Mitsuo Kuratani

Tertiary structure checkpoint at anticodon loop modification in tRNA functional maturation

tRNA precursors undergo a maturation process, involving nucleotide modifications and folding into the L-shaped tertiary structure. The N1-methylguanosine at position 37 (m1G37), 3′ adjacent to the anticodon, is essential for translational fidelity and efficiency. In archaea and eukaryotes, Trm5 introduces the m1G37 modification into all tRNAs bearing G37. Here we report the crystal structures of archaeal Trm5 (aTrm5) in complex with tRNALeu or tRNACys. The D2-D3 domains of aTrm5 discover and modify G37, independently of the tRNA sequences. D1 is connected to D2-D3 through a flexible linker and is designed to recognize the shape of the tRNA outer corner, as a hallmark of the completed L shape formation. This interaction by D1 lowers the Km value for tRNA, enabling the D2-D3 catalysis. Thus, we propose that aTrm5 provides the tertiary structure checkpoint in tRNA maturation.

A Pyrrolo-Pyrimidine Derivative Targets Human Primary AML Stem Cells in Vivo

A pyrrolo-pyrimidine kinase inhibitor, RK-20449, eliminates chemotherapy-resistant human primary AML stem cells in vivo. Taking AML Head-On Like the mythic Lernaean Hydra, acute myeloid leukemia (AML) is hard to kill. It seems like every time one head is cut off, another two—meaner—grow back in its place. Leukemia stem cells (LSCs) are thought to contribute to this resilience; they may survive conventional chemotherapy and increase the risk of relapse. However, it has been difficult to specifically target these cells without also hitting the normal hematopoietic stem cells (HSCs) required for maintaining healthy blood cells. Now, Saito et al. find a new candidate drug that can specifically target LSCs. The authors performed a chemical library screen to target hematopoietic cell kinase (HCK), which they had previously found to be differentially expressed in human LSCs compared with HSCs. They found a candidate HCK inhibitor, RK-20449, which is a pyrrolo-pyrimidine derivative that could bind the active pocket of HCK. In a mouse xenograft of aggressive human AML, RK-20449 greatly reduced LSC burden. If these studies hold true in patients, RK-20449 could accomplish the Herculean task of decreasing the risk of relapse in AML. Leukemia stem cells (LSCs) that survive conventional chemotherapy are thought to contribute to disease relapse, leading to poor long-term outcomes for patients with acute myeloid leukemia (AML). We previously identified a Src-family kinase (SFK) member, hematopoietic cell kinase (HCK), as a molecular target that is highly differentially expressed in human primary LSCs compared with human normal hematopoietic stem cells (HSCs). We performed a large-scale chemical library screen that integrated a high-throughput enzyme inhibition assay, in silico binding prediction, and crystal structure determination and found a candidate HCK inhibitor, RK-20449, a pyrrolo-pyrimidine derivative with an enzymatic IC50 (half maximal inhibitory concentration) in the subnanomolar range. A crystal structure revealed that RK-20449 bound the activation pocket of HCK. In vivo administration of RK-20449 to nonobese diabetic (NOD)/severe combined immunodeficient (SCID)/IL2rgnull mice engrafted with highly aggressive therapy-resistant AML significantly reduced human LSC and non-stem AML burden. By eliminating chemotherapy-resistant LSCs, RK-20449 may help to prevent relapse and lead to improved patient outcomes in AML.

Crystal Structure of tRNA Adenosine Deaminase (TadA) from Aquifex aeolicus

The bacterial tRNA adenosine deaminase (TadA) generates inosine by deaminating the adenosine residue at the wobble position of tRNAArg-2. This modification is essential for the decoding system. In this study, we determined the crystal structure of Aquifex aeolicus TadA at a 1.8-Å resolution. This is the first structure of a deaminase acting on tRNA. A. aeolicus TadA has an α/β/α three-layered fold and forms a homodimer. The A. aeolicus TadA dimeric structure is completely different from the tetrameric structure of yeast CDD1, which deaminates mRNA and cytidine, but is similar to the dimeric structure of yeast cytosine deaminase. However, in the A. aeolicus TadA structure, the shapes of the C-terminal helix and the regions between the β4 and β5 strands are quite distinct from those of yeast cytosine deaminase and a large cavity is produced. This cavity contains many conserved amino acid residues that are likely to be involved in either catalysis or tRNA binding. We made a docking model of TadA with the tRNA anticodon stem loop.

Crystallographic and mutational studies on the tRNA thiouridine synthetase TtuA.

