Seiichiro Kishishita

The University of Tokyo, Tokyo, Tokyo-to, Japan

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Publications (12)81.99 Total impact

  • [show abstract] [hide abstract]
    ABSTRACT: AMP-activated protein kinase (AMPK) is a serine/threonine kinase that functions as a sensor to maintain energy balance at both the cellular and the whole-body levels and is therefore a potential target for drug design against metabolic syndrome, obesity and type 2 diabetes. Here, the crystal structure of the phosphorylated-state mimic T172D mutant kinase domain from the human AMPK α2 subunit is reported in the apo form and in complex with a selective inhibitor, compound C. The AMPK α2 kinase domain exhibits a typical bilobal kinase fold and exists as a monomer in the crystal. Like the wild-type apo form, the T172D mutant apo form adopts the autoinhibited structure of the `DFG-out' conformation, with the Phe residue of the DFG motif anchored within the putative ATP-binding pocket. Compound C binding dramatically alters the conformation of the activation loop, which adopts an intermediate conformation between DFG-out and DFG-in. This induced fit forms a compound-C binding pocket composed of the N-lobe, the C-lobe and the hinge of the kinase domain. The pocket partially overlaps with the putative ATP-binding pocket. These three-dimensional structures will be useful to guide drug discovery.
    Acta Crystallographica Section D Biological Crystallography 05/2011; 67(Pt 5):480-7. · 12.67 Impact Factor
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    ABSTRACT: The 70 kDa heat-shock proteins (Hsp70s) are highly conserved chaperones that are involved in several cellular processes, such as protein folding, disaggregation and translocation. In this study, the crystal structures of the human Hsp70 nucleotide-binding domain (NBD) fragment were determined in the nucleotide-free state and in complex with adenosine 5'-(beta,gamma-imido)triphosphate (AMPPNP). The structure of the nucleotide-free NBD fragment is similar to that of the AMPPNP-bound NBD fragment and is designated as the ;closed form'. In the nucleotide-free NBD fragment the closed form is intrinsically supported through interactions between Tyr15, Lys56 and Glu268 which connect subdomains IA, IB and IIB at the centre of the protein. Interaction with the substrate-binding domain (SBD) of Hsp70 or the BAG domain of BAG1 impairs this subdomain connection and triggers the rotation of subdomain IIA around a hydrophobic helix from subdomain IA. The subdomain rotation is limited by Asp199 and Asp206 from subdomain IIA and clearly defines the open form of the NBD. The open form is further stabilized by a new interaction between Gly230 from subdomain IIB and Ser340 from subdomain IIA. The structure of the NBD in the nucleotide-free state is determined by switching of the inter-subdomain interactions.
    Acta Crystallographica Section D Biological Crystallography 03/2010; 66(Pt 3):223-32. · 12.67 Impact Factor
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    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 2.5A, 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-x(L). Therefore, the Bak dimer structure reported here may represent a pro-apoptotic mode under oxidized conditions.
    Journal of Structural Biology 01/2009; 166(1):32-7. · 3.36 Impact Factor
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    ABSTRACT: Cyclic nucleotide phosphodiesterases (PDEs) catalyze the degradation of the cyclic nucleotides cAMP and cGMP, which are important second messengers. Five of the 11 mammalian PDE families have tandem GAF domains at their N termini. PDE10A may be the only mammalian PDE for which cAMP is the GAF domain ligand, and it may be allosterically stimulated by cAMP. PDE10A is highly expressed in striatal medium spiny neurons. Here we report the crystal structure of the C-terminal GAF domain (GAF-B) of human PDE10A complexed with cAMP at 2.1-angstroms resolution. The conformation of the PDE10A GAF-B domain monomer closely resembles those of the GAF domains of PDE2A and the cyanobacterium Anabaena cyaB2 adenylyl cyclase, except for the helical bundle consisting of alpha1, alpha2, and alpha5. The PDE10A GAF-B domain forms a dimer in the crystal and in solution. The dimerization is mainly mediated by hydrophobic interactions between the helical bundles in a parallel arrangement, with a large buried surface area. In the PDE10A GAF-B domain, cAMP tightly binds to a cNMP-binding pocket. The residues in the alpha3 and alpha4 helices, the beta6 strand, the loop between 3(10) and alpha4, and the loop between alpha4 and beta5 are involved in the recognition of the phosphate and ribose moieties. This recognition mode is similar to those of the GAF domains of PDE2A and cyaB2. In contrast, the adenine base is specifically recognized by the PDE10A GAF-B domain in a unique manner, through residues in the beta1 and beta2 strands.
