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PubMed2006

Nucleotide-mimetic synthetic ligands for DNA-recognizing enzymes One-step purification of Pfu DNA polymerase.

The commercial availability of DNA polymerases has revolutionized molecular biotechnology and certain sectors of the bio-industry. Therefore, the development of affinity adsorbents for purification of DNA polymerases is of academic interest and practical importance. In the present study we describe the design, synthesis and evaluation of a combinatorial library of novel affinity ligands for the purification of DNA polymerases (Pols). Pyrococcus furiosus DNA polymerase (Pfu Pol) was employed as a proof-of-principle example. Affinity ligand design was based on mimicking the natural interactions between deoxynucleoside-triphosphates (dNTPs) and the B-motif, a conserved structural moiety found in Pol-I and Pol-II family of enzymes. Solid-phase 'structure-guided' combinatorial chemistry was used to construct a library of 26 variants of the B-motif-binding 'lead' ligand X-Trz-Y (X is a purine derivative and Y is an aliphatic/aromatic sulphonate or phosphonate derivative) using 1,3,5-triazine (Trz) as the scaffold for assembly. The 'lead' ligand showed complementarity against a Lys and a Tyr residue of the polymerase B-motif. The ligand library was screened for its ability to bind and purify Pfu Pol from Escherichia coli extract. One immobilized ligand (oABSAd), bearing 9-aminoethyladenine (AEAd) and sulfanilic acid (oABS) linked on the triazine scaffold, displayed the highest purifying ability and binding capacity (0,55 mg Pfu Pol/g wet gel). Adsorption equilibrium studies with this affinity ligand and Pfu Pol determined a dissociation constant (K(D)) of 83 nM for the respective complex. The oABSAd affinity adsorbent was exploited in the development of a facile Pfu Pol purification protocol, affording homogeneous enzyme (>99% purity) in a single chromatography step. Quality control tests showed that Pfu Pol purified on the B-motif-complementing ligand is free of nucleic acids and contaminating nuclease activities, therefore, suitable for experimental use.

Amino Acid Motifs↗
PubMed2005

Production of a thermostable archaeal superoxide reductase in plant cells.

Pyrococcus furiosus superoxide reductase (SOR) is a thermostable archaeal enzyme that reduces superoxide without producing oxygen. When produced as a fusion protein with the green fluorescent protein in plant cells, P. furiosus SOR is located in the cytosol and nucleus. The recombinant SOR enzyme retains its function and heat stability when assayed in vitro. Importantly, expressing SOR in plant cells enhances their survival at high temperature indicating that it functions in vivo. The archaeal SOR provides a novel mechanism to reduce superoxide and demonstrates the potential for using archaeal genes to alter eukaryotic metabolism.

Archaeal Proteins↗
PubMed2005

A novel ATP/ADP hydrolysis activity of hyperthermostable group II chaperonin in the presence of cobalt or manganese ion.

A novel ATPase activity that was strongly activated in the presence of either cobalt or manganese ion was discovered in the chaperonin from hyperthermophilic Pyrococcus furiosus (Pfu-cpn). Surprisingly, a significant ADPase activity was also detected under the same conditions. A more extensive search revealed similar nucleotide hydrolysis activities in other thermostable chaperonins. Chaperonin activity, i.e., thermal stabilization and refolding of malate dehydrogenase from the guanidine-hydrochloride unfolded state were also detected for Pfu-cpn under the same conditions. We propose that the novel cobalt/manganese-dependent ATP/ADPase activity may be a common trait of various thermostable chaperonins.

Adenosine Triphosphatases↗
PubMed2006

Hyper-thermostability of CutA1 protein, with a denaturation temperature of nearly 150 degrees C.

We found that the CutA1 protein, from Pyrococcus horikoshii (PhCutA1), has an extremely high denaturation temperature (T(d)) of nearly 150 degrees C, which exceeds the highest record determined by DSC by about 30 degrees C. To elucidate the mechanism of the ultra-high stability of PhCutA1, we analyzed the crystal structures of CutA1 proteins from three different sources, P. horikoshii, Thermus thermophilus, and Escherichia coli, with different growth temperatures (98, 75, and 37 degrees C). This analysis revealed that the remarkably increased number of ion pairs in the monomeric structure contributes to the stabilization of the trimeric structure and plays an important role in enhancing the T(d), up to 150 degrees C, for PhCutA1.

Archaeal Proteins↗
PubMed2004

Heat capacity effects of water molecules and ions at a protein-DNA interface.

