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Osamu Nureki

Publications and source records attributed to Osamu Nureki.

At least 19 recordsLinked to original sources
PubMed2025

General and robust sample preparation strategies for cryo-EM studies of CRISPR-Cas9 and Cas12 enzymes.

Cas9 and Cas12 are RNA-guided DNA endonucleases derived from prokaryotic CRISPR-Cas adaptive immune systems that have been repurposed as versatile genome-engineering tools. Computational mining of genomes and metagenomes has expanded the diversity of Cas9 and Cas12 enzymes that can be used to develop versatile, orthogonal molecular toolboxes. Structural information is pivotal to uncovering the precise molecular mechanisms of newly discovered Cas enzymes and providing a foundation for their application in genome editing. In this chapter, we describe detailed protocols for the preparation of Cas9 and Cas12 enzymes for cryo-electron microscopy. These methods will enable fast and robust structural determination of newly discovered Cas9 and Cas12 enzymes, which will enhance the understanding of diverse CRISPR-Cas effectors and provide a molecular framework for expanding CRISPR-based genome-editing technologies.

Cryoelectron Microscopy↗
PubMed2006

Crystal structures of leucyl/phenylalanyl-tRNA-protein transferase and its complex with an aminoacyl-tRNA analog.

Eubacterial leucyl/phenylalanyl-tRNA protein transferase (L/F-transferase), encoded by the aat gene, conjugates leucine or phenylalanine to the N-terminal Arg or Lys residue of proteins, using Leu-tRNA(Leu) or Phe-tRNA(Phe) as a substrate. The resulting N-terminal Leu or Phe acts as a degradation signal for the ClpS-ClpAP-mediated N-end rule protein degradation pathway. Here, we present the crystal structures of Escherichia coli L/F-transferase and its complex with an aminoacyl-tRNA analog, puromycin. The C-terminal domain of L/F-transferase consists of the GCN5-related N-acetyltransferase fold, commonly observed in the acetyltransferase superfamily. The p-methoxybenzyl group of puromycin, corresponding to the side chain of Leu or Phe of Leu-tRNA(Leu) or Phe-tRNA(Phe), is accommodated in a highly hydrophobic pocket, with a shape and size suitable for hydrophobic amino-acid residues lacking a branched beta-carbon, such as leucine and phenylalanine. Structure-based mutagenesis of L/F-transferase revealed its substrate specificity. Furthermore, we present a model of the L/F-transferase complex with tRNA and substrate proteins bearing an N-terminal Arg or Lys.

Amino Acid Sequence↗
PubMed2006

Complete crystallographic analysis of the dynamics of CCA sequence addition.

CCA-adding polymerase matures the essential 3'-CCA terminus of transfer RNA without any nucleic-acid template. However, it remains unclear how the correct nucleotide triphosphate is selected in each reaction step and how the polymerization is driven by the protein and RNA dynamics. Here we present complete sequential snapshots of six complex structures of CCA-adding enzyme and four distinct RNA substrates with and without CTP (cytosine triphosphate) or ATP (adenosine triphosphate). The CCA-lacking RNA stem extends by one base pair to force the discriminator nucleoside into the active-site pocket, and then tracks back after incorporation of the first cytosine monophosphate (CMP). Accommodation of the second CTP clamps the catalytic cleft, inducing a reorientation of the turn, which flips C74 to allow CMP to be accepted. In contrast, after the second CMP is added, the polymerase and RNA primer are locked in the closed state, which directs the subsequent A addition. Between the CTP- and ATP-binding stages, the side-chain conformation of Arg 224 changes markedly; this is controlled by the global motion of the enzyme and position of the primer terminus, and is likely to achieve the CTP/ATP discrimination, depending on the polymerization stage. Throughout the CCA-adding reaction, the enzyme tail domain firmly anchors the TPsiC-loop of the tRNA, which ensures accurate polymerization and termination.

Adenosine Monophosphate↗
PubMed2006

Crystallization and preliminary X-ray crystallographic analysis of the catalytic domain of pyrrolysyl-tRNA synthetase from the methanogenic archaeon Methanosarcina mazei.