In thermophilic bacteria, specific 2‐thiolation occurs on the conserved ribothymidine at position 54 (T54) in tRNAs, which is necessary for survival at high temperatures. T54 2‐thiolation is achieved by the tRNA thiouridine synthetase TtuA and sulfur‐carrier proteins. TtuA has five conserved CXXC/H motifs and the signature PP motif, and belongs to the TtcA family of tRNA 2‐thiolation enzymes, for which there is currently no structural information. In this study, we determined the crystal structure of a TtuA homolog from the hyperthermophilic archeon Pyrococcus horikoshii at 2.1 Å resolution. The P. horikoshii TtuA forms a homodimer, and each subunit contains a catalytic domain and unique N‐ and C‐terminal zinc fingers. The catalytic domain has much higher structural similarity to that of another tRNA modification enzyme, TilS (tRNAIle2 lysidine synthetase), than to the other type of tRNA 2‐thiolation enzyme, MnmA. Three conserved cysteine residues are clustered in the putative catalytic site, which is not present in TilS. An in vivo mutational analysis in the bacterium Thermus thermophilus demonstrated that the three conserved cysteine residues and the putative ATP‐binding residues in the catalytic domain are important for the TtuA activity. A positively charged surface that includes the catalytic site and the two zinc fingers is likely to provide the tRNA‐binding site. Proteins 2013; 81:1232–1244.

Crystal structure of Sulfolobus tokodaii Sua5 complexed with L-threonine and AMPPNP.

The hypermodified nucleoside N6‐threonylcarbamoyladenosine resides at position 37 of tRNA molecules bearing U at position 36 and maintains translational fidelity in the three kingdoms of life. The N6‐threonylcarbamoyl moiety is composed of L‐threonine and bicarbonate, and its synthesis was genetically shown to require YrdC/Sua5. YrdC/Sua5 binds to tRNA and ATP. In this study, we analyzed the L‐threonine‐binding mode of Sua5 from the archaeon Sulfolobus tokodaii. Isothermal titration calorimetry measurements revealed that S. tokodaii Sua5 binds L‐threonine more strongly than L‐serine and glycine. The Kd values of Sua5 for L‐threonine and L‐serine are 9.3 μM and 2.6 mM, respectively. We determined the crystal structure of S. tokodaii Sua5, complexed with AMPPNP and L‐threonine, at 1.8 Å resolution. The L‐threonine is bound next to AMPPNP in the same pocket of the N‐terminal domain. Thr118 and two water molecules form hydrogen bonds with AMPPNP in a unique manner for adenine‐specific recognition. The carboxyl group and the side‐chain hydroxyl and methyl groups of L‐threonine are buried deep in the pocket, whereas the amino group faces AMPPNP. The L‐threonine is located in a suitable position to react together with ATP for the synthesis of N6‐threonylcarbamoyladenosine. Proteins 2011.

Crystal structure of Methanocaldococcus jannaschii Trm4 complexed with sinefungin.

tRNA:m(5)C methyltransferase Trm4 generates the modified nucleotide 5-methylcytidine in archaeal and eukaryotic tRNA molecules, using S-adenosyl-l-methionine (AdoMet) as methyl donor. Most archaea and eukaryotes possess several Trm4 homologs, including those related to diseases, while the archaeon Methanocaldococcus jannaschii has only one gene encoding a Trm4 homolog, MJ0026. The recombinant MJ0026 protein catalyzed AdoMet-dependent methyltransferase activity on tRNA in vitro and was shown to be the M. jannaschii Trm4. We determined the crystal structures of the substrate-free M. jannaschii Trm4 and its complex with sinefungin at 1.27 A and 2.3 A resolutions, respectively. This AdoMet analog is bound in a negatively charged pocket near helix alpha8. This helix can adopt two different conformations, thereby controlling the entry of AdoMet into the active site. Adjacent to the sinefungin-bound pocket, highly conserved residues form a large, positively charged surface, which seems to be suitable for tRNA binding. The structure explains the roles of several conserved residues that were reportedly involved in the enzymatic activity or stability of Trm4p from the yeast Saccharomyces cerevisiae. We also discuss previous genetic and biochemical data on human NSUN2/hTrm4/Misu and archaeal PAB1947 methyltransferase, based on the structure of M. jannaschii Trm4.

Structure of an archaeal homologue of the bacterial Fmu/RsmB/RrmB rRNA cytosine 5-methyltransferase

One of the modified nucleosides that frequently occurs in rRNAs and tRNAs is 5-methylcytidine (m⁵C). Escherichia coli Fmu/RsmB/RrmB is an S-adenosyl-L-methionine (AdoMet)-dependent methyltransferase that forms m⁵C967 in 16S rRNA. Fmu/RsmB/RrmB homologues exist not only in bacteria but also in archaea and eukarya and constitute a large orthologous group in the RNA:m⁵C methyltransferase family. In the present study, the crystal structure of a homologue of E. coli Fmu/RsmB/RrmB from the archaeon Pyrococcus horikoshii (PH0851) complexed with an AdoMet analogue was determined at 2.55 Å resolution. The structure and sequence of the C-terminal catalytic domain are highly conserved compared with those of E. coli Fmu/RsmB/RrmB. In contrast, the sequence of the N-terminal domain is negligibly conserved between the bacterial and archaeal subfamilies. Nevertheless, the N-terminal domains of PH0851 and E. coli Fmu/RsmB/RrmB are both α-helical and adopt a similar topology. Next to the AdoMet-binding site, a positively charged cleft is formed between the N- and C-terminal domains. This cleft is conserved in the archaeal PH0851 homologues and seems to be suitable for binding the RNA substrate.