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Journal Articles

Structural basis for acceptor-substrate recognition of UDP-glucose: anthocyanidin 3-${it O}$-glucosyltransferase from ${it Clitoria ternatea}$

Hiromoto, Takeshi; Honjo, Eijiro*; Noda, Hisanobu*; Tamada, Taro; Kazuma, Kohei*; Suzuki, Masahiko*; Blaber, M.; Kuroki, Ryota

Protein Science, 24(3), p.395 - 407, 2015/03

 Times Cited Count:66 Percentile:89.27(Biochemistry & Molecular Biology)

UDP-glucose: anthocyanidin 3-${it O}$-glucosyltransferase (UGT78K6) from ${it Clitoria ternatea}$ catalyzes the transfer of glucose from UDP-glucose to anthocyanidins such as delphinidin. To understand the acceptor-recognition scheme of UGT78K6, the crystal structure of UGT78K6 and its complex forms with anthocyanidin delphinidin and petunidin, and flavonol kaempferol were determined to resolutions of 1.85 ${AA}$, 2.55 ${AA}$, 2.70 ${AA}$ and 1.75 ${AA}$ respectively. The anthocyanidin- and flavonol-acceptor binding details are almost identical in each complex structure, although the glucosylation activities against each acceptor were significantly different. The acceptor substrates in UGT78K6 are reversely bound to its binding site by a 180$$^{circ}$$ rotation about the O1-O3 axis of the flavonoid backbones observed in ${it Vv}$GT1 and UGT78G1. These substrate recognition schemes suggest the potential for controlled synthesis of natural pigments.

Journal Articles

Crystal structure of UDP-glucose:anthocyanidin 3-${it O}$-glucosyltransferase from ${it Clitoria ternatea}$

Hiromoto, Takeshi; Honjo, Eijiro*; Tamada, Taro; Noda, Hisanobu*; Kazuma, Kohei*; Suzuki, Masahiko*; Kuroki, Ryota

Journal of Synchrotron Radiation, 20(6), p.894 - 898, 2013/11

 Times Cited Count:39 Percentile:86.30(Instruments & Instrumentation)

Flowers of the butterfly pea (${it Clitoria ternatea}$) accumulate a group of polyacylated anthocyanins, named ternatins, in their petals. The first step in ternatin biosynthesis is the transfer of glucose from UDP-glucose to anthocyanidins such as delphinidin, a reaction catalyzed in ${it C. ternatea}$ by UDP-glucose:anthocyanidin 3-${it O}$-glucosyltransferase (${it Ct}$3GT-A; AB185904). To elucidate the structure-function relationship of ${it Ct}$3GT-A, recombinant ${it Ct}$3GT-A was expressed in ${it Escherichia coli}$ and its tertiary structure was determined to 1.85 ${AA}$, resolution by using X-ray crystallography. The structure of ${it Ct}$3GT-A shows a common folding topology, the GT-B fold, comprised of two Rossmann-like $$beta$$/$$alpha$$/$$beta$$ domains and a cleft located between the N- and C-domains containing two cavities that are used as binding sites for the donor (UDP-Glc) and acceptor substrates. By comparing the structure of ${it Ct}$3GT-A with that of the flavonoid glycosyltransferase ${it Vv}$GT1 from red grape (${it Vitis vinifera}$) in complex with UDP-2-deoxy-2-fluoro glucose and kaempferol, locations of the catalytic His-Asp dyad and the residues involved in recognizing UDP-2-deoxy-2-fluoro glucose were essentially identical in ${it Ct}$3GT-A, but certain residues of ${it Vv}$GT1 involved in binding kaempferol were found to be substituted in ${it Ct}$3GT-A. These findings are important for understanding the differentiation of acceptor-substrate recognition in these two enzymes.