    Journal of Biological Chemistry 08/2008; 283(28):19657-64. · 4.65 Impact Factor
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    ABSTRACT: The target of diphtheria toxin is the diphthamide residue in translation elongation factor 2 (EF-2), which is generated by a three-step post-translational modification of a specific histidine residue in the EF-2 precursor. In the second modification step, an S-adenosylmethionine-dependent methyltransferase, diphthine synthase (DS), catalyzes the trimethylation of the EF-2 precursor. The homodimeric crystal structures of the archaeal diphthine synthases from Pyrococcus horikoshii OT3 and Aeropyrum pernix K1 have been determined. These structures share essentially the same overall fold as the cobalt-precorrin-4 methyltransferase CbiF, confirming that DS belongs to the dimeric class III family of methyltransferases. In the P. horikoshii DS dimer, only one of the two active sites binds the reaction product S-adenosyl-L-homocysteine (AdoHcy), while the other active site contains no ligand. This asymmetric AdoHcy binding may be a consequence of intra-domain and inter-domain movements upon binding of AdoHcy at one of the two sites. These movements disrupt the twofold dimeric symmetry of the DS dimer and probably cause lower AdoHcy affinity at the other binding site.
    Acta Crystallographica Section D Biological Crystallography 05/2008; 64(Pt 4):397-406. · 14.10 Impact Factor
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    ABSTRACT: The gene encoding TTHA1544 is a singleton found in the Thermus thermophilus HB8 genome and encodes a 131-amino-acid protein. The crystal structure of TTHA1544 has been determined at 2.0 A resolution by the single-wavelength anomalous dispersion method in order to elucidate its function. There are two molecules in the asymmetric unit. Each molecule consists of four alpha-helices and six beta-strands, with the beta-strands composing a central beta-sheet. A structural homology search revealed that the overall structure of TTHA1544 resembles the alpha/beta-hydrolase fold, although TTHA1544 lacks the catalytic residues of a hydrolase. These results suggest that TTHA1544 represents the minimized alpha/beta-hydrolase fold and that an additional component would be required for its activity.
    Acta Crystallographica Section F Structural Biology and Crystallization Communications 01/2008; 63(Pt 12):993-7. · 0.55 Impact Factor
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    ABSTRACT: Interleukin (IL)-15 is a pleiotropic cytokine that plays a pivotal role in both innate and adaptive immunity. IL-15 is unique among cytokines due to its participation in a trans signaling mechanism in which IL-15 receptor alpha (IL-15Ralpha) from one subset of cells presents IL-15 to neighboring IL-2Rbeta/gammac-expressing cells. Here we present the crystal structure of IL-15 in complex with the sushi domain of IL-15Ralpha. The structure reveals that the alpha receptor-binding epitope of IL-15 adopts a unique conformation, which, together with amino acid substitutions, permits specific interactions with IL-15Ralpha that account for the exceptionally high affinity of the IL-15.IL-15Ralpha complex. Interestingly, analysis of the topology of IL-15 and IL-15Ralpha at the IL-15.IL-15Ralpha interface suggests that IL-15 should be capable of participating in a cis signaling mechanism similar to that of the related cytokine IL-2. Indeed, we present biochemical data demonstrating that IL-15 is capable of efficiently signaling in cis through IL-15Ralpha and IL-2Rbeta/gammac expressed on the surface of a single cell. Based on our data we propose that cis presentation of IL-15 may be important in certain biological contexts and that flexibility of IL-15Ralpha permits IL-15 and its three receptor components to be assembled identically at the ligand-receptor interface whether IL-15 is presented in cis or trans. Finally, we have gained insights into IL-15.IL-15Ralpha.IL-2Rbeta.gammac quaternary complex assembly through the use of molecular modeling.
