Tsuyoshi Inoue - Academia.edu (original) (raw)

Papers by Tsuyoshi Inoue

The Journal of Biochemistry

The continuous emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants... more The continuous emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants associated with the adaptive evolution of the virus is prolonging the global coronavirus disease 2019 (COVID-19) pandemic. The modification of neutralizing antibodies based on structural information is expected to be a useful approach to rapidly combat emerging variants. A dimerized variable domain of heavy chain of heavy chain antibody (VHH) P17 that has highly potent neutralizing activity against SARS-CoV-2 has been reported but the mode of interaction with the epitope remains unclear. Here, we report the X-ray crystal structure of the complex of monomerized P17 bound to the SARS-CoV-2 receptor binding domain (RBD) and investigated the binding activity of P17 toward various variants of concern (VOCs) using kinetics measurements. The structure revealed details of the binding interface and showed that P17 had an appropriate linker length to have an avidity effect and recognize a wide ra...

The FEBS Journal, 2018

Tardigrades, also known as water bears, can survive extreme conditions. For example, tardigrades ... more Tardigrades, also known as water bears, can survive extreme conditions. For example, tardigrades have high tolerance to extreme desiccation because they can enter an anhydrobiotic state, in which they show no or nearly undetectable metabolic processes. Proteins from anhydrobiotic tardigrades with low homology to known proteins from other organisms are new potential targets for structural genomics. Here, we present spectroscopic and structural characterization of an unprecedented globin protein (Kumaglobin: Kgb) found in an anhydrobiotic tardigrade. Spectroscopy reveals that Kgb contains hexacoordinated low-spin heme, which is not capable of binding to hydrogen sulfide (H 2 S) unlike other globin proteins, such as neuroglobin. Interestingly however, when distal histidine is replaced with alanine, H 2 S is capable of binding to heme, implying that the distal histidine of Kgb binds tightly to heme. The overall structure of Kgb at 1.5 A resolution shows high resemblance to well-characterized eukaryotic globin proteins, such as myoglobin and cytoglobin. However, the heme coordination geometry in Kgb is unique because the distal histidinyl ligand is located at the 11th position of helix E while it is found at 7th position on helix E in many known globin proteins. The unusual conformation of distal histidine in Kgb is stabilized by a hydrogen bond with the carbonyl O atom of A103. Furthermore, bulky residues exist around the heme cofactor, resulting in a ruffling conformation of the porphyrin ring. Based on our study, Kgb is thought to be involved in electron transfer or enzymatic reactions rather than transporting or storing ligands. Database Structural data are available in the Protein Data Bank under the accession numbers 5ZIQ (Kgb4-SR) and 5ZM9 (Kgb7-house).

ACS Chemical Biology, 2017

In the effort to combat antibiotic resistance, inhibitors of the essential bacterial protein FtsZ... more In the effort to combat antibiotic resistance, inhibitors of the essential bacterial protein FtsZ have emerged as a promising new class of compounds with clinical potential. One such FtsZ inhibitor (TXA707) is associated with potent activity against clinical isolates of methicillinresistant Staphylococcus aureus (MRSA) that are resistant to current standard-of-care antibiotics. However, mutations in S. aureus FtsZ (SaFtsZ) that confer resistance to TXA707 have been observed, with mutations in the Gly196 and Gly193 residues being among the most prevalent. Here, we describe structural studies of an FtsZ inhibitor, TXA6101, which retains activity against MRSA isolates that express either G196S or G193D mutant FtsZ. We present the crystal structures of TXA6101 in complex with both wild-type SaFtsZ and G196S mutant SaFtsZ, as well the crystal structure of TXA707 in complex with wild-type SaFtsZ. Comparison of the three structures reveals a molecular basis for the differential targeting abilities of TXA6101 and TXA707. The greater structural flexibility of TXA6101 relative to TXA707 enables TXA6101 to avoid steric clashes with Ser196 and Asp193. Our structures also demonstrate that the binding of TXA6101 induces previously unobserved conformational rearrangements of SaFtsZ residues in the binding pocket. In aggregate, the structures reported in this work reveal key factors for overcoming drug resistance mutations in SaFtsZ and offer a structural basis for the design of FtsZ inhibitors with enhanced antibacterial potency and reduced susceptibility to mutational resistance.