Journal Articles

Distinct structural requirements for interleukin-4 (IL-4) and IL-13 binding to the shared IL-13 receptor facilitate cellular tuning of cytokine responsiveness

Ito, Takachika*; Suzuki, Shoichi*; Kanaji, Sachiko*; Shiraishi, Hiroshi*; Ota, Shoichiro*; Arima, Kazuhiko*; Tanaka, Go*; Tamada, Taro; Honjo, Eijiro*; Garcia, K. C.*; et al.

Journal of Biological Chemistry, 284(36), p.24289 - 24296, 2009/09

 Times Cited Count:24 Percentile:46.21(Biochemistry & Molecular Biology)

Both IL-4 and IL-13 can bind to the shared receptor composed of the IL-4 receptor $$alpha$$ chain and the IL-13 receptor $$alpha$$-1 chain (IL-13R$$alpha$$1); however, the assembly mechanisms of these ligands to the receptor is different, enabling the principal functions of these ligands to be different. We have previously shown that the N-terminal Ig-like domain in IL-13R$$alpha$$1, called the D1 domain, is the specific and critical binding unit for IL-13. However, it has still remained obscure which the amino acid has specific binding capacity to IL-13 and why the D1 domain acts as the binding site for IL-13, but not IL-4. To address these questions, in this study, we performed the mutational analyses for the D1 domain, combining the structural data to identify the amino acids critical for binding to IL-13. Mutations of Lys76, Lys77, or Ile78 in c' strand in which the crystal structure showed interact with IL-13 and those of Trp65 and Ala79 adjacent to the interacting site, resulted in significant impairment of IL-13 binding, demonstrating that these amino acids generate the binding site. Furthermore, mutations of Val35, Leu38, or Val42 at N-terminal $$beta$$-strand also resulted in loss of IL-13 binding, probably from decrease structural stability. None of the mutations employed here affected IL-4 binding. These results demonstrate that the hydrophobic patch composed of Lys76, Lys77, and Ile78 is the IL-13 recognition site and solidify our understanding that the differential requirements of the D1 domain in IL-13R$$alpha$$1 allows the shared receptor to respond differentially to IL-4 and IL-13.

Journal Articles

Structure of HIV-1 protease in complex with potent inhibitor KNI-272 determined by high-resolution X-ray and neutron crystallography

Adachi, Motoyasu; Ohara, Takashi; Kurihara, Kazuo; Tamada, Taro; Honjo, Eijiro; Okazaki, Nobuo; Arai, Shigeki; Shoyama, Yoshinari; Kimura, Kaname*; Matsumura, Hiroyoshi*; et al.

Proceedings of the National Academy of Sciences of the United States of America, 106(12), p.4641 - 4646, 2009/03

 Times Cited Count:113 Percentile:90.57(Multidisciplinary Sciences)

To further understand the catalytic mechanism and inhibitor recognition of HIV-1 protease, we need to determine the locations of key hydrogen atoms in the catalytic aspartates Asp25 and Asp125. The structure of HIV-1 protease in complex with transition-state analog KNI-272 was determined by combined neutron crystallography at 1.9 ${AA}$ resolution and X-ray crystallography at 1.4 ${AA}$ resolution. The resulting structural data shows that the catalytic residue Asp25 is protonated and that Asp125 is deprotonated. The proton on Asp25 makes a hydrogen bond with the carbonyl group of the allophenylnorstatine group in KNI-272. The deprotonated Asp125 bonds to the hydroxyl proton of Apns. The results provide direct experimental evidence for proposed aspects of the catalytic mechanism of HIV-1 protease; and can therefore contribute substantially to the development of specific inhibitors for therapeutic application.

