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The 2′-5′ RNA ligase family members are bacterial and archaeal RNA ligases that ligate 5′ and 3′ half-tRNA molecules with 2′,3′-cyclic phosphate and 5′-hydroxyl termini, respectively, to the product containing the 2′-5′ phosphodiester linkage. Here, the crystal structure of the 2′-5′ RNA ligase protein from an extreme thermophile, Thermus thermophilus HB8, was solved at 2.5 A˚ resolution. The structure of the 2′-5′ RNA ligase superimposes well on that of the Arabidopsis thaliana cyclic phosphodiesterase (CPDase), which hydrolyzes ADP-ribose 1″,2″-cyclic phosphate (a product of the tRNA splicing reaction) to the monoester ADP-ribose 1″-phosphate. Although the sequence identity between the two proteins is remarkably low (9.3%), the 2′-5′ RNA ligase and CPDase structures have two HX(T/S)X motifs in their corresponding positions. The HX(T/S)X motifs play important roles in the CPDase activity, and are conserved in both the CPDases and 2′-5′ RNA ligases. Therefore, the catalytic mechanism of the 2′-5′ RNA ligase may be similar to that of the CPDase. On the other hand, the electrostatic potential of the cavity of the 2′-5′ RNA ligase is positive, but that of the CPDase is negative. Furthermore, in the CPDase, two loops with low B-factors cover the cavity. In contrast, in the 2′-5′ RNA ligase, the corresponding loops form an open conformation and are flexible. These characteristics may be due to the differences in the substrates, tRNA and ADP-ribose 1″,2″-cyclic phosphate. [Copyright &y& Elsevier]

Since phosphorylation is involved in various physiological events, kinases and interacting factors can be potential targets for drug discovery. For the development and improvement of inhibitors from the point of view of mechanistic enzymology, a cell-free protein synthesis system would be advantageous, since it could prepare mutant proteins easily. However, especially in the case of protein kinase, product solubility remains one of the major challenges. To overcome this problem, we prepared a chaperone-supplemented extract from Escherichia coli BL21 cells harboring a plasmid encoding a set of chaperone genes, dnaK , dnaJ , and grpE . We explored cell-disruption procedures and constructed an efficient protein synthesis system. Employing this system, we produced the kinase domain of human hematopoietic cell kinase (HCK) to obtain further structural information about its molecular interaction with one of its inhibitors, previously developed by our group (RK-20449). Lower reaction temperature improved the solubility, and addition of a protein phosphatase (YpoH) facilitated the homogeneous production of the non-phosphorylated kinase domain. Crystals of the purified product were obtained and the kinase-inhibitor complex structure was solved at 1.7 Å resolution. In addition, results of kinase activity measurement, using a synthetic substrate, showed that the kinase activity was facilitated by autophosphorylation at Tyr416, as confirmed by the peptide mass mapping. [ABSTRACT FROM AUTHOR]

Abstract: The vacuolar-type H+-ATPase (V-ATPase) is a multi-subunit proton pump that is involved in both intra- and extracellular acidification processes throughout human body. Subunits constituting the peripheral stalk of the V-ATPase are known to have several isoforms responsible for tissue/cell specific different physiological roles. To study the different interaction of these isoforms, we expressed and purified the isoforms of human V-ATPase peripheral stalk subunits using Escherichia coli cell-free protein synthesis system: E1, E2, G1, G2, G3, C1, C2, H and N-terminal soluble part of a1 and a2 isoforms. The purification conditions were different depending on the isoforms, maybe reflecting the isoform specific biochemical characteristics. The purified proteins are expected to facilitate further experiments to study about the cell specific interaction and regulation and thus provide insight into physiological meaning of the existence of several isoforms of each subunit in V-ATPase. [Copyright &y& Elsevier]