The interaction of the TATA-box binding protein from the thermophilic and halophilic archaea Pyrococcus woesei (PwTBP) with an oligonucleotide containing a specific binding site is stable over a very broad range of temperatures and ionic strengths, and is consequently an outstanding system for characterising general features of protein-DNA thermodynamics. In common with other specific protein-DNA recognition events, the PwTBP-TATA box interaction is accompanied by a large negative change in heat capacity (deltaCp) arising from the total change in solvation that occurs upon binding, which in this case involves a net uptake of cations. Contrary to previous hypotheses, we find no overall effect of ionic strength on this heat capacity change. We investigate the local contributions of site-specific ion and water binding to the overall change in heat capacity by means of a series of site-directed mutations of PwTBP. We find that although changes in the local ion binding capacity affect the enthalpic and entropic contributions to the free energy of the interaction, they do not affect the change in heat capacity. In contrast, we find remarkably large heat capacity effects arising from two particular symmetry-related mutations. The great magnitude of these effects is not explicable in terms of current semi-empirical models of heat capacity change. Previously reported X-ray crystal structures show that these mutated residues are at the centre of an evolutionarily conserved network of water-mediated hydrogen bonds between the protein and the DNA backbone. Consequently, we conclude that, in addition to water molecules buried in the protein-DNA interface that have been previously shown to influence heat capacity, bridging water molecules in a highly polar surface environment can also contribute substantially to negative heat capacity change on formation of a protein-DNA complex.

Archaeal Proteins↗
PubMed2004

The archaeal sRNA binding protein L7Ae has a 3D structure very similar to that of its eukaryal counterpart while having a broader RNA-binding specificity.

The ribosomal L7Ae protein of archaea has the peculiarity to be a component of the C/D and H/ACA snRNPs, that guide rRNA post-transcriptional modifications. Its yeast (Snu13p) and human (15.5kDa protein) homologs are only found in C/D snoRNPs and the (U4/U6, U5) spliceosomal tri-snRNP. By using a large variety of RNAs, we compared the RNA-binding specificities of the recombinant Pyrococcus abyssi L7Ae and Saccharomyces cerevisiae Snu13 proteins. Unlike Snu13p, protein L7Ae binds terminal loops closed by two A:G and G:A pairs and canonical K-turn structures with similar efficiencies, provided that the terminal loop contains at least 5nt. In contrast to Snu13p, binding of protein L7Ae to canonical K-turn structures is not dependent on the identity of the residue at position 2 in the bulge. The peculiar KT-15 motif of P. abyssi 23S rRNA, that is recognized by L7Ae, does not associate with Snu13p. To get more information on the P. abyssi L7Ae protein, we solved its X-ray structure at 1.9A resolution. In spite of their sequence divergence, the free P. abyssi and bound H. marismortui proteins were found to have highly similar structures. Only a limited number of side-chain conformational changes occur at the protein-RNA interface upon RNA binding. In particular, one ion pair that is formed by residues Glu43 and Lys46 in the free protein is disrupted in the ribosomal 50S subunit, so that, residue Glu43 can interact with the RNA residue G264. The Glu43-Lys46 ion pair of protein L7Ae belongs to a complex network of ion pairs that may participate to protein thermostability.

Amino Acid Sequence↗
PubMed2005

Crystal structure of TET protease reveals complementary protein degradation pathways in prokaryotes.

Protein degradation is an essential and strictly controlled process with proteasome and functionally related proteases representing its central part. Tricorn protease (TRI) has been shown to act downstream of the proteasome, degrading produced peptides. Recently, a novel large prokaryotic aminopeptidase oligomeric complex, named TET, has been identified. This complex degrades peptides of different length in organisms where TRI is not present. We determined the crystal structure of TET from the thermophilic archaeon Pyrococcus horikoshii at 1.6 A resolution in native form and in complex with the inhibitor amastatin. We demonstrate that, beside the novel tetrahedral oligomerisation pattern, TET possesses a unique mechanism of substrate attraction and orientation. TET sequentially degrades peptides produced by the proteasome to single amino acids. Furthermore, we reconstituted in vitro the minimal protein degradation system from initial unfolding of labelled protein substrates, up to release of free amino acids. We propose that TET and TRI act as functional analogues in different organisms, with TET being more widely distributed. Thus, TET and TRI represent two evolutionarily diverged pathways of peptide degradation in prokaryotes.

Aminopeptidases↗
PubMed2005

Crystal structures of tyrosyl-tRNA synthetases from Archaea.