Pyrrolysyl-tRNA synthetase (PylRS) from Methanosarcina mazei was overexpressed in an N-terminally truncated form PylRS(c270) in Escherichia coli, purified to homogeneity and crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol as a precipitant. The native PylRS(c270) crystals in complex with an ATP analogue belonged to space group P6(4), with unit-cell parameters a = b = 104.88, c = 70.43 A, alpha = beta = 90, gamma = 120 degrees , and diffracted to 1.9 A resolution. The asymmetric unit contains one molecule of PylRS(c270). Selenomethionine-substituted protein crystals were prepared in order to solve the structure by the MAD phasing method.

Amino Acyl-tRNA Synthetases↗
PubMed2006

Functional categorization of the conserved basic amino acid residues in TrmH (tRNA (Gm18) methyltransferase) enzymes.

Transfer RNA (Gm18) methyltransferase (TrmH) catalyzes the methyl transfer from S-adenosyl-L-methionine (AdoMet) to the 2'-OH group of the G18 ribose in tRNA. To identify amino acid residues responsible for the tRNA recognition, we have carried out the alanine substitution mutagenesis of the basic amino acid residues that are conserved only in TrmH enzymes and not in the other SpoU proteins. We analyzed the mutant proteins by S-adenosyl-L-homocysteine affinity column chromatography, gel mobility shift assay, and kinetic assay of the methyl transfer reaction. Based on these biochemical studies and the crystal structure of TrmH, we found that the conserved residues can be categorized according to their role (i) in the catalytic center (Arg-41), (ii) in the initial site of tRNA binding (Lys-90, Arg-166, Arg-168, and Arg-176), (iii) in the tRNA binding site required for continuation the catalytic cycle (Arg-8, Arg-19, and Lys-32), (iv) in the structural element involved in release of S-adenosyl-L-homocysteine (Arg-11-His-71-Met-147 interaction), (v) in the assisted phosphate binding site (His-34), or (vi) in an unknown function (Arg-109). Furthermore, the difference between the Kd and Km values for tRNA suggests that the affinity for tRNA is enhanced in the presence of AdoMet. To confirm this idea, we carried out the kinetic studies, a gel mobility shift assay with a mutant protein disrupted in the catalytic center, and the analytical gel-filtration chromatography. Our experimental results clearly show that the enzyme has a semi-ordered sequential mechanism in which AdoMet both enhances the affinity for tRNA and induces formation of the tetramer structure.

Amino Acid Motifs↗
PubMed2006

Snapshots of tRNA sulphuration via an adenylated intermediate.

Uridine at the first anticodon position (U34) of glutamate, lysine and glutamine transfer RNAs is universally modified by thiouridylase into 2-thiouridine (s2U34), which is crucial for precise translation by restricting codon-anticodon wobble during protein synthesis on the ribosome. However, it remains unclear how the enzyme incorporates reactive sulphur into the correct position of the uridine base. Here we present the crystal structures of the MnmA thiouridylase-tRNA complex in three discrete forms, which provide snapshots of the sequential chemical reactions during RNA sulphuration. On enzyme activation, an alpha-helix overhanging the active site is restructured into an idiosyncratic beta-hairpin-containing loop, which packs the flipped-out U34 deeply into the catalytic pocket and triggers the activation of the catalytic cysteine residues. The adenylated RNA intermediate is trapped. Thus, the active closed-conformation of the complex ensures accurate sulphur incorporation into the activated uridine carbon by forming a catalytic chamber to prevent solvent from accessing the catalytic site. The structures of the complex with glutamate tRNA further reveal how MnmA specifically recognizes its three different tRNA substrates. These findings provide the structural basis for a general mechanism whereby an enzyme incorporates a reactive atom at a precise position in a biological molecule.

Anticodon↗
PubMed2006

Structural basis of RNA-dependent recruitment of glutamine to the genetic code.

