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.     

Crystal structure of protein Ph1481p in complex with protein Ph1877p of archaeal RNase P from Pyrococcus horikoshii OT3: implication of dimer formation of the holoenzyme

Ribonuclease P (RNase P) in the hyperthermophilic archaeon Pyrococcus horikoshii OT3 consists of a catalytic RNA and five protein subunits. We previously determined crystal structures of four protein subunits. Ph1481p, an archaeal homologue for human hPop5, is the protein component of the P.horikoshii RNase P for which no structural information is available. Here we report the crystal structure of Ph1481p in complex with another protein subunit, Ph1877p, determined at 2.0 A resolution. Ph1481p consists of a five-stranded antiparallel beta-sheet and five helices, which fold in a way that is topologically similar to the ribonucleoprotein (RNP) domain. Ph1481p is, however, distinct from the typical RNP domain in that it has additional helices at the C terminus, which pack against one face of the beta-sheet. The presence of two complexes in the asymmetric unit, together with gel filtration chromatography indicates that the heterotetramer is stable in solution and represents a fundamental building block in the crystals. In the heterotetrameric structure (Ph1877p-(Ph1481p)(2)-Ph1877p), a homodimer of Ph1481p sits between two Ph1877p monomers. Ph1481p dimerizes through hydrogen bonding interaction from the loop between alpha1 and alpha2 helices, and each Ph1481p interacts with two Ph1877p molecules, where alpha2 and alpha3 in Ph1481p interact with alpha7 in one Ph1877p and alpha8 in the other Ph1877p molecule, respectively. Deletion of the alpha1-alpha2 loop in Ph1481p caused heterodimerization with Ph1877p, and abolished ability to homodimerize itself and heterotetramerize with Ph1877p. Furthermore, the reconstituted particle containing the deletion mutant Ph1481p (mPh1481p) exhibited significantly reduced nuclease activity. These results suggest the presence of the heterotetramer of Ph1481p and Ph1877p in P.horikoshii RNase P.     

Structure based hyperthermostability of archaeal histone HPhA from Pyrococcus horikoshii

The histone protein HPhA from the hyperthermophilic archaeon Pyrococcus horikoshii, shows hyperthermostability, as required for optimal growth of the organism at 95 degrees C. The structure of recombinant P.horikoshii HPhA has been determined to 2.3A resolution by molecular replacement, and refined to R(work) and R(free) values of 20.7% and 27.3%, respectively. The HPhA monomer structure is characterized by the histone fold and assembles into a homodimer like other archaeal histones. There are four anions found in the dimer structure, giving rise to clues as to where DNA might bind. A detailed comparison of four known structures of archaeal histones, which evolve and exist under different temperatures, shows that the thermophilic archaeal histone HPhA has a larger hydrophobic contact area, an increased number of hydrogen bonds and a reduced solvent-accessible area. We also observe a unique network of tyrosine residues located at the crossover point of the two HPhA monomers, which locks them together and stabilizes the dimer. These factors together account for the increased thermal stability.     

A fifth protein subunit Ph1496p elevates the optimum temperature for the ribonuclease P activity from Pyrococcus horikoshii OT3