    Journal of Biological Chemistry 01/2008; 282(51):37191-204. · 4.65 Impact Factor
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    ABSTRACT: Familial oncocytic thyroid carcinoma is associated with a missense mutation, P308Q, in the C-terminal domain of Tim44. Tim44 is the mitochondrial inner-membrane translocase subunit and it functions as a membrane anchor for the mitochondrial heat-shock protein 70 (mtHsp70). Here, the crystal structure of the human Tim44 C-terminal domain complexed with pentaethylene glycol has been determined at 1.9 A resolution. The overall structure resembles that of the nuclear transport factor 2-like domain. In the crystal structure, pentaethylene glycol molecules are associated at two potential membrane-binding sites: the large hydrophobic cavity and the highly conserved loop between the alpha1 and alpha2 helices near Pro308. A comparison with the yeast homolog revealed that lipid binding induces conformational changes around the alpha1-alpha2 loop, leading to slippage of the alpha1 helix along the large beta-sheet. These changes may play important roles in the translocation of polypeptides across the mitochondrial inner membrane.
    Acta Crystallographica Section D Biological Crystallography 01/2008; 63(Pt 12):1225-34. · 14.10 Impact Factor
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    ABSTRACT: Interleukin (IL)-15 is a pleiotropic cytokine that plays a pivotal role in both innate and adaptive immunity. IL-15 is unique among cytokines due to its participation in a trans signaling mechanism in which IL-15 receptorα (IL-15Rα) from one subset of cells presents IL-15 to neighboring IL-2Rβ/γc-expressing cells. Here we present the crystal structure of IL-15 in complex with the sushi domain of IL-15Rα. The structure reveals that theα receptor-binding epitope of IL-15 adopts a unique conformation, which, together with amino acid substitutions, permits specific interactions with IL-15Rα that account for the exceptionally high affinity of the IL-15·IL-15Rα complex. Interestingly, analysis of the topology of IL-15 and IL-15Rα at the IL-15·IL-15Rα interface suggests that IL-15 should be capable of participating in a cis signaling mechanism similar to that of the related cytokine IL-2. Indeed, we present biochemical data demonstrating that IL-15 is capable of efficiently signaling in cis through IL-15Rα and IL-2Rβ/γc expressed on the surface of a single cell. Based on our data we propose that cis presentation of IL-15 may be important in certain biological contexts and that flexibility of IL-15Rα permits IL-15 and its three receptor components to be assembled identically at the ligand-receptor interface whether IL-15 is presented in cis or trans. Finally, we have gained insights into IL-15·IL-15Rα·IL-2Rβ·γc quaternary complex assembly through the use of molecular modeling.
    Journal of Biological Chemistry 12/2007; 282(51):37191-37204. · 4.65 Impact Factor
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    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 A resolution. The Nup35 RRM domain monomer adopts the characteristic betaalphabetabetaalphabeta 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 beta-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 beta-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 beta4 strand of one Nup35 molecule is sandwiched by the aromatic side-chains of Phe178 in the beta1 strand and Trp209 in the beta3 strand of the other Nup35 molecule in the dimer. This structure reveals a new homodimerization mode of the RRM domain.
    Journal of Molecular Biology 11/2006; 363(1):114-24. · 3.91 Impact Factor
  • Proteins Structure Function and Bioinformatics 08/2006; 64(1):284-7. · 3.34 Impact Factor
  • Proteins Structure Function and Bioinformatics 01/2005; 57(4):883-7. · 3.34 Impact Factor