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.     

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.     

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.     

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 and structural stability of acylphosphatase from hyperthermophilic archaeon Pyrococcus horikoshii OT3

To elucidate the structural basis for the high stability of acylphosphatase (AcP) from Pyrococcus horikoshii OT3, we determined its crystal structure at 1.72 A resolution. P. horikoshii AcP possesses high stability despite its approximately 30% sequence identity with eukaryotic enzymes that have moderate thermostability. The overall fold of P. horikoshii AcP was very similar to the structures of eukaryotic counterparts. The crystal structure of P. horikoshii AcP shows the same fold betaalphabetabetaalphabeta topology and the conserved putative catalytic residues as observed in eukaryotic enzymes. Comparison with the crystal structure of bovine common-type AcP and that of D. melanogaster AcP (AcPDro2) as representative of eukaryotic AcP revealed some significant characteristics in P. horikoshii AcP that likely play important roles in structural stability: (1) shortening of the flexible N-terminal region and long loop; (2) an increased number of ion pairs on the protein surface; (3) stabilization of the loop structure by hydrogen bonds. In P. horikoshii AcP, two ion pair networks were observed one located in the loop structure positioned near the C-terminus, and other on the beta-sheet. The importance of ion pairs for structural stability was confirmed by site-directed mutation and denaturation induced by guanidium chloride.     

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.     

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.     

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.     

Characterization of thermostable aminoacylase from hyperthermophilic archaeon Pyrococcus horikoshii

The gene encoding putative aminoacylase (ORF: PH0722) in the genome sequence of a hyperthermophilic archaeon, Pyrococcus horikoshii, was cloned and overexpressed in Escherichia coli. The recombinant enzyme was determined to be thermostable aminoacylase (PhoACY), forming a homotetramer. Purified PhoACY showed the ability to release amino acid molecules from the substrates N-acetyl-L-Met, N-acetyl-L-Gln and N-acetyl-L-Leu, but had a lower hydrolytic activity towards N-acetyl-L-Phe. The kinetic parameters K(m) and k(cat) were determined to be 24.6 mm and 370 s(-1), respectively, for N-acetyl-l-Met at 90 degrees C. Purified PhoACY contained one zinc atom per subunit. EDTA treatment resulted in the loss of PhoACY activity. Enzyme activity was fully recovered by the addition of divalent metal ions (Zn(2+), Mn(2+) and Ni(2+)), and Mn(2+) addition caused an alteration in substrate specificity. Site-directed mutagenesis analysis and structural modeling of PhoACY, based on Arabidopsis thaliana indole-3-acetic acid amino acid hydrolase as a template, revealed that, amongst the amino acid residues conserved in PhoACY, His106, Glu139, Glu140 and His164 were related to the metal-binding sites critical for the expression of enzyme activity. Other residues, His198 and Arg260, were also found to be involved in the catalytic reaction, suggesting that PhoACY obeys a similar reaction mechanism to that proposed for mammalian aminoacylases.     

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.     

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.     

X-ray structure of a membrane-bound beta-glycosidase from the hyperthermophilic archaeon Pyrococcus horikoshii

The beta-glycosidase of the hyperthermophilic Archaeon Pyrococcus horikoshii is a membrane-bound enzyme with the preferred substrate of alkyl-beta-glycosides. In this study, the unusual structural features that confer the extreme thermostability and substrate preferences of this enzyme were investigated by X-ray crystallography and docking simulation. The enzyme was crystallized in the presence of a neutral surfactant, and the crystal structure was solved by the molecular replacement method and refined at 2.5 A. The main-chain fold of the enzyme belongs to the (betaalpha)8 barrel structure common to the Family 1 glycosyl hydrolases. The active site is located at the center of the C-termini of the barrel beta-strands. The deep pocket of the active site accepts one sugar unit, and a hydrophobic channel extending radially from there binds the nonsugar moiety of the substrate. The docking simulation for oligosaccharides and alkylglucosides indicated that alkylglucosides with a long aliphatic chain are easily accommodated in the hydrophobic channel. This sparingly soluble enzyme has a cluster of hydrophobic residues on its surface, situated at the distal end of the active site channel and surrounded by a large patch of positively charged residues. We propose that this hydrophobic region can be inserted into the membrane while the surrounding positively charged residues make favorable contacts with phosphate groups on the inner surface of the membrane. The enzyme could thus adhere to the membrane in the proximity of its glycolipid substrate.     