Journal Articles

Expression of the extracellular region of the human interleukin-4 receptor $$alpha$$ chain and interleukin-13 receptor $$alpha$$1 chain by a silkworm-baculovirus system

Honjo, Eijiro; Shoyama, Yoshinari; Tamada, Taro; Shigematsu, Hideki*; Hatanaka, Takaaki*; Kanaji, Sachiko*; Arima, Kazuhiko*; Ito, Yuji*; Izuhara, Kenji*; Kuroki, Ryota

Protein Expression and Purification, 60(1), p.25 - 30, 2008/07

 Times Cited Count:13 Percentile:33.05(Biochemical Research Methods)

The receptor binding to Interleukin (IL)-13 is composed of the IL-13 receptor $$alpha$$1 chain (IL-13R $$alpha$$1) and the IL-4 receptor $$alpha$$ chain (IL-4R $$alpha$$). In order to investigate the interaction of IL-13 with IL-13R $$alpha$$1 and IL-4R $$alpha$$, the DNA fragments coding the extracellular regions of human IL-13R $$alpha$$1 and the IL-4R $$alpha$$ were fused with mouse Fc and expressed by a silkworm-baculovirus system. The expressed receptors were successfully purified by affinity chromatography using protein A, and the Fc region was removed by thrombin digestion. Size exclusion chromatography and SPR analysis revealed that mixture of IL-13 and IL-13R$$alpha$$1 showed predominant affinity to IL-4R$$alpha$$, although neither detectable affinity of IL-13 nor IL-13R$$alpha$$1 was observed against IL-4R$$alpha$$. Combining these data with the moderate affinity of IL-13 to IL-13R$$alpha$$1, this indicates that IL-13 first binds to IL-13R$$alpha$$1 and recruits consequently to IL-4R.

Journal Articles

Mutagenesis of the crystal contact of acidic fibroblast growth factor

Honjo, Eijiro; Tamada, Taro; Adachi, Motoyasu; Kuroki, Ryota; Meher, A.*; Blaber, M.*

Journal of Synchrotron Radiation, 15(3), p.285 - 287, 2008/05

 Times Cited Count:5 Percentile:29.40(Instruments & Instrumentation)

We attempt to improve a crystal contact of human acidic fibroblast growth factor (haFGF1) to control the crystal growth because haFGF crystallizes only as a thin-plate form. X-ray crystal analysis of haFGF showed that side chain Glu81, located at a crystal contact between haFGF molecules related by crystallographic symmetry, were in close proximity, suggesting that charge-repulsion may disrupt suitable crystal-packing interaction. To investigate whether the Glu residue affects crystal packing, we constructed haFGF mutants. Although crystals of Ala, Val and Leu mutants were grown as a thin-plate form by the same precipitant (formate) as wild type, crystals of Ser and Thr mutants were grown as a more bulky form. X-ray structural analysis of Ser and Thr mutants determined at 1.5${AA}$ resolution revealed that hydroxyl groups of Ser and Thr were linked by hydrogen bonds mediated by formate used as a precipitant.

Journal Articles

Know-how for recombinant protein expression and purification

Honjo, Eijiro; Kuroki, Ryota; Kobori, Hiroshi*; Takakura, Hikaru*; Yahata, Kazuhide*; Sone, Takefumi*; Imamoto, Fumio*; Moriyama, Tatsuya*

Tampakushitsu Seisei To Toriatsukai No Kotsu, p.135 - 178, 2007/10

Protein expression and purification are important techniques for protein scientists. Especially in the field of molecular biology, the researchers often requires for some know-how on protein handling based on biochemical information. This book is aimed at introducing an overview of recombinant protein expression and purification, and furthermore providing experimental know-how about the selection of analytical reagents or instruments.

Journal Articles

"Crystal lattice engineering", an approach to engineer protein crystal contacts by creating intermolecular symmetry; Crystallization and structure determination of a mutant human RNase 1 with a hydrophobic interface of leucines

Yamada, Hidenori*; Tamada, Taro; Kosaka, Megumi*; Miyata, Kohei*; Fujiki, Shinya*; Tano, Masaru*; Moriya, Masayuki*; Yamanishi, Mamoru*; Honjo, Eijiro; Tada, Horiko*; et al.