Tyrosyl-tRNA synthetase (TyrRS) catalyzes the tyrosylation of tRNA(Tyr) in a two-step reaction. TyrRS has the "HIGH" and "KMSKS" motifs, which play essential roles in the formation of the tyrosyl-adenylate from tyrosine and ATP. Here, we determined the crystal structures of Archaeoglobus fulgidus and Pyrococcus horikoshii TyrRSs in the l-tyrosine-bound form at 1.8A and 2.2A resolutions, respectively, and that of Aeropyrum pernix TyrRS in the substrate-free form at 2.2 A. The conformation of the KMSKS motif differs among the three TyrRSs. In the A.pernix TyrRS, the KMSKS loop conformation corresponds to the ATP-bound "closed" form. In contrast, the KMSKS loop of the P.horikoshii TyrRS forms a novel 3(10) helix, which appears to correspond to the "semi-closed" form. This conformation enlarges the entrance to the tyrosine-binding pocket, which facilitates the pyrophosphate ion release after the tyrosyl-adenylate formation, and probably is involved in the initial tRNA binding. The KMSSS loop of the A.fulgidus TyrRS is somewhat farther from the active site and is stabilized by hydrogen bonds. Based on the three structures, possible structural changes of the KMSKS motif during the tyrosine activation reaction are discussed. We suggest that the insertion sequence just before the KMSKS motif, which exists in some archaeal species, enhances the binding affinity of the TyrRS for its cognate tRNA. In addition, a non-proline cis peptide bond, which is involved in the tRNA binding, is conserved among the archaeal TyrRSs.

Adenosine Monophosphate↗
PubMed2006

Structural basis of the water-assisted asparagine recognition by asparaginyl-tRNA synthetase.

Asparaginyl-tRNA synthetase (AsnRS) is a member of the class-II aminoacyl-tRNA synthetases, and is responsible for catalyzing the specific aminoacylation of tRNA(Asn) with asparagine. Here, the crystal structure of AsnRS from Pyrococcus horikoshii, complexed with asparaginyl-adenylate (Asn-AMP), was determined at 1.45 A resolution, and those of free AsnRS and AsnRS complexed with an Asn-AMP analog (Asn-SA) were solved at 1.98 and 1.80 A resolutions, respectively. All of the crystal structures have many solvent molecules, which form a network of hydrogen-bonding interactions that surrounds the entire AsnRS molecule. In the AsnRS/Asn-AMP complex (or the AsnRS/Asn-SA), one side of the bound Asn-AMP (or Asn-SA) is completely covered by the solvent molecules, which complement the binding site. In particular, two of these water molecules were found to interact directly with the asparagine amide and carbonyl groups, respectively, and to contribute to the formation of a pocket highly complementary to the asparagine side-chain. Thus, these two water molecules appear to play a key role in the strict recognition of asparagine and the discrimination against aspartic acid by the AsnRS. This water-assisted asparagine recognition by the AsnRS strikingly contrasts with the fact that the aspartic acid recognition by the closely related aspartyl-tRNA synthetase is achieved exclusively through extensive interactions with protein amino acid residues. Furthermore, based on a docking model of AsnRS and tRNA, a single arginine residue (Arg83) in the AsnRS was postulated to be involved in the recognition of the third position of the tRNA(Asn) anticodon (U36). We performed a mutational analysis of this particular arginine residue, and confirmed its significance in the tRNA recognition.

Adenosine Monophosphate↗
PubMed2006

The first crystal structure of L-threonine dehydrogenase.

L-threonine dehydrogenase (TDH) is an enzyme that catalyzes the oxidation of L-threonine to 2-amino-3-ketobutyrate. We solved the first crystal structure of a medium chain L-threonine dehydrogenase from a hyperthermophilic archaeon, Pyrococcus horikoshii (PhTDH), by the single wavelength anomalous diffraction method using a selenomethionine-substituted enzyme. This recombinant PhTDH is a homo-tetramer in solution. Three monomers of PhTDHs were located in the crystallographic asymmetric unit, however, the crystal structure exhibits a homo-tetramer structure with crystallographic and non-crystallographic 222 symmetry in the cell. Despite the low level of sequence identity to a medium-chain NAD(H)-dependent alcohol dehydrogenase (ADH) and the different substrate specificity, the overall folds of the PhTDH monomer and tetramer are similar to those of the other ADH. Each subunit is composed of two domains: a nicotinamide cofactor (NAD(H))-binding domain and a catalytic domain. The NAD(H)-binding domain contains the alpha/beta Rossmann fold motif, characteristic of the NAD(H)-binding protein. One molecule of PhTDH contains one zinc ion playing a structural role. This metal ion exhibits coordination with four cysteine ligands and some of the ligands are conserved throughout the structural zinc-containing ADHs and TDHs. However, the catalytic zinc ion that is coordinated at the bottom of the cleft in the case of ADH was not observed in the crystal of PhTDH. There is a significant difference in the orientation of the catalytic domain relative to the coenzyme-binding domain that results in a larger interdomain cleft.