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.     

Characterization of a TET-like aminopeptidase complex from the hyperthermophilic archaeon Pyrococcus horikoshii

Pyrococcus horikoshii open reading frame PH1527 encodes a 39014 Da protein that shares about 30% identity with endoglucanases and members of the M42 peptidase family. Analytical ultracentrifugation and electron microscopy studies showed that the purified recombinant protein forms stable, large dodecameric complexes with a tetrahedral shape similar to the one described for DAP, a deblocking aminopeptidase that was characterized in the same organism. The two related proteins were named PhTET1 (for DAP) and PhTET2 (for PH1527). The substrate specificity and the mode of action of the PhTET2 complex were studied in detail and compared to those of PhTET1 and other assigned M42 peptidases. When assayed with short chromogenic peptides, PhTET2 was found to be an aminopeptidase, with a clear preference for leucine as the N-terminal amino acid. However, the enzyme can cleave moderately long polypeptide substrates of various compositions in a fairly unspecific manner. The hydrolytic mechanism was found to be nonprocessive. The enzyme has neither carboxypeptidase nor endoproteolytic activities, and it is devoid of N-terminal deblocking activity. PhTET2 was inhibited in the presence of EDTA and bestatin, and cobalt was found to be an activating metal. The PhTET2 protein is a highly thermostable enzyme that displays optimal activity around 100 degrees C over a broad pH array.     

Recombinant expression and characterization of an extremely hyperthermophilic archaeal histone from Pyrococcus horikoshii OT3

A histone-like gene, PHS051 from hyperthermophilic archaeon Pyrococcus horikoshii OT3 strain, was cloned, sequenced, and expressed in Escherichia coli. The recombinant histone, HPhA, encodes a protein of 70 amino acids with a molecular weight of 7868Da. Amino acid sequence analysis of HPhA showed high homology with other archaeal histones and eukaryal core histones. The HPhA was purified to homogeneity by heat precipitation and affinity chromatography. Gel electrophoresis mobility shift assays demonstrate that the purified HPhA has high affinity to DNA. The complex of the HPhA and DNA allows DNA to be protected from cleavage by the restriction enzyme TaqI at 65 degrees C. Circular dichroism spectra reveal that the conformation of the recombinant histone HPhA becomes looser when temperatures increase from 25 to 90 degrees C. The HPhA has inherited a remarkable thermostability especially in the presence of 1M KCl and retained DNA binding activity at extreme temperature, which is consistent with our previous report about its structure stability analyzed by X-ray crystallography.     

Novel substrate specificity of a membrane-bound beta-glycosidase from the hyperthermophilic archaeon Pyrococcus horikoshii

A beta-glycosidase gene homolog of Pyrococcus horikoshii (BGPh) was successfully expressed in Escherichia coli. The enzyme was localized in a membrane fraction and solubilized with 2.5% Triton X-100 at 85 degrees C for 15 min. The optimum pH was 6.0 and the optimum temperature was over 100 degrees C, respectively. BGPh stability was dependent on the presence of Triton X-100, the enzyme's half-life at 90 degrees C (pH 6.0) was 15 h. BGPh has a novel substrate specificity with k(cat)/K(m) values high enough for hydrolysis of beta-D-Glcp derivatives with long alkyl chain at the reducing end and low enough for the hydrolysis of beta-linked glucose dimer more hydrophilic than aryl- or alkyl-beta-D-Glcp.