Protein Science, 16(7), p.1389 - 1397, 2007/07

 Times Cited Count:41 Percentile:60.77(Biochemistry & Molecular Biology)

In an attempt to control protein incorporation in a crystal lattice, a leucine zipper-like hydrophobic interface (comprising four leucine residues) was introduced into a helical region (helix 2) of the human pancreatic ribonuclease 1 (RNase 1) that was predicted to form a suitable crystallization interface. Although crystallization of wild type RNase 1 has not yet been reported, the RNase 1 mutant having four leucines (4L-RNase 1) was successfully crystallized under several different conditions. The crystal structures were subsequently determined by X-ray crystallography by molecular replacement using the structure of bovine RNase A. The overall structure of 4L-RNase 1 is quite similar to that of the bovine RNase A, and the introduced leucine residues formed the designed crystal interface. To further characterize the role of the introduced leucine residues in crystallization of RNase 1, the number of leucines was reduced to three or two (3L- and 2L-RNase 1, respectively). Both mutants crystallized and a similar hydrophobic interface as in 4L-RNase 1 was observed. A related approach to engineer crystal contacts at helix 3 of RNase 1 (N4L-RNase 1) was also evaluated. N4L-RNase 1 also successfully crystallized, and formed the expected hydrophobic packing interface. These results suggest that appropriate introduction of a leucine zipper-like hydrophobic interface can promote intra molecular symmetry for more efficient protein crystallization in crystal lattice engineering efforts.

Journal Articles

Activation mechanism of granulocyte colony-stimulating factor receptor

Tamada, Taro; Honjo, Eijiro; Kuroki, Ryota

Nihon Kessho Gakkai-Shi, 48(6), p.429 - 435, 2006/12

The crystal structure of the signaling complex of human granulocyte colony-stimulating factor (GCSF) and ligand binding region of human GCSF receptor (GCSF-R) has been determined to 2.8 ${AA}$ resolution. The GCSF:GCSF-R complex formed a 2:2 stoichiometry via a cross-over interaction between the Ig-like domains of GCSF-R and GCSF. The conformation of the complex is quite different to that between human GCSF and the CRH domain of mouse GCSF-R. This cross-over homodimerization necessary for GCSF-R activation is consistent with previously reported thermodynamic and mutational studies.

Journal Articles

Homodimeric cross-over structure of the human granulocyte colony-stimulating factor (GCSF) receptor signaling complex

Tamada, Taro; Honjo, Eijiro; Maeda, Yoshitake*; Okamoto, Tomoyuki*; Ishibashi, Matsujiro*; Tokunaga, Masao*; Kuroki, Ryota

Proceedings of the National Academy of Sciences of the United States of America, 103(9), p.3135 - 3140, 2006/02

 Times Cited Count:96 Percentile:84.60(Multidisciplinary Sciences)

A crystal structure of the signaling complex between human granulocyte colony-stimulating factor (GCSF), and a ligand binding region of GCSF receptor (GCSF-R), has been determined to 2.8${AA}$ resolution. The GCSF:GCSF-R complex formed a 2:2 stoichiometry via a cross-over interaction between the Ig-like domains of GCSF-R and GCSF. The conformation of the complex is quite different to that between human GCSF and the CRH domain of mouse GCSF-R, but similar to that of the interleukin-6 (IL-6)/gp130 signaling complex. The Ig-like domain cross-over structure necessary for GCSF-R activation is consistent with previously reported thermodynamic and mutational analyses.

Journal Articles

Crystallization of a 2:2 complex of Granulocyte-Colony Stimulating Factor (GCSF) with the ligand-binding region of the GCSF receptor

Honjo, Eijiro; Tamada, Taro; Maeda, Yoshitake*; Koshiba, Takumi*; Matsukura, Yasuko*; Okamoto, Tomoyuki*; Ishibashi, Matsujiro*; Tokunaga, Masao*; Kuroki, Ryota

Acta Crystallographica Section F, 61(8), p.788 - 790, 2005/08

 Times Cited Count:7 Percentile:54.91(Biochemical Research Methods)