The Journal of biological chemistry, Jan 24, 2017

Tumor necrosis factor-α (TNF) exerts its biological effect through two types of receptors, p55 TN... more Tumor necrosis factor-α (TNF) exerts its biological effect through two types of receptors, p55 TNF receptor (TNFR1) and p75 TNF receptor (TNFR2). An inflammatory response is known to be induced mainly by TNFR1, whereas an anti-inflammatory reaction is thought to be mediated by TNFR2 in some autoimmune diseases. We have been investigating the use of an antagonistic TNF mutant (R1antTNF) to reveal the pharmacological effect of TNFR1-selective inhibition as a new therapeutic modality. Here, we aimed to further improve and optimize the activity and behavior of this mutant protein both in vitro and in vivo. Specifically, we examined a trimeric structural fusion of R1antTNF, formed via the introduction of short peptide linkers, as a strategy to enhance bioactivity and molecular stability. By comparative analysis with R1antTNF, the trimeric fusion, referred to as single-chain R1antTNF (scR1antTNF), was found to retain in vitro molecular properties of receptor selectivity and antagonistic a...

PloS one, 2016

Posttranslational modifications (PTMs) of proteins play a crucial role in regulating protein-prot... more Posttranslational modifications (PTMs) of proteins play a crucial role in regulating protein-protein interactions, enzyme activity, subcellular localization, and stability of the protein. SET domain, bifurcated 1 (SETDB1) is a histone methyltransferase that regulates the methylation of histone H3 on lysine 9 (H3K9), gene silencing, and transcriptional repression. The C-terminal region of SETDB1 is a key site for PTMs, and is essential for its enzyme activity in mammalian and insect cells. In this study, we aimed to evaluate more precisely the effect of PTMs on the H3K9 methyltransferase activity of SETDB1. Using mass spectrometry analysis, we show that the C-terminal region of human SETDB1 purified from insect cells is ubiquitinated. We also demonstrate that the ubiquitination of lysine 867 of the human SETDB1 is necessary for full H3K9 methyltransferase activity in mammalian cells. Finally, we show that SETDB1 ubiquitination regulates the expression of its target gene, serpin pepti...

Proceedings of the National Academy of Sciences, 2016

Significance Copper nitrite reductase (CuNiR) is involved in denitrification of the nitrogen cycl... more Significance Copper nitrite reductase (CuNiR) is involved in denitrification of the nitrogen cycle. Synchrotron X-rays rapidly reduce copper sites and decompose the substrate complex structure, which has made crystallographic studies of CuNiR difficult. Using femtosecond X-ray free electron lasers, we determined intact structures of CuNiR with and without nitrite. Based on the obtained structures, we proposed a redox-coupled proton switch model, which provides an explanation for proton-coupled electron transfer (PCET) in CuNiR. PCET is widely distributed through biogenic processes including respiratory and photosynthetic systems and is highly expected to be incorporated into bioinspired molecular devices. Our study also establishes the foundation for future studies on PCET in other systems.

The Journal of biological chemistry, 2015

Epiregulin (EPR) is a ligand of the epidermal growth factor (EGF) family that upon binding to its... more Epiregulin (EPR) is a ligand of the epidermal growth factor (EGF) family that upon binding to its epidermal growth factor receptor (EGFR) stimulates proliferative signaling, especially in colon cancer cells. Here, we describe the three-dimensional structure of the EPR antibody (the 9E5(Fab) fragment) in the presence and absence of EPR. Among the six complementarity-determining regions (CDRs), CDR1-3 in the light chain and CDR2 in the heavy chain predominantly recognize EPR. In particular, CDR3 in the heavy chain dramatically moves with cis-trans isomerization of Pro103. A molecular dynamics simulation and mutational analyses revealed that Arg40 in EPR is a key residue for the specific binding of 9E5 IgG. From ITC analysis, the dissociation constant is determined to be 6.5 nM. Surface plasmon resonance analysis revealed that the dissociation rate of 9E5 IgG is extremely slow. The superimposed structure of 9E5(Fab)-EPR on the known complex structure of EGF-EGFR showed that the 9E5(Fab...

Acta crystallographica. Section F, Structural biology communications, 2015

β-Conglycinin is a major seed storage protein in soybeans, which are an important source of prote... more β-Conglycinin is a major seed storage protein in soybeans, which are an important source of protein. The major subunits (α, α' and β) of β-conglycinin are sorted to protein-storage vacuoles in seed cells. Vacuolar sorting receptor (VSR) is an integral membrane protein that recognizes the sorting determinant of vacuolar proteins, including β-conglycinin, and regulates their sorting process. Vacuolar sorting determinants of the α' and β subunits of β-conglycinin exist in their C-terminal peptides. Here, the preliminary X-ray diffraction analysis of the binding domain of soybean VSR crystallized with the peptide responsible for the sorting determinant in β-conglycinin is reported. X-ray diffraction data were collected to a resolution of 3.5 Å. The crystals belonged to space group P3121, with unit-cell parameters a = b = 116.4, c = 86.1 Å.

