Molecular characterization of the group II chaperonin from the hyperthermophilic archaeum Pyrococcus horikoshii OT3

The group II chaperonin from the hyperthermophilic archaeum Pyrococcus horikoshii OT3 (PhCPN) and its functional cooperation with the cognate prefoldin were investigated. PhCPN existed as a homo-oligomer in a double-ring structure, which protected the citrate synthase of a porcine heart from thermal aggregation at 45°C, and did the same on the isopropylmalate dehydrogenase (IPMDH) of a thermophilic bacterium, Thermus thermophilus HB8, at 90°C. PhCPN also enhanced the refolding of green fluorescent protein (GFP), which had been unfolded by low pH, in an ATP-dependent manner. Unexpectedly, functional cooperation between PhCPN and Pyrococcus prefoldin (PhPFD) in the refolding of GFP was not observed. Instead, cooperation between PhCPN and PhPFD was observed in the refolding of IPMDH unfolded with guanidine hydrochloride. Although PhCPN alone was not effective in the refolding of IPMDH, the refolding efficiency was enhanced by the cooperation of PhCPN with PhPFD.     

Overexpression, purification, and functional characterization of the group II chaperonin from the hyperthermophilic archaeum Pyrococcus horikoshii OT3

Overexpression in Escherichia coli and functional characterization of the group II chaperonin from the hyperthermophilic archaeum Pyrococcus horikoshii OT3 were investigated in this study. PhCpn, the chaperonin gene from the P. horikoshii OT3, was amplified by PCR from the P. horikoshii genomic DNA, subcloned into pET21a vector, and expressed in three E. coli host cells such as BL21, Rosetta, and Codonplus (DE3). Among these host cells, E. coli Rosetta showed the highest expression level of recombinant PhCpn at induction with 1 mM IPTG. The recombinant PhCpn was purified to 91% by heat-shock treatment and anion-exchange chromatography. The ATPase activity of the purified PhCpn increased in a PhCpn concentration-dependent manner. Also, PhCpn protected the inorganic phosphatase from thermal inactivation at 85 and 110°C, speculating that PhCpn is effective in in vitro holding of the protein. The holding efficiency was enhanced by the addition of Mg²⁺ ion. Through the coexpression of pro-carboxypeptidase B (pro-CPB) and PhCpn in E. coli Rosetta, pro-CPB was produced as a soluble and active form with a marked yield. This result indicated that PhCpn facilitated the in vivo correct folding of pro-CPB and could be used as powerful and novel molecular machinery for the production of recombinant proteins as soluble and active forms in E. coli.     

Protein-protein interactions of the hyperthermophilic archaeon Pyrococcus horikoshii OT3

Background

Although 2,061 proteins of Pyrococcus horikoshii OT3, a hyperthermophilic archaeon, have been predicted from the recently completed genome sequence, the majority of proteins show no similarity to those from other organisms and are thus hypothetical proteins of unknown function. Because most proteins operate as parts of complexes to regulate biological processes, we systematically analyzed protein-protein interactions in Pyrococcus using the mammalian two-hybrid system to determine the function of the hypothetical proteins.

Results

We examined 960 soluble proteins from Pyrococcus and selected 107 interactions based on luciferase reporter activity, which was then evaluated using a computational approach to assess the reliability of the interactions. We also analyzed the expression of the assay samples by western blot, and a few interactions by in vitro pull-down assays. We identified 11 hetero-interactions that we considered to be located at the same operon, as observed in Helicobacter pylori. We annotated and classified proteins in the selected interactions according to their orthologous proteins. Many enzyme proteins showed self-interactions, similar to those seen in other organisms.

Conclusion

We found 13 unannotated proteins that interacted with annotated proteins; this information is useful for predicting the functions of the hypothetical Pyrococcus proteins from the annotations of their interacting partners. Among the heterogeneous interactions, proteins were more likely to interact with proteins within the same ortholog class than with proteins of different classes. The analysis described here can provide global insights into the biological features of the protein-protein interactions in P. horikoshii.     

Over-expression and characterization of recombinant prefoldin from hyperthermophilic archaeum Pyrococcus furiosus in E. coli

Prefoldin is a hexameric chaperone that has been identified in eukaryotic cells and in Archaea. E. coli cells over-expressing the prefoldin gene from the hyperthermophilic, archaeum, Pyrococcus furiosus, grew well at 48°C while control cells were unable to grow above 46°C. The isolated and purified Pfu-prefoldin (specially the β subunit and the prefoldin) thermally protected the activity of lysozyme.     