Glutaminyl-transfer RNA (Gln-tRNA(Gln)) in archaea is synthesized in a pretranslational amidation of misacylated Glu-tRNA(Gln) by the heterodimeric Glu-tRNA(Gln) amidotransferase GatDE. Here we report the crystal structure of the Methanothermobacter thermautotrophicus GatDE complexed to tRNA(Gln) at 3.15 angstroms resolution. Biochemical analysis of GatDE and of tRNA(Gln) mutants characterized the catalytic centers for the enzyme's three reactions (glutaminase, kinase, and amidotransferase activity). A 40 angstrom-long channel for ammonia transport connects the active sites in GatD and GatE. tRNA(Gln) recognition by indirect readout based on shape complementarity of the D loop suggests an early anticodon-independent RNA-based mechanism for adding glutamine to the genetic code.

Acylation↗
PubMed2006

Structural basis for RNA unwinding by the DEAD-box protein Drosophila Vasa.

DEAD-box RNA helicases, which regulate various processes involving RNA, have two RecA-like domains as a catalytic core to alter higher-order RNA structures. We determined the 2.2 A resolution structure of the core of the Drosophila DEAD-box protein Vasa in complex with a single-stranded RNA and an ATP analog. The ATP analog intensively interacts with both of the domains, thereby bringing them into the closed form, with many interdomain interactions of conserved residues. The bound RNA is sharply bent, avoiding a clash with a conserved alpha helix in the N-terminal domain. This "wedge" helix should disrupt base pairs by bending one of the strands when a duplex is bound. Mutational analyses indicated that the interdomain interactions couple ATP hydrolysis to RNA unwinding, probably through fine positioning of the duplex relative to the wedge helix. This mechanism, which differs from those for canonical translocating helicases, may enable the targeted modulation of intricate RNA structures.

Adenosine Triphosphate↗
PubMed2006

Purification, crystallization and preliminary X-ray diffraction of SecDF, a translocon-associated membrane protein, from Thermus thermophilus.

Thermus thermophilus has a multi-path membrane protein, TSecDF, as a single-chain homologue of Escherichia coli SecD and SecF, which form a translocon-associated complex required for efficient preprotein translocation and membrane-protein integration. Here, the cloning, expression in E. coli, purification and crystallization of TSecDF are reported. Overproduced TSecDF was solubilized with dodecylmaltoside, chromatographically purified and crystallized by vapour diffusion in the presence of polyethylene glycol. The crystals yielded a maximum resolution of 4.2 angstroms upon X-ray irradiation, revealing that they belonged to space group P4(3)2(1)2. Attempts were made to improve the diffraction quality of the crystals by combinations of micro-stirring, laser-light irradiation and dehydration, which led to the eventual collection of complete data sets at 3.74 angstroms resolution and preliminary success in the single-wavelength anomalous dispersion analysis. These results provide information that is essential for the determination of the three-dimensional structure of this important membrane component of the protein-translocation machinery.

Bacterial Proteins↗
PubMed2006

Crystallization and preliminary X-ray analysis of the tRNA thiolation enzyme MnmA from Escherichia coli complexed with tRNA(Glu).

MnmA catalyzes a sulfuration reaction to synthesize 2-thiouridine at the wobble positions of tRNA(Glu), tRNA(Gln) and tRNA(Lys) in Escherichia coli. The binary complex of MnmA and tRNA(Glu) was crystallized in two different crystal forms: forms I and II. Cocrystallization of MnmA-tRNA(Glu) with ATP yielded form III crystals. The three crystal forms diffracted to 3.1, 3.4 and 3.4 angstroms resolution, respectively, using synchrotron radiation at SPring-8. These crystals belong to space groups C2, I2(1)2(1)2(1) and C2, with unit-cell parameters a = 225.4, b = 175.8, c = 53.0 angstroms, beta = 101.6 degrees, a = 101.5, b = 108.0, c = 211.2 A and a = 238.1, b = 102.1, c = 108.2 angstroms, beta = 117.0 degrees, respectively. The asymmetric units of these crystals are expected to contain two, one and two MnmA-tRNA(Glu) complexes, respectively.

Cloning, Molecular↗
PubMed2006

Structural basis for sulfur relay to RNA mediated by heterohexameric TusBCD complex.

