Crystal structure of the ADP-dependent glucokinase from Pyrococcus horikoshii at 2.0-A resolution: a large conformational change in ADP-dependent glucokinase

Although ATP is the most common phosphoryl group donor for kinases, some kinases from certain hyperthermophilic archaea such as Pyrococcus horikoshii and Thermococcus litoralis use ADP as the phosphoryl donor. Those are ADP-dependent glucokinases (ADPGK) and phosphofructokinases in their glycolytic pathway. Here, we succeeded in gene cloning the ADPGK from P. horikoshii OT3 (phGK) in Escherichia coli,and in easy preparation of the enzyme, crystallization, and the structure determination of the apo enzyme. Recently, the three-dimensional structure of the ADPGK from T. litoralis (tlGK) in a complex with ADP was reported. The overall structure of two homologous enzymes (56.7%) was basically similar: This means that they consisted of large alpha/beta-domains and small domains. However, a marked adjustment of the two domains, which is a 10-A translation and a 20 degrees rotation from the conserved GG sequence located at the center of the hinge, was observed between the apo-phGK and ADP-tlGK structures. The ADP-binding loop (430-439) was disordered in the apo form. It is suggested that a large conformational change takes place during the enzymatic reaction.     

Crystal structure of an archaeal DNA sliding clamp: proliferating cell nuclear antigen from Pyrococcus furiosus

The proliferating cell nuclear antigen (PCNA) is now recognized as one of the key proteins in DNA metabolic events because of its direct interactions with many proteins involved in important cellular processes. We have determined the crystal structure of PCNA from a hyperthermophilic archaeon, Pyrococcus furiosus (pfuPCNA), at 2.1 A resolution. pfuPCNA forms a toroidal, ring-shaped structure consisting of homotrimeric molecules, which is also observed in the PCNA crystals from human and yeast. The overall structure of pfuPCNA is highly conserved with other PCNA proteins, as well as with the bacterial ss clamp and the bacteriophage gp45. This result shows that the three-dimensional structure of the sliding clamp is conserved in the three domains of life. pfuPCNA has two remarkable features compared with the human and yeast PCNA molecules: it has more ion pairs and fewer intermolecular main chain hydrogen bonds. The former may contribute to the thermal stability of pfuPCNA, and the latter may be the cause of the stimulatory effect of pfuPCNA on the DNA synthesizing activity of P. furiosus DNA polymerases in the absence of the clamp loader replication factor C in vitro.     

Crystal structure of an archaeal Ski2p-like protein from Pyrococcus horikoshii OT3

The Ski complex composed of Ski2p, Ski3p, and Ski8p plays an essential role in the 3' to 5' cytoplasmic mRNA degradation pathway in yeast. Ski2p is a putative RNA helicase, belonging in the DExD/H-box protein families and conserved in eukarya as well as in archaea. The gene product (Ph1280p) from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 shows sequence homology with Ski2p, sharing 22.6% identical amino acids with a central region of Ski2p. In order to gain structural information about the Ski2p-like RNA helicase, we overproduced Ph1280p in Escherichia coli cells, and purified it to apparent homogeneity. Ph1280p exhibits DNA/RNA-dependent ATPase activity with an optimal temperature at approximately 90 degrees C. The crystal structure of Ph1280p has been solved at a resolution of 3.5 A using single-wavelength anomalous dispersion (SAD) and selenomethionyl (Se-Met)-substituted protein. Ph1280p comprises four subdomains; the two N-terminal subdomains (N1 and N2) fold into an RecA-like architecture with the conserved helicase motifs, while the two C-terminal subdomains (C1 and C2) fold into alpha-helical structures containing a winged helix (WH)-fold and helix-hairpin-helix (HhH)-fold, respectively. Although the structure of each of the Ph1280p subdomains can be individually superimposed on the corresponding domains in other helicases, such as the Escherichia coli DNA helicase RecQ, the relative orientation of the helicase and C-terminal subdomains in Ph1280p is significantly different from that of other helicases. This structural feature is implicated in substrate specificity for the Ski2-like helicase and would play a critical role in the 3' to 5' cytoplasmic mRNA degradation in the Ski complex.     

