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.

Switch of the interactions between the ribosomal stalk and EF1A in the GTP- and GDP-bound conformations.

Translation elongation factor EF1A delivers aminoacyl-tRNA to the ribosome in a GTP-bound form, and is released from the ribosome in a GDP-bound form. This association/dissociation cycle proceeds efficiently via a marked conformational change in EF1A. EF1A function is dependent on the ribosomal "stalk" protein of the ribosomal large subunit, although the precise mechanism of action of the stalk on EF1A remains unclear. Here, we clarify the binding mode of archaeal stalk aP1 to GTP-bound aEF1A associated with aPelota. Intriguingly, the C-terminal domain (CTD) of aP1 binds to aEF1A•GTP with a similar affinity to aEF1A•GDP. We have also determined the crystal structure of the aP1-CTD•aEF1A•GTP•aPelota complex at 3.0 Šresolution. The structure shows that aP1-CTD binds to a space between domains 1 and 3 of aEF1A. Biochemical analyses show that this binding is crucial for protein synthesis. Comparison of the structures of aP1-CTD•aEF1A•GTP and aP1-CTD•aEF1A•GDP demonstrates that the binding mode of aP1 changes markedly upon a conformational switch between the GTP- and GDP-bound forms of aEF1A. Taking into account biochemical data, we infer that aP1 employs its structural flexibility to bind to aEF1A before and after GTP hydrolysis for efficient protein synthesis.

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.

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.

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.

Alteration of molecular assembly of peroxiredoxins from hyperthermophilic archaea.

Peroxiredoxin from Pyrococcus horikoshii (PhPrx) is a decameric protein formed by ring-type assembly of five dimers. To engineer the quaternary structure of PhPrx, we created a mutant PhPrx (PhPrx6m) by introducing six point mutations designed to dissociate PhPrx into dimers. Although PhPrx6m was a dimer in solution, the six dimers assembled into a dodecamer following crystallization. In the crystal structure, PhPrx6m was overoxidized, and the peroxidatic cysteine was in sulfonic acid form and two cysteines in the C-terminal region were linked by an intramolecular disulfide bond. Thus, we characterized the wild-type PhPrx overoxidized by hydrogen peroxide (PhPrxPer). Analytical ultracentrifugation showed that PhPrxPer had a higher molecular mass in solution than PhPrx. This was confirmed by analysis of the crystal structure of PhPrxPer, which was found to form a ring-type dodecamer composed of six dimers. The monomeric structures of PhPrx6m and PhPrxPer differed from that of PhPrx in the relative orientation of two domains, reflecting the number of dimers in the ring-type assembly. Unlike PhPrx, homologous peroxiredoxin from Aeropyrum pernix (ApPrx) did not undergo hexameric association. This property can be explained by the stronger connection between the two domains in ApPrx due to its C-terminal extension relative to PhPrx.

Specific electrical capacitance and voltage breakdown as a function of temperature for different planar lipid bilayers.

The breakdown voltage and specific electrical capacitance of planar lipid bilayers formed from lipids isolated from the membrane of archaeon Aeropyrum pernix K1 as a function of temperature were studied and compared with data obtained previously in MD simulation studies. Temperature dependence of breakdown voltage and specific electrical capacitance was measured also for dipalmitoylphosphatidylcholine (DPPC) bilayers and bilayers formed from mixture of diphytanoylphosphocholine (DPhPC) and DPPC in ratio 80:20. The breakdown voltage of archaeal lipids planar lipid bilayers is more or less constant until 50°C, while at higher temperatures a considerable drop is observed, which is in line with the results from MD simulations. The breakdown voltage of DPPC planar lipid bilayer at melting temperature is considerably higher than in the gel phase. Specific electrical capacitance of planar lipid bilayers formed from archaeal lipids is approximately constant for temperatures up to 40°C and then gradually decreases. The difference with MD simulation predictions is discussed. Specific electrical capacitance of DPPC planar lipid bilayers in fluid phase is 1.75 times larger than that of the gel phase and it follows intermediated phases before phase transition. Increase in specific electrical capacitance while approaching melting point of DPPC is visible also for DPhPC:DPPC mixture.

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.

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.

Mobility Measurements Probe Conformational Changes in Membrane Proteins due to Tension.

The function of membrane-embedded proteins such as ion channels depends crucially on their conformation. We demonstrate how conformational changes in asymmetric membrane proteins may be inferred from measurements of their diffusion. Such proteins cause local deformations in the membrane, which induce an extra hydrodynamic drag on the protein. Using membrane tension to control the magnitude of the deformations, and hence the drag, measurements of diffusivity can be used to infer-via an elastic model of the protein-how conformation is changed by tension. Motivated by recent experimental results [Quemeneur et al., Proc. Natl. Acad. Sci. U.S.A. 111, 5083 (2014)], we focus on KvAP, a voltage-gated potassium channel from Aeropyrum pernix. The conformation of KvAP is found to change considerably due to tension, with its "walls," where the protein meets the membrane, undergoing significant angular strains. The torsional stiffness is determined to be 26.8k(B)T per radian at room temperature. This has implications for both the structure and the function of such proteins in the environment of a tension-bearing membrane.

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

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.

Reconstitution of a Kv channel into lipid membranes for structural and functional studies.

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels.

Cytotoxicity and uptake of archaeosomes prepared from Aeropyrum pernix lipids.