Ribonuclease P (RNase P) is a ribonucleoprotein complex involved in the processing of the 5' leader sequence of precursor tRNA. We previously found that the reconstituted particle (RP) composed of RNase P RNA and four proteins (Ph1481p, Ph1601p, Ph1771p, and Ph1877p) in the hyperthermophilic archaeon Pyrococcus horikoshii OT3 exhibited the RNase P activity, but had a lower optimal temperature (around at 55 degrees C), as compared with 70 degrees C of the authentic RNase P from P. horikoshii [Kouzuma et al., Biochem. Biophys. Res. Commun. 306 (2003) 666-673]. In the present study, we found that addition of a fifth protein Ph1496p, a putative ribosomal protein L7Ae, to RP specifically elevated the optimum temperature to about 70 degrees C comparable to that of the authentic RNase P. Characterization using gel shift assay and chemical probing localized Ph1496p binding sites on two stem-loop structures encompassing nucleotides A116-G201 and G229-C276 in P. horikoshii RNase P RNA. Moreover, the crystal structure of Ph1496p was determined at 2.0 A resolution by the molecular replacement method using ribosomal protein L7Ae from Haloarcula marismortui as a search model. Ph1496p comprises five alpha-helices and a four stranded beta-sheet. The beta-sheet is sandwiched by three helices (alpha1, alpha4, and alpha5) at one side and two helices (alpha2 and alpha3) at other side. The archaeal ribosomal protein L7Ae is known to be a triple functional protein, serving as a protein component in ribosome and ribonucleoprotein complexes, box C/D, and box H/ACA. Although we have at present no direct evidence that Ph1496p is a real protein component in the P. horikoshii RNase P, the present result may assign an RNase P protein to L7Ae as a fourth function.     

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.     

Crystal structures of biotin protein ligase from Pyrococcus horikoshii OT3 and its complexes: structural basis of biotin activation

Biotin protein ligase (EC 6.3.4.15) catalyses the synthesis of an activated form of biotin, biotinyl-5'-AMP, from substrates biotin and ATP followed by biotinylation of the biotin carboxyl carrier protein subunit of acetyl-CoA carboxylase. The three-dimensional structure of biotin protein ligase from Pyrococcus horikoshii OT3 has been determined by X-ray diffraction at 1.6A resolution. The structure reveals a homodimer as the functional unit. Each subunit contains two domains, a larger N-terminal catalytic domain and a smaller C-terminal domain. The structural feature of the active site has been studied by determination of the crystal structures of complexes of the enzyme with biotin, ADP and the reaction intermediate biotinyl-5'-AMP at atomic resolution. This is the first report of the liganded structures of biotin protein ligase with nucleotide and biotinyl-5'-AMP. The structures of the unliganded and the liganded forms are isomorphous except for an ordering of the active site loop upon ligand binding. Catalytic binding sites are suitably arranged to minimize the conformational changes required during the reaction, as the pockets for biotin and nucleotide are located spatially adjacent to each other in a cleft of the catalytic domain and the pocket for biotinyl-5'-AMP binding mimics the combination of those of the substrates. The exact locations of the ligands and the active site residues allow us to propose a general scheme for the first step of the reaction carried out by biotin protein ligase in which the positively charged epsilon-amino group of Lys111 facilitates the nucleophilic attack on the ATP alpha-phosphate group by the biotin carboxyl oxygen atom and stabilizes the negatively charged intermediates.     

Crystal structure of aspartate racemase from Pyrococcus horikoshii OT3 and its implications for molecular mechanism of PLP-independent racemization

There exists a d-enantiomer of aspartic acid in lactic acid bacteria and several hyperthermophilic archaea, which is biosynthesized from the l-enantiomer by aspartate racemase. Aspartate racemase is a representative pyridoxal 5'-phosphate (PLP)-independent amino acid racemase. The "two-base" catalytic mechanism has been proposed for this type of racemase, in which a pair of cysteine residues are utilized as the conjugated catalytic acid and base. We have determined the three-dimensional structure of aspartate racemase from the hyperthermophilic archaeum Pyrococcus horikoshii OT3 at 1.9 A resolution by X-ray crystallography and refined it to a crystallographic R factor of 19.4% (R(free) of 22.2%). This is the first structure reported for aspartate racemase, indeed for any amino acid racemase from archaea. The crystal structure revealed that this enzyme forms a stable dimeric structure with a strong three-layered inter-subunit interaction, and that its subunit consists of two structurally homologous alpha/beta domains, each containing a four-stranded parallel beta-sheet flanked by six alpha-helices. Two strictly conserved cysteine residues (Cys82 and Cys194), which have been shown biochemically to act as catalytic acid and base, are located on both sides of a cleft between the two domains. The spatial arrangement of these two cysteine residues supports the "two-base" mechanism but disproves the previous hypothesis that the active site of aspartate racemase is located at the dimeric interface. The structure revealed a unique pseudo mirror-symmetry in the spatial arrangement of the residues around the active site, which may explain the molecular recognition mechanism of the mirror-symmetric aspartate enantiomers by the non-mirror-symmetric aspartate racemase.     