S-adenosylhomocysteine hydrolase from the archaeon Pyrococcus furiosus: biochemical characterization and analysis of protein structure by comparative molecular modeling

S-adenosylhomocysteine hydrolase (AdoHcyHD) is an ubiquitous enzyme that catalyzes the breakdown of S-adenosylhomocysteine, a powerful inhibitor of most transmethylation reactions, to adenosine and L-homocysteine. AdoHcyHD from the hyperthermophilic archaeon Pyrococcus furiosus (PfAdoHcyHD) was cloned, expressed in Escherichia coli, and purified. The enzyme is thermoactive with an optimum temperature of 95 degrees C, and thermostable retaining 100% residual activity after 1 h at 90 degrees C and showing an apparent melting temperature of 98 degrees C. The enzyme is a homotetramer of 190 kDa and contains four cysteine residues per subunit. Thiol groups are not involved in the catalytic process whereas disulfide bond(s) could be present since incubation with 0.8 M dithiothreitol reduces enzyme activity. Multiple sequence alignment of hyperthermophilic AdoHcyHD reveals the presence of two cysteine residues in the N-terminus of the enzyme conserved only in members of Pyrococcus species, and shows that hyperthermophilic AdoHcyHD lack eight C-terminal residues, thought to be important for structural and functional properties of the eukaryotic enzyme. The homology-modeled structure of PfAdoHcyHD shows that Trp220, Tyr181, Tyr184, and Leu185 of each subunit and Ile244 from a different subunit form a network of hydrophobic and aromatic interactions in the central channel formed at the subunits interface. These contacts partially replace the interactions of the C-terminal tail of the eukaryotic enzyme required for tetramer stability. Moreover, Cys221 and Lys245 substitute for Thr430 and Lys426, respectively, of the human enzyme in NAD-binding. Interestingly, all these residues are fairly well conserved in hyperthermophilic AdoHcyHDs but not in mesophilic ones, thus suggesting a common adaptation mechanism at high temperatures.     

Crystal structure of domain‐swapped STE20 OSR1 kinase domain

OSR1 (oxidative stress-responsive-1) and SPAK (Ste20/Sps1-related proline/alanine-rich kinase) belong to the GCK-VI subfamily of Ste20 group kinases. OSR1 and SPAK are key regulators of NKCCs (Na⁺/K⁺/2Cl⁻ cotransporters) and activated by WNK family members (with-no-lysine kinase), mutations of which are known to cause Gordon syndrome, an autosomal dominant form of inherited hypertension. The crystal structure of OSR1 kinase domain has been solved at 2.25 Å. OSR1 forms a domain-swapped dimer in an inactive conformation, in which P+1 loop and αEF helix are swapped between dimer-related monomers. Structural alignment with nonswapped Ste20 TAO2 kinase indicates that the integrity of chemical interactions in the kinase domain is well preserved in the domain-swapped interfaces. The OSR1 kinase domain has now been added to a growing list of domain-swapped protein kinases recently reported, suggesting that the domain-swapping event provides an additional layer of complexity in regulating protein kinase activity.     

Crystal structure of a phosphatase with a unique substrate binding domain from Thermotoga maritima

We have determined the crystal structure of a phosphatase with a unique substrate binding domain from Thermotoga maritima, TM0651 (gi 4981173), at 2.2 A resolution by selenomethionine single-wavelength anomalous diffraction (SAD) techniques. TM0651 is a member of the haloacid dehalogenase (HAD) superfamily, with sequence homology to trehalose-6-phosphate phosphatase and sucrose-6(F)-phosphate phosphohydrolase. Selenomethionine labeled TM0651 crystallized in space group C2 with three monomers per asymmetric unit. Each monomer has approximate dimensions of 65 x 40 x 35 A(3), and contains two domains: a domain of known hydrolase fold characteristic of the HAD family, and a domain with a new tertiary fold consisting of a six-stranded beta-sheet surrounded by four alpha-helices. There is one disulfide bond between residues Cys35 and Cys265 in each monomer. One magnesium ion and one sulfate ion are bound in the active site. The superposition of active site residues with other HAD family members indicates that TM0651 is very likely a phosphatase that acts through the formation of a phosphoaspartate intermediate, which is supported by both NMR titration data and a biochemical assay. Structural and functional database searches and the presence of many aromatic residues in the interface of the two domains suggest the substrate of TM0651 is a carbohydrate molecule. From the crystal structure and NMR data, the protein likely undergoes a conformational change upon substrate binding.     