Alcohol Oxidoreductases↗
PubMed2006

Dimeric core structure of modular stator subunit E of archaeal H+ -ATPase.

Archaeal H(+)-ATPase (A-ATPase) is composed of an A(1) region that hydrolyzes ATP and an integral membrane part A(0) that conducts protons. Subunit E is a component of peripheral stator(s) that physically links A(1) and A(0) parts of the A-ATPase. Here we report the first crystal structure of subunit E of A-ATPase from Pyrococcus horikoshii OT3 at 1.85 A resolution. The protomer structure of subunit E represents a novel fold. The quaternary structure of subunit E is a homodimer, which may constitute the core part of the stator. To investigate the relationship with other stator subunit H, the complex of subunits EH was prepared and characterized using electrophoresis, mass spectrometry, N-terminal sequencing and circular dichroism spectroscopy, which revealed the polymeric and highly helical nature of the EH complex with equimolar stoichiometry of both the subunits. On the basis of the modular architecture of stator subunits, it is suggested that both cytoplasm and membrane sides of the EH complex may interact with other subunits to link A(1) and A(0) parts.

Circular Dichroism↗
PubMed2005

Direct proteolysis-based purification of an overexpressed hyperthermophile protein from Escherichia coli lysate: a novel exploitation of the link between structural stability and proteolytic resistance.

The susceptibility of a peptide bond to cleavage by a protease is determined by: (a) the flexibility of the protein chain region in which it is located, (b) the extent to which the bond is exposed, and (c) the nature of the local interactions made by the sidechains of its flanking residues. Each of these parameters is known to be influenced by the overall structural stability of the protein; thus, proteins of higher structurally stability commonly show higher resistance to proteolysis. Extrapolating this relationship to 'ultrastable' proteins, our intention here was to investigate whether a hyperthermophile protein expressed and folded within Escherichia coli could prove to be so resistant to proteolysis as to allow direct purification from complex mixtures of E. coli cytoplasmic and/or membrane proteins, through proteolytic means. Thus, we cloned the gene encoding the triosephosphate isomerase enzyme of Pyrococcus furiosus (PfuTIM) and overexpressed it in E. coli in fusion with glutathione S-transferase (GST). The GST-PfuTIM fusion product partitioned mainly into the insoluble fraction of the whole cell lysate. Upon exposure of the E. coli cell lysate precipitate fractions to the non-specific protease, subtilisin, all polypeptides barring PfuTIM (including the GST affinity tag cloned in fusion with PfuTIM) were found to be degraded to undetectable levels. Trace residual amounts of an E. coli protein, OmpF, survived proteolytic digestion, together with an extremely pure population of PfuTIM. Either autonomously or in combination with the more conventional method of heating solutions to enrich heat-stable proteins through the thermal unfolding and aggregation of all other proteins, such proteolysis-based purification could prove to be useful.

Amino Acid Sequence↗
PubMed2005

Structural and functional analyses of an archaeal XPF/Rad1/Mus81 nuclease: asymmetric DNA binding and cleavage mechanisms.

XPF/Rad1/Mus81/Hef proteins recognize and cleave branched DNA structures. XPF and Rad1 proteins cleave the 5' side of nucleotide excision repair bubble, while Mus81 and Hef cleave similar sites of the nicked Holliday junction, fork, or flap structure. These proteins all function as dimers and consist of catalytic and helix-hairpin-helix DNA binding (HhH) domains. We have determined the crystal structure of the HhH domain of Pyrococcus furiosus Hef nuclease (HefHhH), which revealed the distinct mode of protein dimerization. Our structural and biochemical analyses also showed that each of the catalytic and HhH domains binds to distinct regions within the fork-structured DNA: each HhH domain from two separate subunits asymmetrically binds to the arm region, while the catalytic domain binds near the junction center. Upon binding to DNA, Hef nuclease disrupts base pairs near the cleavage site. It is most likely that this bipartite binding mode is conserved in the XPF/Rad1/Mus81 nuclease family.