Crystal structure of the regulatory subunit of archaeal initiation factor 2B (aIF2B) from hyperthermophilic archaeon Pyrococcus horikoshii OT3: a proposed structure of the regulatory subcomplex of eukaryotic IF2B

Eukaryotic translation initiation factor 2B (eIF2B) is the guanine-nucleotide exchange factor for eukaryotic initiation factor 2 (eIF2). eIF2B is a heteropentameric protein composed of alpha- subunits. The alpha, beta, and delta subunits form a regulatory subcomplex, while the gamma and form a catalytic subcomplex. Archaea possess homologues of alpha, beta, and delta subunits of eIF2B. Here, we report the three-dimensional structure of an archaeal regulatory subunit (aIF2Balpha) from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 determined by X-ray crystallography at 2.2A resolution. aIF2Balpha consists of two subdomains, an N-domain (residues 1-95) and a C-domain (residues 96-276), connected by a long alpha-helix (alpha5: 78-106). The N-domain contains a five helix bundle structure, while the C-domain folds into the alpha/beta structure, thus showing similarity to D-ribose-5-phosphate isomerase structure. The presence of two molecules in the crystallographic asymmetric unit and the gel filtration analysis suggest a dimeric structure of aIF2Balpha in solution, interacting with each other by C-domains. Furthermore, the crystallographic 3-fold symmetry generates a homohexameric structure of aIF2Balpha; the interaction is primarily mediated by the long alpha-helix at the N-domains. This structure suggests an architecture of the three subunits, alpha, beta, and delta, in the regulatory subcomplex within eIF2B.     

Crystal structure of tRNA N2,N2-guanosine dimethyltransferase Trm1 from Pyrococcus horikoshii

Trm1 catalyzes a two-step reaction, leading to mono- and dimethylation of guanosine at position 26 in most eukaryotic and archaeal tRNAs. We report the crystal structures of Trm1 from Pyrococcus horikoshii liganded with S-adenosyl-l-methionine or S-adenosyl-l-homocysteine. The protein comprises N-terminal and C-terminal domains with class I methyltransferase and novel folds, respectively. The methyl moiety of S-adenosyl-l-methionine points toward the invariant Phe27 and Phe140 within a narrow pocket, where the target G26 might flip in. Mutagenesis of Phe27 or Phe140 to alanine abolished the enzyme activity, indicating their role in methylating G26. Structural analyses revealed that the movements of Phe140 and the loop preceding Phe27 may be involved in dissociation of the monomethylated tRNA•Trm1 complex prior to the second methylation. Moreover, the catalytic residues Asp138, Pro139, and Phe140 are in a different motif from that in DNA 6-methyladenosine methyltransferases, suggesting a different methyl transfer mechanism in the Trm1 family.     

Molecular structure of a novel membrane protease specific for a stomatin homolog from the hyperthermophilic archaeon Pyrococcus horikoshii