Structural modeling of RNase P RNA of the hyperthermophilic archaeon Pyrococcus horikoshii OT3

Ribonuclease P (RNase P) is a ubiquitous trans-acting ribozyme that processes the 5′ leader sequence of precursor tRNA (pre-tRNA). The RNase P RNA (PhopRNA) of the hyperthermophilic archaeon Pyrococcus horikoshii OT3 is central to the catalytic process and binds five proteins (PhoPop5, PhoRpp21, PhoRpp29, PhoRpp30, and PhoRpp38) which contribute to the enzymatic activity of the holoenzyme. Despite significant progress in determining the crystal structure of the proteins, the structure of PhopRNA remains elusive. Comparative analysis of the RNase P RNA sequences and existing crystallographic structural information of the bacterial RNase P RNAs were combined to generate a phylogenetically supported three-dimensional (3-D) model of the PhopRNA. The model structure shows an essentially flat disk with 16 tightly packed helices and a conserved face suitable for the binding of pre-tRNA. Moreover, the structure in solution was investigated by enzymatic probing and small-angle X-ray scattering (SAXS) analysis. The low resolution model derived from SAXS and the comparative 3-D model have similar overall shapes. The 3-D model provides a framework for a better understanding of structure–function relationships of this multifaceted primordial ribozyme.     

Characterization of a thermostable enzyme with phosphomannomutase/phosphoglucomutase activities from the hyperthermophilic archaeon Pyrococcus horikoshii OT3

The phosphomannomutase/phosphoglucomutase (PMM/PGM) enzyme catalyzes reversibly the intra-molecular phosphoryl interconverting reaction of mannose-6-phosphate and mannose-1-phosphate or glucose-6-phosphate and glucose-1-phosphate. Glucose-6-phosphate and glucose-1-phosphate are known to be utilized for energy metabolism and cell surface construction, respectively. PMM/PGM has been isolated from many microorganisms. By performing similarity searches using existing PMM/PGM sequences, the homologous ORFs PH0923 and PH1210 were identified from the genomic data of Pyrococcus horikoshii OT3. Since PH0923 appears to be part of an operon consisting of four carbohydrate metabolic enzymes, PH0923 was selected as the first target for the investigation of PMM/PGM activity in P. horikoshii OT3. The coding region of PH0923 was cloned and the purified recombinant protein was utilized for an examination of its biochemical properties. The enzyme retained half its initial activity after treatment at 95 degrees C for 90 min. Detailed analyses of activities showed that this protein is capable of utilizing a variety of metal ions that are not utilized by previously characterized PMM/PGM proteins. A mutated protein with an alanine residue replacing the active site serine residue indicated that this residue plays an important but non-essential role in PMM/PGM activity.     

Identification of nucleotide residues essential for RNase P activity from the hyperthermophilic archaeon Pyrococcus horikoshii OT3

Ribonuclease P (RNase P) is involved in the processing of the 5' leader sequence of precursor tRNA (pre-tRNA). We have found that RNase P RNA (PhopRNA) and five proteins (PhoPop5, PhoRpp21, PhoRpp29, PhoRpp30, and PhoRpp38) reconstitute RNase P activity with enzymatic properties similar to those of the authentic ribozyme from the hyperthermophilic archaeon Pyrococcus horikoshii OT3. We report here that nucleotides A40, A41, and U44 at helix P4, and G269 and G270 located at L15/16 in PhopRNA, are, like the corresponding residues in Esherichia coli RNase P RNA (M1RNA), involved in hydrolysis by coordinating catalytic Mg(2+) ions, and in the recognition of the acceptor end (CCA) of pre-tRNA by base-pairing, respectively. The information reported here strongly suggests that PhopRNA catalyzes the hydrolysis of pre-tRNA in approximately the same manner as eubacterial RNase P RNAs, even though it has no enzymatic activity in the absence of the proteins.     