Journal of biochemistry, Jan 2, 2015

For a multistep pre-targeting method using antibodies, a streptavidin mutant with low immunogenic... more For a multistep pre-targeting method using antibodies, a streptavidin mutant with low immunogenicity, termed low immunogenic streptavidin mutant No. 314 (LISA-314), was produced previously as a drug delivery tool. However, endogenous biotins (BTNs) with high affinity (Kd < 10(-10) M) for the binding pocket of LISA-314 prevents access of exogenous BTN-labelled anticancer drugs. In this study, we improve the binding pocket of LISA-314 to abolish its affinity for endogenous BTN species, therefore ensuring that the newly designed LISA-314 binds only artificial BTN analogue. The replacement of three amino acid residues was performed in two steps to develop a mutant termed V212, which selectively binds to 6-(5-((3aS,4S,6aR)-2-iminohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanamido)hexanoic acid (iminobiotin long tail, IMNtail). Surface plasmon resonance results showed that V212 has a Kd value of 5.9 × 10(-7) M towards IMNtail, but no binding affinity for endogenous BTN species. This V...

Protein science : a publication of the Protein Society, 2015

ROBO1, fibronectin Type-III domain (Fn)-containing protein, is a novel immunotherapeutic target f... more ROBO1, fibronectin Type-III domain (Fn)-containing protein, is a novel immunotherapeutic target for hepatocellular carcinoma in humans. The crystal structure of the antigen-binding fragment (Fab) of B2212A, the monoclonal antibody against the third Fn domain (Fn3) of ROBO1, was determined in pursuit of antibody drug for hepatocellular carcinoma. This effort was conducted in the presence or absence of the antigen, with the chemical features being investigated by determining the affinity of the antibody using molecular dynamics (MD) and thermodynamics. The structural comparison of B2212A Fab between the complex and the free form revealed that the interfacial Tyr(L) 50 (superscripts L, H, and F stand for the residues in the light chain, heavy chain, and Fn3, respectively) played important roles in Fn3 recognition. That is, the aromatic ring of Tyr(L) 50 pivoted toward Phe(F) 68, forming a CH/π interaction and a new hydrogen bond with the carbonyl O atom of Phe(F) 68. MD simulations pre...

Journal of bioscience and bioengineering, Jan 26, 2014

We previously created a low-immunogenic core streptavidin mutant No. 314 (LISA-314) by replacing ... more We previously created a low-immunogenic core streptavidin mutant No. 314 (LISA-314) by replacing six amino-acid residues for use as a delivery tool for an antibody multistep pre-targeting process (Yumura et al., Protein science, 22, 213-221, 2013). Here, we performed high-resolution X-ray structural analyses of LISA-314 and wild-type streptavidin to investigate the effect of substitutions on the protein function and the three-dimensional structure. LISA-314 forms a tetramer in the same manner as wild-type streptavidin. The binding mode of d-biotin in LISA-314 is also completely identical to that in wild-type streptavidin, and conformational changes were observed mostly at the side chains of substituted sites. Any large conformational changes corresponding to the reduction of B factors around the substituted sites were not observed. These results demonstrated the LISA-314 acquired low immunogenicity without losing structural properties of original wild-type streptavidin.

Proceedings of the National Academy of Sciences of the United States of America, Jan 19, 2014

The tomato mosaic virus (ToMV) resistance gene Tm-1 encodes a protein that shows no sequence homo... more The tomato mosaic virus (ToMV) resistance gene Tm-1 encodes a protein that shows no sequence homology to functionally characterized proteins. Tm-1 binds ToMV replication proteins and thereby inhibits replication complex formation. ToMV mutants that overcome this resistance have amino acid substitutions in the helicase domain of the replication proteins (ToMV-Hel). A small region of Tm-1 in the genome of the wild tomato Solanum habrochaites has been under positive selection during its antagonistic coevolution with ToMV. Here we report crystal structures for the N-terminal inhibitory domains of Tm-1 and a natural Tm-1 variant with an I91-to-T substitution that has a greater ability to inhibit ToMV RNA replication and their complexes with ToMV-Hel. Each complex contains a Tm-1 dimer and two ToMV-Hel monomers with the interfaces between Tm-1 and ToMV-Hel bridged by ATP. Residues in ToMV-Hel and Tm-1 involved in antagonistic coevolution are found at the interface. The structural differen...