Membrane-bound proteases are involved in various regulatory functions. Our previous study indicated that the N-terminal region of an open reading frame, PH1510 (residues 16-236, designated as 1510-N) from the hyperthermophilic archaeon Pyrococcus horikoshii, is a serine protease with a catalytic Ser-Lys dyad that specifically cleaves the C-terminal hydrophobic residues of a membrane protein, the stomatin-homolog PH1511. In humans, an absence of stomatin is associated with a form of hemolytic anemia known as hereditary stomatocytosis, but the function of stomatin is not fully understood. Here, we report the crystal structure of 1510-N in dimeric form. Each active site of 1510-N is rich in hydrophobic residues, which accounts for the substrate-specificity. The monomer of 1510-N shows structural similarity to one monomer of Escherichia coli ClpP, an ATP-dependent tetradecameric protease. But, their oligomeric forms are different. Major contributors to dimeric interaction in 1510-N are the alpha7 helix and beta9 strand, both of which are missing from ClpP. While the long handle region of ClpP contributes to the stacking of two heptameric rings, the corresponding L2 loop of 1510-N is disordered because the region has little interaction with other residues of the same molecule. The catalytic Ser97 of 1510-N is in almost the same location as the catalytic Ser97 of E.coli ClpP, whereas another residue, Lys138, presumably forming the catalytic dyad, is located in the disordered L2 region of 1510-N. These findings suggest that the binding of the substrate to the catalytic site of 1510-N induces conformational changes in a region that includes loop L2 so that Lys138 approaches the catalytic Ser97.     

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.     

Heat effect on the structure and activity of the recombinant glutamate dehydrogenase from a hyperthermophilic archaeon Pyrococcus horikoshii

Glutamate dehydrogenase from Pyrococcus horikoshii (Pho-GDH) was cloned and overexpressed in Escherichia coli. The cloned enzyme with His-tag was purified to homogeneity by affinity chromatography and shown to be a hexamer enzyme of 290+/-8 kDa (subunit mass 48 kDa). Its optimal pH and temperature were 7.6 and 90 degrees C, respectively. The purified enzyme has outstanding thermostability (the half-life for thermal inactivation at 100 degrees C was 4 h). The enzyme shows strict specificity for 2-oxoglutarate and L-glutamate and requires NAD(P)H and NADP as cofactors but it does not reveal activity on NAD as cofactor. K(m) values of the recombinant enzyme are comparable for both substrates: 0.2 mM for L-glutamate and 0.53 mM for 2-oxoglutarate. The enzyme was activated by heating at 80 degrees C for 1 h, which was accompanied by the formation of its active conformation. Circular dichroism and fluorescence spectra show that the active conformation is heat-inducible and time-dependent.     

Structures of dimeric nonstandard nucleotide triphosphate pyrophosphatase from Pyrococcus horikoshii OT3: functional significance of interprotomer conformational changes

Nonstandard nucleotide triphosphate pyrophosphatase (NTPase) can efficiently hydrolyze nonstandard purine nucleotides in the presence of divalent cations. The crystal structures of the NTPase from Pyrococcus horikoshii OT3 (PhNTPase) have been determined in two unliganded forms and in three liganded forms with inosine 5′-monophosphate (IMP), ITP and Mn²⁺, which visualize the recognition of these ligands unambiguously. The overall structure of PhNTPase is similar to that of previously reported crystal structures of the NTPase from Methanococcus jannaschii and the human ITPase. They share a similar protomer folding with two domains and a similar homodimeric quaternary structure. The dimeric interface of NTPase is well conserved, and the dimeric state might be important to constitute the active site of this enzyme. A conformational analysis of the five snapshots of PhNTPase structures using the multiple superposition method reveals that IMP, ITP and Mn²⁺ bind to the active site without inducing large local conformational changes, indicating that a combination of interdomain and interprotomer rigid-body shifts mainly describes the conformational change of PhNTPase. The interdomain and interprotomer conformations of the ITP liganded form are essentially the same as those observed in the unliganded form 1, indicating that ITP binding to PhNTPase in solution may follow the selection mode in which ITP binds to the subunit that happens to be in the conformation observed in the unliganded form 1. In contrast to the human ITPase inducing a large domain closure upon ITP binding, the interdomain active site cleft is generally closed in PhNTPase and only the IMP binding form shows a remarkable domain opening by 14° only in the B subunit. The interprotomer rigid-body rotation of PhNTPase has a tendency to keep the dimeric 2-fold symmetry, which is also true in human ITPase, thereby suggesting its relevance to the positive cooperativity of the dimeric NTPases. The exception of this rule is observed in the IMP liganded form in which the dimeric 2-fold symmetry is broken by a 3° interprotomer rotation in an unusual direction. A combination of the exceptional interdomain and interprotomer relocations is most likely the reason for the observed asymmetric IMP binding that might be necessary for PhNTPase to release the reaction product IMP.     