Crystal structure of the ribonuclease P protein Ph1877p from hyperthermophilic archaeon Pyrococcus horikoshii OT3

Ribonuclease P (RNase P) is a ribonucleoprotein complex involved in the processing of pre-tRNA. Protein Ph1877p is one of essential components of the hyperthermophilic archaeon Pyrococcus horikoshii OT3 RNase P [Biochem. Biophys. Res. Commun. 306 (2003) 666]. The crystal structure of Ph1877p was determined at 1.8A by X-ray crystallography and refined to a crystallographic R factor of 22.96% (Rfree of 26.77%). Ph1877p forms a TIM barrel structure, consisting of ten alpha-helices and seven beta-strands, and has the closest similarity to the TIM barrel domain of Escherichia coli cytosine deaminase with a root-mean square deviation of 3.0A. The protein Ph1877p forms an oblate ellipsoid, approximate dimensions being 45Ax43Ax39A, and the electrostatic representation indicated the presence of several clusters of positively charged amino acids present on the molecular surface. We made use of site-directed mutagenesis to assess the role of twelve charged amino acids, Lys42, Arg68, Arg87, Arg90, Asp98, Arg107, His114, Lys123, Lys158, Arg176, Asp180, and Lys196 related to the RNase P activity. Individual mutations of Arg90, Arg107, Lys123, Arg176, and Lys196 by Ala resulted in reconstituted particles with reduced enzymatic activities (32-48%) as compared with that reconstituted RNase P by wild-type Ph1877p. The results presented here provide an initial step for definite understanding of how archaeal and eukaryotic RNase Ps mediate substrate recognition and process 5'-leader sequence of pre-tRNA.     

Protein-protein interactions in the subunits of ribonuclease P in the hyperthermophilic archaeon Pyrococcus horikoshii OT3

Ribonuclease P (RNase P) is a ribonucleoprotein complex involved in the processing of the 5' leader sequence of precursor tRNA (pre-tRNA). RNase P in the hyperthermophilic archaeon Pyrococcus horikoshii OT3 consists of RNA and five protein subunits (Ph1481p, Ph1496p, Ph1601p, Ph1771p, and Ph1877p). In vivo interactions among five protein subunits of RNase P in P. horikoshii OT3 were examined using a yeast two-hybrid system. The analysis indicates that proteins Ph1481p and Ph1601p interact strongly with Ph1877p and Ph1771p respectively, whereas Ph1481p interacts moderately with Ph1601p. In contrast, no interaction was detected between Ph1496p and the other four proteins. Co-immunoprecipitation analysis confirmed the interactions obtained by yeast two-hybrid assay.     

Characterization of the Family I inorganic pyrophosphatase from Pyrococcus horikoshii OT3

A gene encoding for a putative Family I inorganic pyrophosphatase (PPase, EC 3.6.1.1) from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 was cloned and the biochemical characteristics of the resulting recombinant protein were examined. The gene (Accession No. 1907) from P. horikoshii showed some identity with other Family I inorganic pyrophosphatases from archaea. The recombinant PPase from P. horikoshii (PhPPase) has a molecular mass of 24.5 kDa, determined by SDS-PAGE. This enzyme specifically catalyzed the hydrolysis of pyrophosphate and was sensitive to NaF. The optimum temperature and pH for PPase activity were 70 degrees C and 7.5, respectively. The half-life of heat inactivation was about 50 min at 105 degrees C. The heat stability of PhPPase was enhanced in the presence of Mg2+. A divalent cation was absolutely required for enzyme activity, Mg2+ being most effective; Zn2+, Co2+ and Mn2+ efficiently supported hydrolytic activity in a narrow range of concentrations (0.05-0.5 mM). The K(m) for pyrophosphate and Mg2+ were 113 and 303 microM, respectively; and maximum velocity, V(max), was estimated at 930 U mg(-1).     

Characterization of the archaeal ribonuclease P proteins from Pyrococcus horikoshii OT3