Amino Acid Sequence↗
PubMed2005

Additivity in both thermodynamic stability and thermal transition temperature for rubredoxin chimeras via hybrid native partitioning.

Given any operational definition of pairwise interaction, the set of residues that differ between two structurally homologous proteins can be uniquely partitioned into subsets of clusters for which no such interactions occur between clusters. Although hybrid protein sequences that preserve such clustering are consistent with tertiary structures composed of only parental native-like interactions, the stability of such predicted structures will depend upon the physical robustness of the assumed interaction potential. A simple distance cutoff criterion was applied to the most thermostable protein known to predict such a seven-residue cluster in the metal binding site region of Pyrococcus furiosus rubredoxin and a mesophile homolog. Both conformational stability and thermal transition temperature measurements demonstrate that 39% of the differential stability arises from these seven residues.

Binding Sites↗
PubMed2005

Structural basis for tRNA-dependent amidotransferase function.

Besides direct charging of tRNAs by aminoacyl-tRNA synthetases, indirect routes also ensure attachment of some amino acids onto tRNA. Such routes may explain how new amino acids entered into protein synthesis. In archaea and in most bacteria, tRNA(Gln) is first misaminoacylated by glutamyl-tRNA synthetase. Glu-tRNA(Gln) is then matured into Gln-tRNA(Gln) by a tRNA-dependent amidotransferase. We report the structure of a tRNA-dependent amidotransferase-that of GatDE from Pyrococcus abyssi. The 3.0 A resolution crystal structure shows a tetramer with two GatD molecules as the core and two GatE molecules at the periphery. The fold of GatE cannot be related to that of any tRNA binding enzyme. The ammonium donor site on GatD and the tRNA site on GatE are markedly distant. Comparison of GatD and L-asparaginase structures shows how the motion of a beta hairpin region containing a crucial catalytic threonine may control the overall reaction cycle of GatDE.

Amino Acid Sequence↗
PubMed2005

Two-step pathway to aminoacylated tRNA.

The 3.0 Angstrom crystal structure of a tRNA-dependent amidotransferase from the hyperthermophilic archaeon Pyrococcus abyssi provides the first detailed insight into how cells lacking canonical tRNA synthetases nonetheless carry out protein synthesis with high fidelity.

Adenosine Diphosphate↗
PubMed2006

Common evolutionary origin of swapped-hairpin and double-psi beta barrels.

The core of swapped-hairpin and double-psi beta barrels is formed by duplication of a conserved betaalphabeta element, suggesting a common evolutionary origin. The path connecting the two folds is unclear as the two barrels are not interconvertible by a simple topological modification, such as circular permutation. We have identified a protein family whose sequence properties are intermediate to the two folds. The structure of one of these proteins, Pyrococcus horikoshii PhS018, is also built by duplication of the conserved betaalphabeta element but shows yet a third topology, which we name the RIFT barrel. This topology is widespread in the structure database and spans three folds of the SCOP classification, including the middle domain of EF-Tu and the N domain of F1-ATPase. We propose that swapped-hairpin beta barrels arose from an ancestral RIFT barrel by strand invasion and double-psi beta barrels by a strand swap. We group the three barrel types into a metafold, the cradle-loop barrels.

Amino Acid Sequence↗
PubMed2007

Divergent substrate-binding mechanisms reveal an evolutionary specialization of eukaryotic prefoldin compared to its archaeal counterpart.

Prefoldin (PFD) is a molecular chaperone that stabilizes and then delivers unfolded proteins to a chaperonin for facilitated folding. The PFD hexamer has undergone an evolutionary change in subunit composition, from two PFDalpha and four PFDbeta subunits in archaea to six different subunits (two alpha-like and four beta-like subunits) in eukaryotes. Here, we show by electron microscopy that PFD from the archaeum Pyrococcus horikoshii (PhPFD) selectively uses an increasing number of subunits to interact with nonnative protein substrates of larger sizes. PhPFD stabilizes unfolded proteins by interacting with the distal regions of the chaperone tentacles, a mechanism different from that of eukaryotic PFD, which encapsulates its substrate inside the cavity. This suggests that although the fundamental functions of archaeal and eukaryal PFD are conserved, their mechanism of substrate interaction have diverged, potentially reflecting a narrower range of substrates stabilized by the eukaryotic PFD.

Archaeal Proteins↗