Granulocyte colony-stimulating factor (GCSF) receptor receives signals for regulating the proliferation and differentiation of the precursor cells of granulocytes. The complex composed of two GCSFs and two GCSF receptors was crystallized. The crystal of the complex was grown in 1.0 M sodium formate and 0.1 M sodium acetate (pH4.6). It belongs to the space group ${it P}$4$$_{1}$$2$$_{1}$$2 (or its enantiomorph ${it P}$4$$_{3}$$2$$_{1}$$2) with unit cell dimensions of ${it a}$ = ${it b}$ = 110.1 ${AA}$, ${it c}$ = 331.8 ${AA}$. However, the diffraction data from the crystal beyond 5 ${AA}$ resolution could not be collected. Since the heterogeneity of GCSF receptor seems to interrupt growth of good quality crystals, the GCSF receptor was fractionated by achromatography. Crystals of GCSF/fractionated GCSF receptor complex were grown as a new crystal form in 0.2 M ammonium phosphate. The new crystal diffracts beyond 3.0 ${AA}$ resolution and belongs to space group ${it P}$3$$_{1}$$21 (or its enantiomorph ${it P}$3$$_{2}$$21) with unit-cell parameters ${it a}$ = ${it b}$ = 134.8, ${it c}$ = 105.7 ${AA}$.

Oral presentation

Challenge to develop neutron crystallography for drug discovery

Tamada, Taro; Arai, Shigeki; Honjo, Eijiro; Kuroki, Ryota

no journal, , 

no abstracts in English

Oral presentation

Practical methods to prepare protein sample for three-dimentional structure analysis

Honjo, Eijiro

no journal, , 

no abstracts in English

Oral presentation

Structural and thermodynamic change upon the antigen binding of human thrombopoietin neutralizing IgG TN1

Arai, Shigeki; Tamada, Taro; Honjo, Eijiro; Maeda, Yoshitake*; Kuroki, Ryota

no journal, , 

The mouse antibody TN1 recognizes the human thrombopoietin (hTPO) that primarily stimulates megakaryocytopoiesis and platelet production. By the structural comparison between TN1-Fab itselt (hTPO unbound form) and hTPO bound form of TN1-Fab (PDB id 1V7M and 1V7N), we found that the CDR of TN1-Fab need not accompany the large conformational changes of upon TPO recognition. Moreover, the isothermal titration calorimetry showed that the conformational entropy change upon the hTPO binding to the TN1-Fab was $$sim$$ 446.4 kJ/mol/K, corresponding to 2,920 $AA $^{2}$$ burying upon hTPO binding. This change of accessible surface area is larger than that of the previous result (1,580 $AA $^{2}$$) estimated from the crystal structure of hTPO / TN1-Fab complex. Since the CDR structure of TN1-Fab did not change, the change in surface area may be from the conformational change of hTPO upon the binding to Fab.

Oral presentation

Engineering of protein crystal lattice

Honjo, Eijiro

no journal, , 

no abstracts in English

Oral presentation

Expression and preparation of the extra cellular region of receptors

Kuroki, Ryota; Honjo, Eijiro; Tamada, Taro

no journal, , 

Silk worm expresssion system is the rapid system for several protein expressions developed by Katakura Industries company. We have used this system for test expression of the extracellular region of receptors. Every cDNA coding extracellular region of receptors were fused to the cDNA of Fc region of antibody, and cloned into the transfer vector and the vector was transformed into vacuro virus that can infect silk worm. So far, expression of 10 extracellular regions of menbrane proteins were tested using this system, all proteins were successfully expressed. Among these proteins, interleukin-13 receptor alpha 1 and 2, interluekin-4 receptor were purified from the silk worm and their functions were comfirmed.

Oral presentation

Improvement of crystal growth of human acidic FGF by protein engineering for neutron crystallography

Adachi, Motoyasu; Honjo, Eijiro; Tamada, Taro; Blaber, M.*; Kuroki, Ryota

no journal, , 

Human acidic fibroblast growth factor (haFGF) is a powerful mitogen and angiogenic factor. Our objective is to reveal structure function relationships of haFGF by protein neutron crystallography. For the determination of neutron crystal structures, large crystal in size is required. Since haFGF forms thin crystals, we tried improvement of crystal growth by changing amino acids at molecular interface. The analysis using X-ray crystal structure showed that the residue of Glu81 is located near Glu81 generated by crystallographic symmetry. Therefore, Glu81 was replaced with Ala, Val, Leu, Ser and Thr residue. The mutants of E81S and E81T provided larger crystal in size than that of WT. The X-ray structures of E81S and E81T showed that a molecule of formic acid bridges between the two Glu81 residues by hydrogen bonding.