Uridine at wobble position 34 of tRNA(Lys), tRNA(Glu), and tRNA(Gln) is exclusively modified into 2-thiouridine (s2U), which is crucial for both precise codon recognition and recognition by the cognate aminoacyl-tRNA synthetases. Recent Escherichia coli genetic studies revealed that the products of five novel genes, tusABCDE, function in the s2U modification. Here, we solved the 2.15 angstroms crystal structure of the E. coli TusBCD complex, a sulfur transfer mediator, forming a heterohexamer composed of a dimer of the heterotrimer. Structure-based sequence alignment suggested two putative active site Cys residues, Cys79 (in TusC) and Cys78 (in TusD), which are exposed on the hexameric complex. In vivo mutant analyses revealed that only Cys78, in the TusD subunit, participates in sulfur transfer during the s2U modification process. Since the single Cys acts as a catalytic residue, we proposed that TusBCD mediates sulfur relay via a putative persulfide state of the TusD subunit.

Amino Acid Sequence↗
PubMed2005

Structural basis for anticodon recognition by methionyl-tRNA synthetase.

In the 2.7-A resolution crystal structure of methionyl-tRNA synthetase (MetRS) in complex with tRNA(Met) and a methionyl-adenylate analog, the tRNA anticodon loop is distorted to form a triple-base stack comprising C34, A35 and A38. A tryptophan residue stacks on C34 to extend the triple-base stack. In addition, C34 forms Watson-Crick-type hydrogen bonds with Arg357. This structure resolves the longstanding question of how MetRS specifically recognizes tRNA(Met).

Amino Acid Sequence↗
PubMed2005

Structural basis for lysidine formation by ATP pyrophosphatase accompanied by a lysine-specific loop and a tRNA-recognition domain.

Lysidine, a lysine-combined modified cytidine, is exclusively located at the anticodon wobble position (position 34) of eubacterial tRNA(Ile)(2) and not only converts the codon specificity from AUG to AUA, but also converts the aminoacylation specificity from recognition by methionyl-tRNA synthetase to that by isoleucyl-tRNA synthetase (IleRS). Here, we report the crystal structure of lysidine synthetase (TilS) from Aquifex aeolicus at 2.42-A resolution. TilS forms a homodimer, and each subunit consists of the N-terminal dinucleotide-binding fold domain (NTD), with a characteristic central hole, and the C-terminal globular domain (CTD) connected by a long alpha-helical linker. The NTD shares striking structural similarity with the ATP-pyrophosphatase domain of GMP synthetase, which reminds us of the two-step reaction by TilS: adenylation of C34 and lysine attack on the C2 carbon. Conserved amino acid residues are clustered around the NTD central hole. Kinetic analyses of the conserved residues' mutants indicated that C34 of tRNA(Ile)(2) is adenylated by an ATP lying across the NTD central hole and that a lysine, which is activated at a loop appended to the NTD, nucleophilically attacks the C2 carbon from the rear. Escherichia coli TilS (called MesJ) has an additional CTD, which may recognize the tRNA(Ile)(2) acceptor stem. In contrast, a mutational study revealed that A. aeolicus TilS does not recognize the tRNA acceptor stem but recognizes the C29.G41 base pair in the anticodon stem. Thus, the two TilS enzymes discriminate tRNA(Ile)(2) from tRNA(Met) by strategies similar to that used by IleRS, but in distinct manners.

Amino Acyl-tRNA Synthetases↗
PubMed2005

Recent progress of structural biology of tRNA processing and modification.

Transfer RNA (tRNA) is a key molecule to decode the genetic information on mRNA to amino aicds (protein), in a ribosome. For tRNA to fulfill its adopter function, tRNA should be processed into the standard length, and be post-transcriptionally modified. This modification step is essential for the tRNA to maintain the canonical L-shaped structure, which is required for the decoding function of tRNA. Otherwise, it has recently been proposed that modification procedure itself contributes to the RNA (re)folding, where the modification enzymes function as a kind of RNA chaperones. Recent genome analyses and post-genome (proteomics and transcriptomics) analyses have identified genes involved in the tRNA processings and modifications. Furthermore, post-genomic structural analysis has elucidated the structural basis for the tRNA maturation mechanism. In this paper, the recent progress of the structural biology of the tRNA processing and modification is reviewed.