Abstract: Enterococcus hirae vacuolar ATPase (V-ATPase) is composed of a soluble catalytic domain (V1; NtpA3-B3-D-G) and an integral membrane domain (V0; NtpI-K10) connected by a central and peripheral stalk(s) (NtpC and NtpE-F). Here we examined the nucleotide binding of NtpA monomer, NtpB monomer or NtpD-G heterodimer purified by using Escherichia coli expression system in vivo or in vitro, and the reconstitution of the V1 portion with these polypeptides. The affinity of nucleotide binding to NtpA was 6.6μM for ADP or 3.1μM for ATP, while NtpB or NtpD-G did not show any binding. The NtpA and NtpB monomers assembled into NtpA3-B3 heterohexamer in nucleotide binding-dependent manner. NtpD-G bound NtpA3-B3 forming V1 (NtpA3-B3-D-G) complex independent of nucleotides. The V1 formation from individual NtpA and NtpB monomers with NtpD-G heterodimer was absolutely dependent on nucleotides. The ATPase activity of reconstituted V1 complex was as high as that of native V1-ATPase purified from the V0V1 complex by EDTA treatment of cell membrane. This in vitro reconstitution system of E. hirae V1 complex will be valuable for characterizing the subunit-subunit interactions and assembly mechanism of the V1-ATPase complex. [Copyright &y& Elsevier]

Abstract: In many microorganisms, menaquinone is an essential lipid-soluble electron carrier. Recently, an alternative menaquinone biosynthetic pathway was found in some microorganisms [Hiratsuka, T., Furihata, K., Ishikawa, J., Yamashita, H., Itoh, N., Seto, H., Dairi, T., 2008. An alternative menaquinone biosynthetic pathway operating in microorganisms. Science 321, 1670–1673]. Here, we report the 1.55Å crystal structure of MqnD (TTHA1568) from Thermus thermophilus HB8, an enzyme within the alternative menaquinone biosynthetic pathway. The structure comprises two domains with α/β structures, a large domain and a small domain. L(+)-Tartaric acid was bound to the pocket between the two domains, suggesting that this pocket is a putative active site. The conserved glycine residues at positions 78, 80 and 82 seem to act as hinges, allowing the substrate to access the pocket. Highly conserved residues, such as Asp14, Asp38, Asn43, Ser57, Thr107, Ile144, His145, Glu146, Leu176 and Tyr234, are located at this pocket, suggesting that these residues are involved in substrate binding and/or catalysis, and especially, His145 could function as a catalytic base. Since humans and their commensal intestinal bacteria, including lactobacilli, lack the alternative menaquinone biosynthetic pathway, this enzyme in pathogenic species, such as Helicobacter pylori and Campylobacter jejuni, is an attractive target for the development of chemotherapeutics. This high-resolution structure may contribute toward the development of its inhibitors. [Copyright &y& Elsevier]

Abstract: Interactions of Bcl-2 family proteins play a regulatory role in mitochondrial apoptosis. The pro-apoptotic protein Bak resides in the outer mitochondrial membrane, and the formation of Bak homo- or heterodimers is involved in the regulation of apoptosis. The previously reported structure of the human Bak protein (residues Glu16–Gly186) revealed that a zinc ion was coordinated with two pairs of Asp160 and His164 residues from the symmetry-related molecules. This zinc-dependent homodimer was regarded as an anti-apoptotic dimer. In the present study, we determined the crystal structure of the human Bak residues Ser23–Asn185 at 2.5Å, and found a distinct type of homodimerization through Cys166 disulfide bridging between the symmetry-related molecules. In the two modes of homodimerization, the molecular interfaces are completely different. In the membrane-targeted model of the S–S bridged dimer, the BH3 motifs are too close to the membrane to interact directly with the anti-apoptotic relatives, such as Bcl-xL. Therefore, the Bak dimer structure reported here may represent a pro-apoptotic mode under oxidized conditions. [Copyright &y& Elsevier]

Abstract: The Sto12a protein, from the thermoacidophilic archaeon Sulfolobus tokodaii, has been identified as a small putative DNA-binding protein. Most of the proteins with a high level of amino acid sequence homology to this protein are derived from members of the Sulfolobaceae family, including a transcriptional regulator. We determined the crystal structure of Sto12a at 2.05 Å resolution by multiple-wavelength anomalous dispersion phasing from the selenomethionine-containing protein crystal. This is the first structure of a member of this family of DNA-binding proteins. The Sto12a protein forms a homodimer, and the structure is composed of an N-terminal α-helix, a winged-helix–turn–helix domain, and a C-terminal α-helix that forms an interchain antiparallel coiled coil. The two winged-helix domains are located at both ends of the coiled coil, with putative DNA-recognition helices separated by approximately 34 Å. A structural homology search indicated that the winged-helix domain shared a high level of homology with those found in B-DNA- or Z-DNA-binding proteins from various species, including archaea, bacteria, and human, despite a low level of sequence similarity. The unique structural features of the Sto12a protein include intrachain and interchain disulfide bonds, which stabilize the chain and homodimer structures. There are three cysteine residues: Cys15 and Cys16 in the N-terminal α-helix, and Cys100 in the C-terminal α-helix. Cys15 is involved in an interchain disulfide bridge with the other Cys15, and Cys16 forms an intrachain disulfide bridge with Cys100. This is a novel fold among winged-helix DNA-binding proteins. Possible DNA-binding interactions of the Sto12a protein are discussed based on the crystal structure of Sto12a and comparisons to other winged-helix DNA-binding proteins. [Copyright &y& Elsevier]