Crystal structure of a c-di-AMP riboswitch reveals an internally pseudo-dimeric RNA

Cyclic diadenosine monophosphate (c-di-AMP) is a second messenger that is essential for growth and homeostasis in bacteria. A recently discovered c-di-AMP-responsive riboswitch controls the expression of genes in a variety of bacteria, including important pathogens. To elucidate the molecular basis for specific binding of c-di-AMP by a gene-regulatory mRNA domain, we have determined the co-crystal structure of this riboswitch. Unexpectedly, the structure reveals an internally pseudo-symmetric RNA in which two similar three-helix-junction elements associate head-to-tail, creating a trough that cradles two c-di-AMP molecules making quasi-equivalent contacts with the riboswitch. The riboswitch selectively binds c-di-AMP and discriminates exquisitely against other cyclic dinucleotides, such as c-di-GMP and cyclic-AMP-GMP, via interactions with both the backbone and bases of its cognate second messenger. Small-angle X-ray scattering experiments indicate that global folding of the riboswitch is induced by the two bound cyclic dinucleotides, which bridge the two symmetric three-helix domains. This structural reorganization likely couples c-di-AMP binding to gene expression.     

Crystal structure of a novel ATPase RadD from Escherichia coli

The helicase superfamily 2 (SF2) proteins are involved in essentially every step in DNA and RNA metabolism. The radD (yejH) gene, which belongs to SF2, plays an important role in DNA repair. The RadD protein includes all seven conserved SF2 motifs and has shown ATPase activity. Here, we first reported the structure of RadD from Escherichia coli containing two RecA-like domains, a zinc finger motif, and a C-terminal domain. Based on the structure of RadD and other SF2 proteins, we then built a model of the RedD-ATP complex.     

Crystal structure of the kinase domain of serum and glucocorticoid-regulated kinase 1 in complex with AMP PNP

Serum and glucocorticoid-regulated kinase 1 (SGK1) is a serine/threonine protein kinase of the AGC family which participates in the control of epithelial ion transport and is implicated in proliferation and apoptosis. We report here the 1.9 A crystal structure of the catalytic domain of inactive human SGK1 in complex with AMP-PNP. SGK1 exists as a dimer formed by two intermolecular disulfide bonds between Cys258 in the activation loop and Cys193. Although most of the SGK1 structure closely resembles the common protein kinase fold, the structure around the active site is unique when compared to most protein kinases. The alphaC helix is not present in this inactive form of SGK1 crystal structure; instead, the segment corresponding to the C helix forms a beta-strand that is stabilized by the N-terminal segment of the activation loop through a short antiparallel beta-sheet. Since the differences from other kinases occur around the ATP binding site, this structure can provide valuable insight into the design of selective and highly potent ATP-competitive inhibitors of SGK1 kinase.     

Crystal structure of Bacillus anthracis ThiI, a tRNA-modifying enzyme containing the predicted RNA-binding THUMP domain

ThiI is an enzyme responsible for the formation of the modified base S(4)U (4-thiouridine) found at position 8 in some prokaryotic tRNAs. This base acts as a sensitive trigger for the response mechanism to UV exposure, providing protection against its damaging effects. We present the crystal structure of Bacillus anthracis ThiI in complex with AMP, revealing an extended tripartite architecture in which an N-terminal ferredoxin-like domain (NFLD) connects the C-terminal catalytic PP-loop pyrophosphatase domain with a THUMP domain, an ancient predicted RNA-binding domain that is widespread in all kingdoms of life. We describe the structure of the THUMP domain, which appears to be unrelated to RNA-binding domains of known structure. Mapping the conserved residues of NFLD and the THUMP domain onto the ThiI structure suggests that these domains jointly form the tRNA-binding surface. The inaccessibility of U8 in the canonical L-shaped form of tRNA, and the existence of a glycine-rich linker joining the catalytic and RNA-binding moieties of ThiI suggest that structural changes may occur in both molecules upon binding.     

Crystal structure of pullulanase: evidence for parallel binding of oligosaccharides in the active site