Archaeon Aeropyrum pernix K1 is an obligate aerobic hyperthermophilic organism with C25,25-archeol membrane lipids with head groups containing inositol. Interactions of archaeosomes, liposomes prepared from lipids of A. pernix, with mammalian cells in vitro were studied. In vitro cytotoxicity was tested on five different cell lines: rodent mouse melanoma cells (B16-F1) and Chinese hamster ovary (CHO) cells, and three human cell lines-epithelial colorectal adenocarcinoma cells (CACO-2), liver hepatocellular carcinoma cell line (Hep G2) and endothelial umbilical vein cell line (EA.hy926). Archaeosomes were nontoxic to human Hep G2, CACO-2 and mildly toxic to rodent CHO and B16-F1 cells but showed strong cytotoxic effect on EA.hy926 cells. Confocal microscopy revealed that archaeosomes are taken up by endocytosis. The uptake of archaeosomes and the release of loaded calcein are more prominent in EA.hy926 cells, which is in line with high toxicity toward these cells. The mechanisms of uptake, release and action in these cells as well as in vivo functioning have to be further studied for possible targeted drug delivery.

Coupled motions during dynamics reveal a tunnel toward the active site regulated by the N-terminal α-helix in an acylaminoacyl peptidase.

Acylaminoacyl peptidase (AAP) subfamily belongs to the prolyl oligopeptidase (POP) family of serine-proteases. There is a great interest in the definition of molecular mechanisms related to the activity and substrate recognition of these complex multi-domain enzymes. The active site relies at the interface between the C-terminal catalytic domain and the ß-propeller domain, whose N-terminal region acts as a bridge to the hydrolase domain. In AAP, the N-terminal extension is characterized by a structurally conserved α1-helix, which is known to affect thermal stability and thermal dependence of the catalytic activity. In the present contribution, results from hundreds nanosecond all-atom molecular dynamics simulations, along with analyses of the networks of cross-correlated motions of a member of the AAP subfamily are discussed. The MD investigation identifies a tunnel that from the surrounding of the N-terminal α1-helix bring to the catalytic site. This cavity seems to be regulated by conformational changes of the α1-helix itself during the dynamics. The evidence here provided can be a useful guide for a better understanding of the mechanistic aspects related to AAP activity, but also for drug design purposes.

Structure of Aeropyrum pernix fibrillarin in complex with natively bound S-adenosyl-L-methionine at 1.7†Šresolution.

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 Šusing 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 π-π interactions with the conserved residue Phe110 and by electrostatic interactions with the Asp134, Ala135 and Gln157 residues. The π-π 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 α-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.

Effect of growth medium pH of Aeropyrum pernix on structural properties and fluidity of archaeosomes.

The influence of pH (6.0; 7.0; 8.0) of the growth medium of Aeropyrum pernix K1 on the structural organization and fluidity of archaeosomes prepared from a polar-lipid methanol fraction (PLMF) was investigated using fluorescence anisotropy and electron paramagnetic resonance (EPR) spectroscopy. Fluorescence anisotropy of the lipophilic fluorofore 1,6-diphenyl-1,3,5-hexatriene and empirical correlation time of the spin probe methylester of 5-doxylpalmitate revealed gradual changes with increasing temperature for the pH. A similar effect has been observed by using the trimethylammonium-6-diphenyl-1,3,5-hexatriene, although the temperature changes were much smaller. As the fluorescence steady-state anisotropy and the empirical correlation time obtained directly from the EPR spectra alone did not provide detailed structural information, the EPR spectra were analysed by computer simulation. This analysis showed that the archaeosome membranes are heterogeneous and composed of several regions with different modes of spin-probe motion at temperatures below 70°C. At higher temperatures, these membranes become more homogeneous and can be described by only one spectral component. Both methods indicate that the pH of the growth medium of A. pernix does not significantly influence its average membrane fluidity. These results are in accordance with TLC analysis of isolated lipids, which show no significant differences between PLMF isolated from A. pernix grown in medium with different pH.

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

BACKGROUND: An 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 FINDINGS: Degradation 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/SIGNIFICANCE: Our 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).

A highly charged voltage-sensor helix spontaneously translocates across membranes.

Moving freely: A recent model for voltage gating of potassium channels proposed that the four arginine residues of the voltage-sensing S4 helix (left) are in direct contact with the membrane lipids and move into the hydrocarbon core of the membrane during gating. It is demonstrated that the physical properties of the isolated S4 sequence (right) are sufficient to allow it to freely translocate across synthetic membranes.

Membrane-perturbing properties of two Arg-rich paddle domains from voltage-gated sensors in the KvAP and HsapBK K(+) channels.

Voltage-gated K(+) channels are gated by displacement of basic residues located in the S4 helix that together with a part of the S3 helix, S3b, forms a "paddle" domain, whose position is altered by changes in the membrane potential modulating the open probability of the channel. Here, interactions between two paddle domains, KvAPp from the K(v) channel from Aeropyrum pernix and HsapBKp from the BK channel from Homo sapiens, and membrane models have been studied by spectroscopy. We show that both paddle domains induce calcein leakage in large unilamellar vesicles, and we suggest that this leakage represents a general thinning of the bilayer, making movement of the whole paddle domain plausible. The fact that HsapBKp induces more leakage than KvAPp may be explained by the presence of a Trp residue in HsapBKp. Trp residues generally promote localization to the hydrophilic-hydrophobic interface and disturb tight packing. In magnetically aligned bicelles, KvAPp increases the level of order along the whole acyl chain, while HsapBKp affects the morphology, also indicating that KvAPp adapts more to the lipid environment. Nuclear magnetic resonance (NMR) relaxation measurements for HsapBKp show that overall the sequence has anisotropic motions. The S4 helix is well-structured with restricted local motion, while the turn between S4 and S3b is more flexible and undergoes slow local motion. Our results indicate that the calcein leakage is related to the flexibility in this turn region. A possibility by which HsapBKp can undergo structural transitions is also shown by relaxation NMR, which may be important for the gating mechanism.