Oral presentation

Several measurements of drug target receptor proteins

Kuroki, Ryota; Tamada, Taro; Honjo, Eijiro; Maeda, Yoshitake*

no journal, , 

Receptor proteins expressed in the surface of the cell are known to be important drug targets for several diseases. The receptor transfer the signal originated from the binding of ligand proteins in to the signal cascades located inside of the cell. The signal transduction usually induced by the association of plural receptor molecules, thus the ligand molecules that help association of receptors are considered to be drug candidates. There are several association schemes are known. For example, the erythropoietin receptors associate with its ligand in 2:1 manner, the granulocyte colony stimulating factor receptor active complex with its ligand in 2:2 manners, and so on. Therefore, it is quite important to investigate the active scheme of receptor-ligand complex to understand how the receptor molecule start signal transduction. In this symposium, we show several examples of the measurements aimed to understand the activation scheme of drug target receptors.

Oral presentation

Expression and purification of extracellular region of interleukin-13 receptor $$alpha$$1 chain and its interaction with its ligand

Kuroki, Ryota; Honjo, Eijiro; Tamada, Taro; Arima, Kazuhiko*; Izuhara, Kenji*

no journal, , 

Interleukin-13 (IL-13) interacts with two types of receptors, the IL-13R$$alpha$$1 chain (IL-13R$$alpha$$1) and the IL-4R$$alpha$$ chain (IL-4R$$alpha$$), that transduce the IL-13 signals. In order to investigate the interaction of IL-13 with IL-13R$$alpha$$1 and IL-4R$$alpha$$, the genes coding the extracellular regions of human IL-13R$$alpha$$1 and the IL-4R$$alpha$$ containing cytokine receptor homologous region (CRH), were fused with human Fc, and expressed by silk worm. After further purification with anion-exchange chromatography, the receptors were used to investigate the ligand-receptor interaction. The size exclusion chromatography showed that association between IL-13 and IL-13Ra1 could not be observed in this condition whereas the clear association of IL-13 and IL-13Ra1 was observed after adding IL-4R$$alpha$$. It indicates that the association between IL-13 and IL-4R$$alpha$$ increase the IL-13 affinity to IL-13R$$alpha$$1.

Oral presentation

Neutron crystallography for investigation of catalytic mechanism of HIV-1 protease

Adachi, Motoyasu; Ohara, Takashi; Kurihara, Kazuo; Tamada, Taro; Honjo, Eijiro; Okazaki, Nobuo; Arai, Shigeki; Shoyama, Yoshinari; Kimura, Kaname*; Matsumura, Hiroyoshi*; et al.

no journal, , 

HIV-1 protease is a dimeric aspartic protease that cleaves the nascent polyproteins of HIV-1 and plays an essential role in viral replication. To further understand the catalytic mechanism of HIV-1 protease, we have determined the crystal structure of HIV-1 protease in complex with a transition state mimetic tripeptide inhibitor, KNI-272 to 1.9 ${AA}$ resolution by neutron crystallography in combination with 1.4 ${AA}$ resolution X-ray diffraction data. Our results indicates that the carbonyl group of allophenylnorstatine in KNI-272 forms a significant hydrogen bond with protonated Asp 25, and the hydrogen atom from the hydroxyl group of Apns forms a remarkable hydrogen bond with the deprotonated Asp125. These results show direct evidence that Asp25 provides a proton to carbonyl group of substrate and Asp125 contributes to activate the attacking water molecule as a nucleophile.

42 (Records 1-20 displayed on this page)