The crystal structures of Klebsiella pneumoniae pullulanase and its complex with glucose (G1), maltose (G2), isomaltose (isoG2), maltotriose (G3), or maltotetraose (G4), have been refined at around 1.7-1.9A resolution by using a synchrotron radiation source at SPring-8. The refined models contained 920-1052 amino acid residues, 942-1212 water molecules, four or five calcium ions, and the bound sugar moieties. The enzyme is composed of five domains (N1, N2, N3, A, and C). The N1 domain was clearly visible only in the structure of the complex with G3 or G4. The N1 and N2 domains are characteristic of pullulanase, while the N3, A, and C domains have weak similarity with those of Pseudomonas isoamylase. The N1 domain was found to be a new type of carbohydrate-binding domain with one calcium site (CBM41). One G1 bound at subsite -2, while two G2 bound at -1 approximately -2 and +2 approximately +1, two G3, -1 approximately -3 and +2 approximately 0', and two G4, -1 approximately -4 and +2 approximately -1'. The two bound G3 and G4 molecules in the active cleft are almost parallel and interact with each other. The subsites -1 approximately -4 and +1 approximately +2, including catalytic residues Glu706 and Asp677, are conserved between pullulanase and alpha-amylase, indicating that pullulanase strongly recognizes branched point and branched sugar residues, while subsites 0' and -1', which recognize the non-reducing end of main-chain alpha-1,4 glucan, are specific to pullulanase and isoamylase. The comparison suggested that the conformational difference around the active cleft, together with the domain organization, determines the different substrate specificities between pullulanase and isoamylase.     

Crystal structure of shikimate kinase from Mycobacterium tuberculosis reveals the dynamic role of the LID domain in catalysis

Shikimate kinase (SK) and other enzymes in the shikimate pathway are potential targets for developing non-toxic antimicrobial agents, herbicides, and anti-parasite drugs, because the pathway is essential in the above species but is absent from mammals. The crystal structure of Mycobacterium tuberculosis SK (MtSK) in complex with MgADP has been determined at 1.8 A resolution, revealing critical information for the structure-based design of novel anti-M. tuberculosis agents. MtSK, with a five-stranded parallel beta-sheet flanked by eight alpha-helices, has three domains: the CORE domain, the shikimate-binding domain (SB), and the LID domain. The ADP molecule is bound with its adenine moiety sandwiched between the side-chains of Arg110 and Pro155, its beta-phosphate group in the P-loop, and the alpha and beta-phosphate groups hydrogen bonded to the guanidinium group of Arg117. Arg117 is located in the LID domain, is strictly conserved in SK sequences, is observed for the first time to interact with any bound nucleotide, and appears to be important in both substrate binding and catalysis. The crystal structure of MtSK (this work) and that of Erwinia chrysanthemi SK suggest a concerted conformational change of the LID and SB domains upon nucleotide binding.     

The structure of lombricine kinase: implications for phosphagen kinase conformational changes

Lombricine kinase is a member of the phosphagen kinase family and a homolog of creatine and arginine kinases, enzymes responsible for buffering cellular ATP levels. Structures of lombricine kinase from the marine worm Urechis caupo were determined by x-ray crystallography. One form was crystallized as a nucleotide complex, and the other was substrate-free. The two structures are similar to each other and more similar to the substrate-free forms of homologs than to the substrate-bound forms of the other phosphagen kinases. Active site specificity loop 309-317, which is disordered in substrate-free structures of homologs and is known from the NMR of arginine kinase to be inherently dynamic, is resolved in both lombricine kinase structures, providing an improved basis for understanding the loop dynamics. Phosphagen kinases undergo a segmented closing on substrate binding, but the lombricine kinase ADP complex is in the open form more typical of substrate-free homologs. Through a comparison with prior complexes of intermediate structure, a correlation was revealed between the overall enzyme conformation and the substrate interactions of His(178). Comparative modeling provides a rationale for the more relaxed specificity of these kinases, of which the natural substrates are among the largest of the phosphagen substrates.     

Crystal structure of a consensus-designed ankyrin repeat protein: implications for stability

Consensus-designed ankyrin repeat (AR) proteins are thermodynamically very stable. The structural analysis of the designed AR protein E3_5 revealed that this stability is due to a regular fold with highly conserved structural motifs and H-bonding networks. However, the designed AR protein E3_19 exhibits a significantly lower stability than E3_5 (9.6 vs. 14.8 kcal/mol), despite 88% sequence identity. To investigate the structural correlations of this stability difference between E3_5 and E3_19, we determined the crystal structure of E3_19 at 1.9 A resolution. E3_19 as well has a regular AR domain fold with the characteristic H-bonding patterns. All structural features of the E3_5 and E3_19 molecules appear to be virtually identical (RMSD(Calpha) approximately 0.7 A). However, clear differences are observed in the surface charge distribution of the two AR proteins. E3_19 features clusters of charged residues and more exposed hydrophobic residues than E3_5. The atomic coordinates of E3_19 have been deposited in the Protein Data Bank. PDB ID: 2BKG.