Amino Acyl-tRNA Synthetases↗
PubMed2005

Roles of conserved amino acid sequence motifs in the SpoU (TrmH) RNA methyltransferase family.

Transfer RNA (Gm18) methyltransferase (TrmH (SpoU)) catalyzes the transfer of a methyl group from S-adenosyl-l-methionine (AdoMet) to the 2'-OH of guanosine 18 in tRNA. This enzyme is a member of the SpoU family of RNA methyltransferases. Recent computational researches have shown that three amino acid sequence motifs are conserved among the SpoU members. Recently, we determined the crystal structures of the apoand AdoMet bound forms of TrmH (Nureki, O., Watanabe, K., Fukai, S., Ishii, R., Endo, Y., Hori, H., and Yokoyama, S. (2004) Structure 12, 593-602). Furthermore, we clarified the AdoMet binding site and proposed the catalytic mechanism. Since the functions of the conserved amino acid residues in the motifs remain unknown, here we have prepared 17 mutants of TrmH and carried out various biochemical studies, including determination of the kinetic parameters for both AdoMet and tRNA, S-adenosyl-l-homocysteine affinity chromatography, gel mobility shift assay, CD spectroscopy, and analytical gel filtration. Our results show that Asn(35), Arg(41), Glu(124), and Asn(152) are involved in binding tRNA and that the Asn(35) residue is involved in the release of S-adenosyl-l-homocysteine. Several residues of TrmH are important for stability of the enzyme. Taken together, our biochemical studies reinforce the previously proposed catalytic mechanism. We also discuss amino acid substitutions in general within the SPOUT superfamily of methyltransferases.

Amino Acid Motifs↗
PubMed2005

Structural change of tRNA (Gm18) methyltransferase by binding of methyl donor analogues.

Transfer RNA (Gm18) methyltransferase (TrmH, EC 2.1.1.34) catalyzes the transfer of a methyl group from S-adenosyl-L-methionine (AdoMet) to the 2'-OH of the ribose of guanosine at position 18 in the D-loop of tRNA. The methyl group donor, AdoMet, is converted to S-adenosyl-L-homocysteine (AdoHcy) in the reaction. Recently, we determined the crystal structures of the AdoMet-bound and apo-forms of TrmH from Thermus thermophilus HB8. Based on the crystal structures, we carried out site-directed mutagenesis to clarify the functions of the conserved amino acid residues among SpoU RNA methyltransferases. During the course of the study, we found that the subunit structure of the wild-type TrmH changed according to the presence or absence of AdoMet analogues. In the absence of AdoMet or AdoHcy, the enzyme forms a homodimer. However, the addition of AdoMet induced the formation of a tetramer structure. Furthermore, the addition of AdoHcy caused the dissociation of subunits and gave rise to a monomer structure. These findings suggest that the dissociation of tRNA from the tRNA-enzyme complex after the methyl-transfer reaction is caused by the dissociation of enzyme subunits. In this meeting, we discuss the relationship between the catalytic mechanism and the change of the subunit structure of TrmH.

Catalysis↗
PubMed2004

Crystallization of leucyl-tRNA synthetase complexed with tRNALeu from the archaeon Pyrococcus horikoshii.

All five tRNALeu isoacceptors from the archaeon Pyrococcus horikoshii have been transcribed in vitro and purified. The leucyl-tRNA synthetase (LeuRS) from P. horikoshii was overexpressed in Escherichia coli and purified, and cocrystallizations with each of the tRNALeu isoacceptors were attempted. Cocrystals were obtained by the hanging-drop vapour-diffusion method, but only when the tRNALeu isoacceptor with the anticodon CAA was used. Electrophoretic analyses revealed that the crystals contain both LeuRS and tRNALeu, suggesting that they are LeuRS-tRNALeu complex crystals. A data set diffracting to 3.3 A resolution was collected from a single crystal at 100 K. The crystal belongs to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 118.18, b = 120.55, c = 231.13 A. The asymmetric unit is expected to contain two complexes of LeuRS-tRNALeu, with a corresponding crystal volume per protein weight of 2.9 A3 Da(-1) and a solvent content of 57.3%.

Archaeal Proteins↗