Abstract: The Wnt/β-catenin signaling pathway plays important roles in animal development and cancer. Pygopus (Pygo) and Legless (Lgs) are recently discovered core components of the Wnt/β-catenin transcription machinery complex, and are crucially involved in the regulation of the transcription of the Arm/β-catenin and T cell factors (TCF). Lgs/Bcl9 functions as an adaptor between Pygo and Arm/β-catenin. Here, we report the first crystal structure of the plant homeodomain (PHD) finger of Pygopus (Pygo1 PHD), a Pygo family member, which is essential for the association with Lgs/Bcl9. The Pygo1 PHD structure forms a canonical PHD finger motif, stabilized by two Zn ions coordinated in a cross-brace scheme. Surprisingly, the Pygo1 PHD domain forms a dimer in both the crystals and solution. This is the first structural evidence for dimerization among the known PHD domain structures. The dimer formation occurs by the interactions of antiparallel β-sheets between the symmetry-related β3 strands of the monomers. The Pygo1 PHD dimer interface mainly comprises hydrophobic residues. Interestingly, some of the interface residues, such as Met372, Thr373, Ala376 and Leu380, are reportedly important for the association with Lgs/Bcl9 and are also critical for transcriptional activation. The M372A and L380D mutants, and several surrounding mutants such as S385A and A386D, showed decreased ability to form dimers and to interact with the homology domain 1 (HD1) of Lgs/Bcl9. These results suggest that the Pygo1 PHD dimerization is functionally important for Lgs/Bcl9 recognition as well as for the regulation of the Wnt/β-catenin signaling pathway. [Copyright &y& Elsevier]

Abstract: SWIRM is a conserved domain found in several chromatin-associated proteins. Based on their sequences, the SWIRM family members can be classified into three subfamilies, which are represented by Swi3, LSD1, and Ada2. Here we report the SWIRM structure of human MYb-like, Swirm and Mpn domain-containing protein-1 (MYSM1). The MYSM1 SWIRM structure forms a compact HTH-related fold comprising five α-helices, which best resembles the Swi3 SWIRM structure, among the known SWIRM structures. The MYSM1 and Swi3 SWIRM structures are more similar to the LSD1 structure than the Ada2α structure. The SWIRM domains of MYSM1 and LSD1 lacked DNA binding activity, while those of Ada2α and the human Swi3 counterpart, SMARCC2, bound DNA. The dissimilarity in the DNA-binding ability of the MYSM1 and SMARCC2 SWIRM domains might be due to a couple of amino acid differences in the last helix. These results indicate that the SWIRM family has indeed diverged into three structural subfamilies (Swi3/MYSM1, LSD1, and Ada2 types), and that the Swi3/MYSM1-type subfamily has further diverged into two functionally distinct groups. We also solved the structure of the SANT domain of MYSM1, and demonstrated that it bound DNA with a similar mode to that of the c-Myb DNA-binding domain. [Copyright &y& Elsevier]

Abstract: The transcription factor NusA functions in transcriptional regulation involving termination in bacteria. A NusA homolog consisting of only the two KH domains is widely conserved in archaea, but its function remains unknown. We have found that Aeropyrum pernix NusA strongly binds to a certain CU-rich sequence near a termination signal. Our crystal structure of A. pernix NusA revealed that its spatial arrangement is quite similar to that of the KH domains of bacterial NusA. Thus, we consider archaeal NusA to have retained some functions of bacterial NusA, including the ssRNA-binding ability. Remarkable structural differences between archaeal and bacterial NusA exist at the interface with RNAP, in connection with the different NusA-binding sites around the termination signals. Transcriptional termination in archaea could differ from all of the known bacterial and eukaryal mechanisms, in terms of the combination of a bacterial factor and a eukaryal-type RNAP. [Copyright &y& Elsevier]