Ribonuclease P (RNase P) is a ribonucleoprotein complex involved in the processing of the 5'-leader sequence of precursor tRNA (pre-tRNA). Our earlier study revealed that RNase P RNA (pRNA) and five proteins (PhoPop5, PhoRpp38, PhoRpp21, PhoRpp29, and PhoRpp30) in the hyperthermophilic archaeon Pyrococcus horikoshii OT3 reconstituted RNase P activity that exhibits enzymatic properties like those of the authentic enzyme. In present study, we investigated involvement of the individual proteins in RNase P activity. Two particles (R-3Ps), in which pRNA was mixed with three proteins, PhoPop5, PhoRpp30, and PhoRpp38 or PhoPop5, PhoRpp30, and PhoRpp21 showed a detectable RNase P activity, and five reconstituted particles (R-4Ps) composed of pRNA and four proteins exhibited RNase P activity, albeit at reduced level compared to that of the reconstituted particle (R-5P) composed of pRNA and five proteins. Time-course analysis of the RNase P activities of R-4Ps indicated that the R-4Ps lacking PhoPop5, PhoRpp21, or PhoRpp30 had virtually reduced activity, while omission of PhoRpp29 or PhoRpp38 had a slight effect on the activity. The results indicate that the proteins contribute to RNase P activity in order of PhoPop5 > PhoRpp30 > PhoRpp21 > PhoRpp29 > PhoRpp38. It was further found that R-4Ps showed a characteristic Mg2+ ion dependency approximately identical to that of R-5P. However, R-4Ps had optimum temperature of around at 55 degrees C which is lower than 70 degrees C for R-5P. Together, it is suggested that the P. horikoshii RNase P proteins are predominantly involved in optimization of the pRNA conformation, though they are individually dispensable for RNase P activity in vitro.     

Crystal structure and functional analysis of an archaeal chromatin protein Alba from the hyperthermophilic archaeon Pyrococcus horikoshii OT3

The crystal structure of the Alba protein (PhoAlba) from a hyperthermophilic archaeon, Pyrococcus horikoshii OT3, was determined at a resolution of 2.8 A. PhoAlba structurally belongs to the alpha/beta proteins and is similar not only to archaeal homologues but also to RNA-binding proteins, including the C-terminal half of initiation factor 3 (IF3-C) from Bacillus stearothermophilus, an Esherichia coli protein implicated in cell division (Yhhp), and an Arabidopsis protein of unknown function. We found by gel shift assay that PhoAlba interacts with both ribonuclease P (RNase P) RNA (PhopRNA) and precursor-tRNA(Tyr) (pre-tRNA(Tyr)) in P. horikoshii. However, the addition of PhoAlba to reconstituted particles composed of PhopRNA and four or five protein subunits had little influence on either the pre-tRNA processing activity or the optimum temperature for the processing activity. These results suggest that PhoAlba contributes little to the catalytic activity of P. horikoshii RNase P.     

FK506 binding protein from the hyperthermophilic archaeon Pyrococcus horikoshii suppresses the aggregation of proteins in Escherichia coli

The 29-kDa FK506 binding protein (FKBP) gene is the only peptidyl-prolyl cis-trans isomerase (PPIase) gene in the genome of Pyrococcus horikoshii. We characterized the function of this FKBP (PhFKBP29) and used it to increase the production yield of soluble recombinant protein in Escherichia coli. The PPIase activity (k(cat)/K(m)) of PhFKBP29 was found to be much lower than that of other archaeal 16- to 18-kDa FKBPs by a chymotrypsin-coupled assay of the oligo-peptidyl substrate at 15 degrees C. Besides this low PPIase activity, PhFKBP29 showed chaperone-like protein folding activity which enhanced the refolding yield of chemically unfolded rhodanese in vitro. In addition, it suppressed thermal protein aggregation in a temperature range of 45 to 100 degrees C. When the PhFKBP29 gene was coexpressed with the recombinant Fab fragment gene of the anti-hen egg lysozyme antibody in the cytoplasm of E. coli, whose expressed product tended to form an inactive aggregate in E. coli, it improved the yield of the soluble Fab fragments with antibody specificity. PhFKBP29 exerted protein folding and aggregation suppression in E. coli cells.     

In vitro phosphorylation of initiation factor 2 alpha (aIF2 alpha) from hyperthermophilic archaeon Pyrococcus horikoshii OT3