The structure of the soluble domain of an archaeal Rieske iron-sulfur protein at 1.1 A resolution

The first crystal structure of an archaeal Rieske iron-sulfur protein, the soluble domain of Rieske iron-sulfur protein II (soxF) from the hyperthermo-acidophile Sulfolobus acidocaldarius, has been solved by multiple wavelength anomalous dispersion (MAD) and has been refined to 1.1 A resolution. SoxF is a subunit of the terminal oxidase supercomplex SoxM in the plasma membrane of S. acidocaldarius that combines features of a cytochrome bc(1) complex and a cytochrome c oxidase. The [2Fe-2S] cluster of soxF is most likely the primary electron acceptor during the oxidation of caldariella quinone by the cytochrome a(587)/Rieske subcomplex. The geometry of the [2Fe-2S] cluster and the structure of the cluster-binding site are almost identical in soxF and the Rieske proteins from eucaryal cytochrome bc(1) and b(6)f complexes, suggesting a strict conservation of the catalytic mechanism. The main domain of soxF and part of the cluster-binding domain, though structurally related, show a significantly divergent structure with respect to topology, non-covalent interactions and surface charges. The divergent structure of soxF reflects a different topology of the soxM complex compared to eucaryal bc complexes and the adaptation of the protein to the extreme ambient conditions on the outer membrane surface of a hyperthermo-acidophilic organism.     

Crystal structure of archaeal ribonuclease P protein Ph1771p from Pyrococcus horikoshii OT3: an archaeal homolog of eukaryotic ribonuclease P protein Rpp29

Ribonuclease P (RNase P) is the endonuclease responsible for the removal of 5' leader sequences from tRNA precursors. The crystal structure of an archaeal RNase P protein, Ph1771p (residues 36-127) from hyperthermophilic archaeon Pyrococcus horikoshii OT3 was determined at 2.0 A resolution by X-ray crystallography. The structure is composed of four helices (alpha1-alpha4) and a six-stranded antiparallel beta-sheet (beta1-beta6) with a protruding beta-strand (beta7) at the C-terminal region. The strand beta7 forms an antiparallel beta-sheet by interacting with strand beta4 in a symmetry-related molecule, suggesting that strands beta4 and beta7 could be involved in protein-protein interactions with other RNase P proteins. Structural comparison showed that the beta-barrel structure of Ph1771p has a topological resemblance to those of Staphylococcus aureus translational regulator Hfq and Haloarcula marismortui ribosomal protein L21E, suggesting that these RNA binding proteins have a common ancestor and then diverged to specifically bind to their cognate RNAs. The structure analysis as well as structural comparison suggested two possible RNA binding sites in Ph1771p, one being a concave surface formed by terminal alpha-helices (alpha1-alpha4) and beta-strand beta6, where positively charged residues are clustered. A second possible RNA binding site is at a loop region connecting strands beta2 and beta3, where conserved hydrophilic residues are exposed to the solvent and interact specifically with sulfate ion. These two potential sites for RNA binding are located in close proximity. The crystal structure of Ph1771p provides insight into the structure and function relationships of archaeal and eukaryotic RNase P.     