Structure, 2002

itive reaction of the carboxylation through the oxygenation of ribulose 1,5-bisphosphate carboxyl... more itive reaction of the carboxylation through the oxygenation of ribulose 1,5-bisphosphate carboxylase/oxy-Osaka University Suita, Osaka 565-0871 genase (Rubisco). The photorespiration decreases the efficiency of CO 2 assimilation and is increased under Japan 2 Department of Public Health environmental conditions such as water stress and high temperature [4]. In contrast, C 4 plants have a unique Graduate School of Medicine Kyoto University atmospheric CO 2 pathway involved in PEPC. Unlike Rubisco, PEPC uses HCO 3 Ϫ , instead of CO 2 , as a sub-Sakyo-ku, Kyoto 606-8501 Japan strate and has a high affinity for the relatively inert bicarbonate ion. C 4 form PEPC is more abundantly expressed 3 Division of Integrated Life Science Graduate School of Biostudies in C 4 plants than in C 3 plants and catalyzes the first committed step for the fixation of atmospheric CO 2 dur-Kyoto University Sakyo-ku, Kyoto 606-8502 ing C 4 photosynthesis [2]. In C 4 plants, after carboxylation by PEPC, the resulting C 4 compounds are trans-Japan ferred into the chloroplast in bandle sheath cells and decarboxylated to supply Rubisco with a high concentration of CO 2 [5]. As a consequence, Rubisco oxygen-Summary ation is more effectively suppressed in C 4 plants. The reaction mechanism of PEPC for carboxylation has re-Phosphoenolpyruvate carboxylase (PEPC) catalyzes ceived much attention because PEPC has a high affinity the first step in the fixation of atmospheric CO 2 during for HCO 3 Ϫ and is not inhibited by O 2. C 4 photosynthesis. The crystal structure of C 4 form Recently, trials for introducing C 4-specific genes into maize PEPC (ZmPEPC), the first structure of the plant C 3 plants have been carried out to improve the efficiency PEPCs, has been determined at 3.0 Å resolution. The of CO 2 fixation in C 3 photosynthesis through recombistructure includes a sulfate ion at the plausible binding nant DNA techniques [6]. PEPC genes were also overexsite of an allosteric activator, glucose 6-phosphate. pressed in C 3 plants, such as rice, potato, and tobacco The crystal structure of E. coli PEPC (EcPEPC) com-[7-10]. In particular, high-level expression of maize plexed with Mn 2؉ , phosphoenolpyruvate analog (3,3-PEPC transgenic rice plants was reported to show a dichloro-2-dihydroxyphosphinoylmethyl-2-propenoate), reduction of O 2 inhibition of photosynthesis [7]. In transand an allosteric inhibitor, aspartate, has also been genic potatos, overexpression of the PEPC gene redetermined at 2.35 Å resolution. Dynamic movements sulted in the induction of other C 4 enzymes [10]. Since were found in the ZmPEPC structure, compared with the activities of PEPC in transgenic plants are also reguthe EcPEPC structure, around two loops near the aclated by metabolites, such as malate and glucose tive site. On the basis of these molecular structures, 6-phosphate, and covalent modification by reversible the mechanisms for the carboxylation reaction and for phosphorylation, as in C 4 plants, the properties of PEPC the allosteric regulation of PEPC are proposed. need to be modified by gene engineering suitable for the transgenic plants. Thus, an understanding of the Introduction molecular mechanism of PEPC has been crucial in elucidating the regulation mechanism. PEPC (EC 4.1.1.31) catalyzes the irreversible HCO 3 Ϫ-All known PEPCs are tetrameric enzymes with molecdependent/biotin-independent carboxylation of phosular weights of ‫000,044ف‬ [3]. The amino acid sequences phoenolpyruvate (PEP) in the presence of a divalent of these PEPCs show significant conservation [11]. For cation, such as Mg 2ϩ or Mn 2ϩ , to form oxaloacetate example, ZmPEPC and EcPEPC share 40% identity (Fig-(OAA) and phosphate (Figure 1A) [1-3]. The enzymes ure 2), suggesting that the reaction mechanisms among have been isolated from various organisms, including the enzymes from various organisms are essentially the plants and a variety of bacteria. PEPC in nonphotosynsame. In most cases, the activity of the enzyme is allothetic tissues takes on anaplerotic functions by replensterically controlled by a variety of positive (e.g., glucose ishing C 4 dicarboxylic acids for the syntheses of various 6-phosphate) and negative (e.g., malate and aspartate) cellular constituents and for the maintenance of the citric metabolite effectors. Despite the knowledge of the crysacid cycle [1-3]. On the other hand, higher plants have tal structure of the EcPEPC complexed with aspartate several isoforms of PEPC with different kinetic and regulatory properties that correlate with their respective roles