Abstract: The nuclear pore complex mediates the transport of macromolecules across the nuclear envelope (NE). The vertebrate nuclear pore protein Nup35, the ortholog of Saccharomyces cerevisiae Nup53p, is suggested to interact with the NE membrane and to be required for nuclear morphology. The highly conserved region between vertebrate Nup35 and yeast Nup53p is predicted to contain an RNA-recognition motif (RRM) domain. Due to its low level of sequence homology with other RRM domains, the RNP1 and RNP2 motifs have not been identified in its primary structure. In the present study, we solved the crystal structure of the RRM domain of mouse Nup35 at 2.7 Å resolution. The Nup35 RRM domain monomer adopts the characteristic βαββαβ topology, as in other reported RRM domains. The structure allowed us to locate the atypical RNP1 and RNP2 motifs. Among the RNP motif residues, those on the β-sheet surface are different from those of the canonical RRM domains, while those buried in the hydrophobic core are highly conserved. The RRM domain forms a homodimer in the crystal, in accordance with analytical ultracentrifugation experiments. The β-sheet surface of the RRM domain, with its atypical RNP motifs, contributes to homodimerization mainly by hydrophobic interactions: the side-chain of Met236 in the β4 strand of one Nup35 molecule is sandwiched by the aromatic side-chains of Phe178 in the β1 strand and Trp209 in the β3 strand of the other Nup35 molecule in the dimer. This structure reveals a new homodimerization mode of the RRM domain. [Copyright &y& Elsevier]

Tyrosyl-tRNA synthetase (TyrRS) catalyzes the tyrosylation of tRNATyr in a two-step reaction. TyrRS has the “HIGH” and “KMSKS” motifs, which play essential roles in the formation of the tyrosyl-adenylate from tyrosine and ATP. Here, we determined the crystal structures of Archaeoglobus fulgidus and Pyrococcus horikoshii TyrRSs in the l-tyrosine-bound form at 1.8Å and 2.2Å resolutions, respectively, and that of Aeropyrum pernix TyrRS in the substrate-free form at 2.2 Å. The conformation of the KMSKS motif differs among the three TyrRSs. In the A.pernix TyrRS, the KMSKS loop conformation corresponds to the ATP-bound “closed” form. In contrast, the KMSKS loop of the P.horikoshii TyrRS forms a novel 310 helix, which appears to correspond to the “semi-closed” form. This conformation enlarges the entrance to the tyrosine-binding pocket, which facilitates the pyrophosphate ion release after the tyrosyl-adenylate formation, and probably is involved in the initial tRNA binding. The KMSSS loop of the A.fulgidus TyrRS is somewhat farther from the active site and is stabilized by hydrogen bonds. Based on the three structures, possible structural changes of the KMSKS motif during the tyrosine activation reaction are discussed. We suggest that the insertion sequence just before the KMSKS motif, which exists in some archaeal species, enhances the binding affinity of the TyrRS for its cognate tRNA. In addition, a non-proline cis peptide bond, which is involved in the tRNA binding, is conserved among the archaeal TyrRSs. [Copyright &y& Elsevier]

Peroxiredoxins (Prxs) are thiol-dependent peroxidases that catalyze the detoxification of various peroxide substrates such as H2O2, peroxinitrite, and hydroperoxides, and control some signal transduction in eukaryotic cells. Prxs are found in all cellular organisms and represent an enormous superfamily. Recent genome sequencing projects and biochemical studies have identified a novel subfamily, the archaeal Prxs. Their primary sequences are similar to those of the 1-Cys Prxs, which use only one cysteine residue in catalysis, while their catalytic properties resemble those of the typical 2-Cys Prxs, which utilize two cysteine residues from adjacent monomers within a dimer in catalysis. We present here the X-ray crystal structure of an archaeal Prx from the aerobic hyperthermophilic crenarchaeon, Aeropyrum pernix K1, determined at 2.3Å resolution (R work of 17.8% and R free of 23.0%). The overall subunit arrangement of the A.pernix archaeal Prx is a toroid-shaped pentamer of homodimers, or an (α2)5 decamer, as observed in the previously reported crystal structures of decameric Prxs. The basic folding topology and the peroxidatic active site structure are essentially the same as those of the 1-Cys Prx, hORF6, except that the C-terminal extension of the A.pernix archaeal Prx forms a unique helix with its flanking loops. The thiol group of the peroxidatic cysteine C50 is overoxidized to sulfonic acid. Notably, the resolving cysteine C213 forms the intra-monomer disulfide bond with the third cysteine, C207, which should be a unique structural characteristic in the many archaeal Prxs that retain two conserved cysteine residues in the C-terminal region. The conformational flexibility near the intra-monomer disulfide linkage might be necessary for the dramatic structural rearrangements that occur in the catalytic cycle. [Copyright &y& Elsevier]