The structure of an archaeal homodimeric ligase which has RNA circularization activity

The genome of Pyrococcus abyssi contains two open reading frames encoding proteins which had been previously predicted to be DNA ligases, Pab2002 and Pab1020. We show that while the former is indeed a DNA ligase, Pab1020 had no effect on the substrate deoxyoligo-ribonucleotides tested. Instead, Pab1020 catalyzes the nucleotidylation of oligo-ribonucleotides in an ATP-dependent reaction, suggesting that it is an RNA ligase. We have solved the structure of Pab1020 in complex with the ATP analog AMPPNP by single-wavelength anomalous dispersion (SAD), elucidating a structure with high structural similarity to the catalytic domains of two RNA ligases from the bacteriophage T4. Additional carboxy-terminal domains are also present, and one of these mediates contacts with a second protomer, which is related by noncrystallographic symmetry, generating a homodimeric structure. These C-terminal domains are terminated by short domain swaps which themselves end within 5 Å of the active sites of the partner molecules. Additionally, we show that the protein is indeed capable of circularizing RNA molecules in an ATP-dependent reaction. These structural and biochemical results provide an insight into the potential physiological roles of Pab1020.     

The closed structure of an archaeal DNA ligase from Pyrococcus furiosus

DNA ligases join single-strand breaks in double-stranded DNA, and are essential to maintain genome integrity in DNA metabolism. Here, we report the 1.8 A resolution structure of Pyrococcus furiosus DNA ligase (PfuLig), which represents the first full-length atomic view of an ATP-dependent eukaryotic-type DNA ligase. The enzyme comprises the N-terminal DNA-binding domain, the middle adenylation domain, and the C-terminal OB-fold domain. The architecture of each domain resembles those of human DNA ligase I, but the domain arrangements differ strikingly between the two enzymes. The closed conformation of the two "catalytic core" domains at the carboxyl terminus in PfuLig creates a small compartment, which holds a non-covalently bound AMP molecule. This domain rearrangement results from the "domain-connecting" role of the helical extension conserved at the C termini in archaeal and eukaryotic DNA ligases. The DNA substrate in the human open-ligase is replaced by motif VI in the Pfu closed-ligase. Both the shapes and electrostatic distributions are similar between motif VI and the DNA substrate, suggesting that motif VI in the closed state mimics the incoming substrate DNA. Two basic residues (R531 and K534) in motif VI reside within the active site pocket and interact with the phosphate group of the bound AMP. The crystallographic and functional analyses of mutant enzymes revealed that these two residues within the RxDK sequence play essential and complementary roles in ATP processing. This sequence is also conserved exclusively among the covalent nucleotidyltransferases, even including mRNA-capping enzymes with similar helical extensions at the C termini.     

Crystal structure of a ribonuclease P protein Ph1601p from Pyrococcus horikoshii OT3: an archaeal homologue of human nuclear ribonuclease P protein Rpp21

Ribonuclease P (RNase P) is a ribonucleoprotein complex involved in the removal of 5' leader sequences from tRNA precursors (pre-tRNA). The human protein Rpp21 is essential for human RNase P activity in tRNA processing in vitro. The crystal structure of Ph1601p from the hyperthermophilic archaeon Pyrococcus horikoshii OT3, the archaeal homologue of Rpp21, was determined using the multiple anomalous dispersion (MAD) method with the aid of anomalous scattering in zinc and selenium at 1.6 A resolution. Ph1601p comprises an N-terminal domain (residues 1-55), a central linker domain (residues 56-79), and a C-terminal domain (residues 80-120), forming an L-shaped structure. The N-terminal domain consists of two long alpha-helices, while the central and C-terminal domains fold in a zinc ribbon domain. The electrostatic potential representation indicates the presence of positively charged clusters along the L arms, suggesting a possible role in RNA binding. A single zinc ion binds the well-ordered binding site that consists of four Cys residues (Cys68, Cys71, Cys97, and Cys100) and appears to stabilize the relative positions of the N- and C-domains. Mutations of Cys68 and Cys71 or Cys97 and Cys100 to Ser destabilize the protein structure, which results in inactivation of the RNase P activity. In addition, site-directed mutagenesis suggests that Lys69 at the central loop and Arg86 and Arg105 at the zinc ribbon domain are strongly involved in the functional activity, while Arg22, Tyr44, Arg65, and Arg84 play a modest role in the activity.