7. DAH7PS(Ap) follows Michaelis-Menten kinetics at 60 °C, with a K(M) for erythrose 4-phosphate of 280 μM, a K(M) for phosphoenolpyruvate of 891 μM, and a k(cat) of 1.0 s(-1). None of the downstream products of the shikimate biosynthetic pathway we tested inhibited the activity of DAH7PS(Ap). The structure of DAH7PS(Ap) is similar to the structures of DAH7PS from Thermatoga maritima (PDB ID: 3PG8) and Pyrococcus furiosus (PDB ID: 1ZCO), and is consistent with its designation as an unregulated DAH7PS.

| S-EPMC3260417 | biostudies-literature

Disassembly of the ring-type decameric structure of peroxiredoxin from Aeropyrum pernix K1 by amino acid mutation.

Project description:The quaternary structure of peroxiredoxin from Aeropyrum pernix K1 (ApPrx) is a decamer, in which five homodimers are assembled in a pentagonal ring through hydrophobic interactions. In this study, we determined the amino acid (AA) residues of ApPrx crucial for forming the decamer using AA mutations. The ApPrx0Cys mutant, wherein all cysteine residues were mutated to serine, was prepared as a model protein to remove the influence of the redox states of the cysteines on its assembling behavior. The boundary between each homodimer of ApPrx0Cys contains characteristic aromatic AA residues forming hydrophobic interactions: F46, F80, W88, W210, and W211. We found that a single mutation of F46, F80, or W210 to alanine completely disassembled the ApPrx0Cys decamer to homodimers, which was clarified by gel-filtration chromatography and dynamic light scattering measurements. F46A, F80A, and W210A mutants lacked only one aromatic ring compared with ApPrx0Cys, indicating that the assembly is very sensitive to the surface structure of the protein. X-ray structures revealed two mechanisms of disassembly of the ApPrx decamer: loss of hydrophobicity between homodimers and flip of the arm domain. The AA residues targeted in this study are well conserved in ring-type Prx proteins, suggesting the importance of these residues in the assembly. This study demonstrates the sensitivity and modifiability of peroxiredoxin assembly by a simple AA mutation.

| S-EPMC7184774 | biostudies-literature

Structure of Aeropyrum pernix fibrillarin in complex with natively bound S-adenosyl-L-methionine at 1.7?A resolution.

Project description:Fibrillarin is the key methyltransferase associated with the C/D class of small nuclear ribonucleoproteins (snRNPs) and participates in the preliminary step of pre-ribosomal rRNA processing. This molecule is found in the fibrillar regions of the eukaryotic nucleolus and is involved in methylation of the 2'-O atom of ribose in rRNA. Human fibrillarin contains an N-terminal GAR domain, a central RNA-binding domain comprising an RNP-2-like superfamily consensus sequence and a catalytic C-terminal helical domain. Here, Aeropyrum pernix fibrillarin is described, which is homologous to the C-terminal domain of human fibrillarin. The protein was crystallized with an S-adenosyl-L-methionine (SAM) ligand bound in the active site. The molecular structure of this complex was solved using X-ray crystallography at a resolution of 1.7?Å using molecular replacement with fibrillarin structural homologs. The structure shows the atomic details of SAM and its active-site interactions; there are a number of conserved residues that interact directly with the cofactor. Notably, the adenine ring of SAM is stabilized by ?-? interactions with the conserved residue Phe110 and by electrostatic interactions with the Asp134, Ala135 and Gln157 residues. The ?-? interaction appears to play a critical role in stabilizing the association of SAM with fibrillarin. Furthermore, comparison of A. pernix fibrillarin with homologous structures revealed different orientations of Phe110 and changes in ?-helix 6 of fibrillarin and suggests key differences in its interactions with the adenine ring of SAM in the active site and with the C/D RNA. These differences may play a key role in orienting the SAM ligand for catalysis as well as in the assembly of other ribonucleoproteins and in the interactions with C/D RNA.

| S-EPMC3412760 | biostudies-literature

Enzymatic degradation of PrPSc by a protease secreted from Aeropyrum pernix K1.

Project description:BackgroundAn R30 fraction from the growth medium of Aeropyrum pernix was analyzed for the protease that can digest the pathological prion protein isoform (PrP(Sc)) from different species (human, bovine, deer and mouse).Methodology/principal findingsDegradation of the PrP(Sc) isoform by the R30 fraction and the purified protease was evaluated using the 6H4 anti-PrP monoclonal antibody. Fragments from the N-terminal and C-terminal of PrP(Sc) were also monitored by Western blotting using the EB8 anti-PrP monoclonal antibody, and by dot blotting using the C7/5 anti-PrP monoclonal antibody, respectively. For detection of smaller peptides from incomplete digestion of PrP(Sc), the EB8 monoclonal antibody was used after precipitation with sodium phosphotungstate. Characterization of the purified active protease from the R30 fraction was achieved, through purification by fast protein liquid chromatography, and identification by tandem mass spectrometry the serine metalloprotease pernisine. SDS-PAGE and zymography show the purified pernisine plus its proregion with a molecular weight of ca. 45 kDa, and the mature purified pernisine as ca. 23 kDa. The purified pernisine was active between 58 °C and 99 °C, and between pH 3.5 and 8.0. The temperature and pH optima of the enzymatic activity of the purified pernisine in the presence of 1 mM CaCl(2) were 105 °C ± 0.5 °C and pH 6.5 ± 0.2, respectively.Conclusions/significanceOur study has identified and characterized pernisine as a thermostable serine metalloprotease that is secreted from A. pernix and that can digest the pathological prion protein PrP(Sc).

| S-EPMC3386259 | biostudies-literature

Outside the unusual cell wall of the hyperthermophilic archaeon Aeropyrum pernix K1.

Project description:In contrast to the extensively studied eukaryal and bacterial protein secretion systems, comparatively less is known about how and which proteins cross the archaeal cell membrane. To identify secreted proteins of the hyperthermophilic archaeon Aeropyrum pernix K1 we used a proteomics approach to analyze the extracellular and cell surface protein fractions. The experimentally obtained data comprising 107 proteins were compared with the in silico predicted secretome. Because of the lack of signal peptide and cellular localization prediction tools specific for archaeal species, programs trained on eukaryotic and/or Gram-positive and Gram-negative bacterial signal peptide data sets were used. PSortB Gram-negative and Gram-positive analysis predicted 21 (1.2% of total ORFs) and 24 (1.4% of total ORFs) secreted proteins, respectively, from the entire A. pernix K1 proteome, 12 of which were experimentally identified in this work. Six additional proteins were predicted to follow non-classical secretion mechanisms using SecP algorithms. According to at least one of the two PSortB predictions, 48 proteins identified in the two fractions possess an unknown localization site. In addition, more than half of the proteins do not contain signal peptides recognized by current prediction programs. This suggests that known mechanisms only partly describe archaeal protein secretion. The most striking characteristic of the secretome was the high number of transport-related proteins identified from the ATP-binding cassette (ABC), tripartite ATP-independent periplasmic, ATPase, small conductance mechanosensitive ion channel (MscS), and dicarboxylate amino acid-cation symporter transporter families. In particular, identification of 21 solute-binding receptors of the ABC superfamily of the 24 predicted in silico confirms that ABC-mediated transport represents the most frequent strategy adopted by A. pernix for solute translocation across the cell membrane.

| S-EPMC2773722 | biostudies-literature

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