Ethylene-insensitive3 (EIN3) and EIN3-like (EIL) proteins are essential transcription factors in the ethylene signaling of higher plants. The EIN3/EIL proteins bind to the promoter regions of the downstream genes and regulate their expression. The location of the DNA-binding domain (DBD) in the primary structure was unclear, since the proteins show no sequence similarity to other known DBDs. Here, we identify the major DBD of an EIN3/EIL protein, Arabidopsis thaliana EIL3, containing a key mutational site for DNA binding and signaling (ein3-3 site), and determine its solution structure by NMR spectroscopy. The structure consists of five α-helices, possessing a novel fold dissimilar to known DBD structures. By a chemical-shift perturbation analysis, a region including the ein3-3 site is suggested to be involved in DNA binding. [Copyright &y& Elsevier]

Abstract: The Dugesia japonica vasa-like gene B (DjVLGB) protein is a DEAD-box RNA helicase of a planarian, which is well known for its strong regenerative capacity. DjVLGB shares sequence similarity to the Drosophila germ-line-specific DEAD-box RNA helicase Vasa, and even higher similarity to its paralogue, mouse PL10. In this study, we solved the crystal structure of the DjVLGB N-terminal RecA-like domain. The overall fold and the structures of the putative ATPase active site of the DjVLGB N-terminal RecA-like domain are similar to those of the previously reported DEAD-box RNA helicase structures. In contrast, the surface structure of the side opposite to the putative ATPase active site is different from those of the other DEAD-box RNA helicases; the characteristic hydrophobic pockets are formed with aromatic and proline residues. These pocket-forming residues are conserved in the PL10-subfamily proteins, but less conserved in the Vasa orthologues and not conserved in the DEAD-box RNA helicases. Therefore, the structural features that we found are characteristic of the PL10-subfamily proteins and might contribute to their biological roles in germ-line development. [Copyright &y& Elsevier]

Abstract: The Thermus thermophilus HB8 genome encodes a signal transducing PII protein, GlnK. The crystal structures of GlnK have been determined in two different space groups, P212121 and P3121. The PII protein has the T-loop, which is essential for interactions with receptor proteins. In both crystal forms, three GlnK molecules form a trimer in the asymmetric unit. In one P212121 crystal form, the three T-loops in the trimer are disordered, while in another P212121 crystal form, the T-loop from one molecule in the trimer is ordered. In the P3121 crystal, one T-loop is ordered while the other two T-loops are disordered. The conformations of the ordered T-loops significantly differ between the two crystal forms; one makes the α-helix in the middle of the T-loop, while the other has an extension of the β-hairpin. Two different conformations are captured by the crystal contacts. The observation of multiple T-loop conformations suggests that the T-loop could potentially exhibit “polysterism,” which would be important for interactions with receptor proteins. The crystal structures of the nucleotide-bound forms, GlnK·ATP and GlnK·ADP, have also been determined. ATP/ADP binding within a cleft at the interface of two adjacent T. thermophilus GlnK monomers might affect the conformation of the T-loop. [Copyright &y& Elsevier]