Eukaryotic initiation factor 2 (eIF2) is a heterotrimeric protein composed of alpha, beta, and gamma subunits, of which the alpha subunit (eIF2 alpha) plays a crucial role in regulation of protein synthesis through phosphorylation at Ser51. All three subunit genes are conserved in Archaea. To examine the properties of archaeal initiation factor 2 alpha (aIF2 alpha), three genes encoding alpha, beta, and gamma subunits of aIF2 from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 were expressed in Escherichia coli cells, and the resulting proteins, aIF2 alpha, aIF2 beta, and aIF2 gamma, were characterized with reference to the properties of eIF2. aIF2 alpha preferentially interacts with aIF2 gamma, but does not interact with aIF2 beta, which is consistent with data obtained with eIF2, of which eIF2 gamma serves as a core subunit, interacting with eIF2 alpha and eIF2 beta. It was found that aIF2 alpha was, albeit to a lower degree, phosphorylated by double-stranded RNA-dependent protein kinase (hPKR) from human, and a primary target site was suggested to be Ser48 within aIF2 alpha. This finding led us to the search for a putative aIF2 specific kinase gene (PH0512) in the P. horikoshii genome. The gene product Ph0512p unambiguously phosphorylated aIF2 alpha, and Ser48, as in the phosphorylation by hPKR, was suggested to be a target amino acid residue for the PKR homologue Ph0152p in P. horikoshii. These findings suggest that aIF2 alpha, like eIF2 alpha in eukaryotes, plays a role in regulation of the protein synthesis in Archaea through phosphorylation and dephosphorylation.     

Thermodynamic analysis of a multifunctional RNA-binding protein, PhoRpp38, in the hyperthermophilic archaeon Pyrococcus horikoshii OT3

The protein component PhoRpp38 of Pyrococcus horikoshii ribonuclease P (RNase P) is known to be a multifunctional RNA-binding protein. Previous biochemical data indicate that it binds to two stem-loops in RNase P RNA (PhopRNA). Thermodynamic analysis revealed that PhoRpp38 and PhopRNA interact with each other with an association constant (Ka) of 1.56×10(7) M(-1). It was further found that PhoRpp38 simultaneously binds two stem-loop structures in PhopRNA with approximately equal affinity. Crystals of PhoRpp38 in complex with the stem-loop were grown and diffracted to a resolution of 7.0 ? on a synchrotron X-ray source.     

The three-dimensional structure of septum site-determining protein MinD from Pyrococcus horikoshii OT3 in complex with Mg-ADP

BACKGROUND: In Escherichia coli, the cell division site is determined by the cooperative activity of min operon products MinC, MinD, and MinE. MinC is a nonspecific inhibitor of the septum protein FtsZ, and MinE is the supressor of MinC. MinD plays a multifunctional role. It is a membrane-associated ATPase and is a septum site-determining factor through the activation and regulation of MinC and MinE. MinD is also known to undergo a rapid pole-to-pole oscillation movement in vivo as observed by fluorescent microscopy. RESULTS: The three-dimensional structure of the MinD-2 from Pyrococcus horikoshii OT3 (PH0612) has been determined at 2.3 A resolution by X-ray crystallography using the Se-Met MAD method. The molecule consists of a beta sheet with 7 parallel and 1 antiparallel strands and 11 peripheral alpha helices. It contains the classical mononucleotide binding loop with bound ADP and magnesium ion, which is consistent with the suggested ATPase activity. CONCLUSIONS: Structure analysis shows that MinD is most similar to nitrogenase iron protein, which is a member of the P loop-containing nucleotide triphosphate hydrolase superfamily of proteins. Unlike nitrogenase or other member proteins that normally work as a dimer, MinD was present as a monomer in the crystal. Both the 31P NMR and Malachite Green method exhibited relatively low levels of ATPase activity. These facts suggest that MinD may work as a molecular switch in the multiprotein complex in bacterial cell division.     

Identification of Nucleotide Residues Essential for RNase P Activity from the Hyperthermophilic Archaeon Pyrococcus horikoshii OT3

Ribonuclease P (RNase P) is involved in the processing of the 5′ leader sequence of precursor tRNA (pre-tRNA). We have found that RNase P RNA (PhopRNA) and five proteins (PhoPop5, PhoRpp21, PhoRpp29, PhoRpp30, and PhoRpp38) reconstitute RNase P activity with enzymatic properties similar to those of the authentic ribozyme from the hyperthermophilic archaeon Pyrococcus horikoshii OT3. We report here that nucleotides A40, A41, and U44 at helix P4, and G269 and G270 located at L15/16 in PhopRNA, are, like the corresponding residues in Esherichia coli RNase P RNA (M1RNA), involved in hydrolysis by coordinating catalytic Mg²⁺ ions, and in the recognition of the acceptor end (CCA) of pre-tRNA by base-pairing, respectively. The information reported here strongly suggests that PhopRNA catalyzes the hydrolysis of pre-tRNA in approximately the same manner as eubacterial RNase P RNAs, even though it has no enzymatic activity in the absence of the proteins.