Ribosomal protein L16 is an essential component of the bacterial ribosome. It organizes the architecture of aminoacyl tRNA binding site in the ribosome 50S subunit. The three-dimensional structure of L16 from Thermus thermophilus HB8 was determined by NMR. In solution, L16 forms an α+β sandwich structure combined with two additional β sheets located at the loop regions connecting the two layers. The terminal regions and a central loop region did not show any specific secondary structure. The structured part of L16 could be superimposed well on the Cα model of L16 determined in the crystal structure of the ribosome 50S subunit. By overlaying the L16 solution structure onto the coordinates of the ribosome crystal structure, we constructed the combined model that represents the ribosome-bound state of L16 in the detailed structure. The model showed that L16 possesses residues in contact with helices 38, 39, 42, 43 and 89 of 23S rRNA and helix 4 of 5S rRNA. This suggests its broad effect on the ribosome architecture. Comparison of L16 with the L10e protein, which is the archaeal counterpart, showed that they share a common fold, but differ in some regions of functional importance, especially in the N-terminal region. All known mutation sites in L16 that confer resistance to avilamycin and evernimicin were positioned so that their side-chains were exposed to solvent in the internal cavity of the ribosome. This suggests the direct participation of L16 as a part of the binding site for antibiotics. [Copyright &y& Elsevier]

Obg comprises a unique family of high-molecular mass GTPases conserved from bacteria to eukaryotes. Bacterial Obg is essential for cellular growth, sporulation, and differentiation. Here, we report the crystal structure of the full-length form of Obg from Thermus thermophilus HB8 at 2.07 A˚ resolution, in the nucleotide-free state. It reveals a three-domain arrangement, composed of the N-terminal domain, the guanine nucleotide-binding domain (G domain), and the C-terminal domain. The N-terminal and G domains have the Obg fold and the Ras-like fold, respectively. These global folds are similar to those of the recently published structure of the C-terminal domain-truncated form of Obg from Bacillus subtilis. On the other hand, the C-terminal domain of Obg was found to have a novel fold (the OCT fold). A comparison of the T. thermophilus and B. subtilis nucleotide-free Obg structures revealed significant conformational changes in the switch-I and switch-II regions of the G domain. Notably, the N-terminal domain is rotated drastically, by almost 180°, around the G domain axis. In the T. thermophilus Obg crystal, the nucleotide-binding site of the G domain interacts with the C-terminal domain of the adjacent molecule. These data suggest a possible domain rearrangement of Obg, and a potential role of the C-terminal domain in the regulation of the nucleotide-binding state. [Copyright &y& Elsevier]

SQUAMOSA promoter binding proteins (SBPs) form a major family of plant-specific transcription factors related to flower development. Although SBPs are heterogeneous in primary structure, they share a highly conserved DNA-binding domain (DBD) that has been suggested to be zinc binding. Here we report the NMR solution structures of DBDs of two SBPs of Arabidopsis thaliana, SPL4 and SPL7. The two share essentially the same structural features. Each structure contains two zinc-binding sites consisting of eight Cys or His residues in a Cys3HisCys2HisCys or Cys6HisCys sequence motif in which the first four residues coordinate to one zinc and the last four coordinate to the other. These structures are dissimilar to other known zinc-binding structures, and thus represent a novel type of zinc-binding motif. The electrostatic profile on the surface suggested that a continuous region, including all the conserved basic residues, is involved in the DNA binding, the mode of which is likely to be novel as well. [Copyright &y& Elsevier]

The thermophilic bacterium Thermus thermophilus synthesizes lysine through the α-aminoadipate pathway, which uses α-aminoadipate as a biosynthetic intermediate of lysine. LysX is the essential enzyme in this pathway, and is believed to catalyze the acylation of α-aminoadipate. We have determined the crystal structures of LysX and its complex with ADP at 2.0 A˚ and 2.38 A˚ resolutions, respectively. LysX is composed of three α+β domains, each composed of a four to five-stranded β-sheet core flanked by α-helices. The C-terminal and central domains form an ATP-grasp fold, which is responsible for ATP binding. LysX has two flexible loop regions, which are expected to play an important role in substrate binding and protection. In spite of the low level of sequence identity, the overall fold of LysX is surprisingly similar to that of other ATP-grasp fold proteins, such as d-Ala:d-Ala ligase, PurT-encoded glycinamide ribonucleotide transformylase, glutathione synthetase, and synapsin I. In particular, they share a similar spatial arrangement of the amino acid residues around the ATP-binding site. This observation strongly suggests that LysX is an ATP-utilizing enzyme that shares a common evolutionary ancestor with other ATP-grasp fold proteins possessing a carboxylate-amine/thiol ligase activity. [Copyright &y& Elsevier]