Proceedings of the National Academy of Sciences, 1999

The crystal structure of phospho enol pyruvate carboxylase (PEPC; EC 4.1.1.31 ) has been determin... more The crystal structure of phospho enol pyruvate carboxylase (PEPC; EC 4.1.1.31 ) has been determined by x-ray diffraction methods at 2.8-Å resolution by using Escherichia coli PEPC complexed with l -aspartate, an allosteric inhibitor of all known PEPCs. The four subunits are arranged in a “dimer-of-dimers” form with respect to subunit contact, resulting in an overall square arrangement. The contents of α-helices and β-strands are 65% and 5%, respectively. All of the eight β-strands, which are widely dispersed in the primary structure, participate in the formation of a single β-barrel. Replacement of a conserved Arg residue (Arg-438) in this linkage with Cys increased the tendency of the enzyme to dissociate into dimers. The location of the catalytic site is likely to be near the C-terminal side of the β-barrel. The binding site for l -aspartate is located about 20 Å away from the catalytic site, and four residues (Lys-773, Arg-832, Arg-587, and Asn-881) are involved in effector bindi...

Nucleic Acids Research, 2010

Aptamers are short single-stranded nucleic acids with high affinity to target molecules and are a... more Aptamers are short single-stranded nucleic acids with high affinity to target molecules and are applicable to therapeutics and diagnostics. Regardless of an increasing number of reported aptamers, the structural basis of the interaction of RNA aptamer with proteins is poorly understood. Here, we determined the 2.15 Å crystal structure of the Fc fragment of human IgG1 (hFc1) complexed with an anti-Fc RNA aptamer. The aptamer adopts a characteristic structure fit to hFc1 that is stabilized by a calcium ion, and the binding activity of the aptamer can be controlled many times by calcium chelation and addition. Importantly, the aptamer-hFc1 interaction involves mainly van der Waals contacts and hydrogen bonds rather than electrostatic forces, in contrast to other known aptamer-protein complexes. Moreover, the aptamer-hFc1 interaction involves human IgG-specific amino acids, rendering the aptamer specific to human IgGs, and not crossreactive to other species IgGs. Hence, the aptamer is a potent alternative for protein A affinity purification of Fc-fusion proteins and therapeutic antibodies. These results demonstrate, from a structural viewpoint, that conformational plasticity and selectivity of an RNA aptamer is achieved by multiple interactions other than electrostatic forces, which is applicable to many protein targets of low or no affinity to nucleic acids.

Journal of Synchrotron Radiation, 2010

Old yellow enzyme (OYE) is an NADPH oxidoreductase which contains flavin mononucleotide as prosth... more Old yellow enzyme (OYE) is an NADPH oxidoreductase which contains flavin mononucleotide as prosthetic group. The X-ray structures of OYE from Trypanosoma cruzi (TcOYE) which produces prostaglandin (PG) F 2 from PGH 2 have been determined in the presence or absence of menadione. The binding motif of menadione, known as one of the inhibitors for TcOYE, should accelerate the structure-based development of novel anti-chagasic drugs that inhibit PGF 2 production specifically.

Journal of Synchrotron Radiation, 2013

Heart-type fatty-acid-binding protein (FABP3), which is a cytosolic protein abundantly found in c... more Heart-type fatty-acid-binding protein (FABP3), which is a cytosolic protein abundantly found in cardiomyocytes, plays a role in trafficking fatty acids throughout cellular compartments by reversibly binding intracellular fatty acids with relatively high affinity. The fluorescent probe 1-anilinonaphthalene-8sulfonate (ANS) is extensively utilized for examining the interaction of ligands with fatty-acid-binding proteins. The X-ray structure of FABP3 was determined in the presence of ANS and revealed the detailed ANS-binding mechanism. Furthermore, four water molecules were clearly identified in the binding cavity. Through these water molecules, the bound ANS molecule forms indirect hydrogen-bond interactions with FABP3. The adipocyte-type fatty-acid-binding protein (FABP4) exhibits 67% sequence identity with FABP3 and its crystal structure is almost the same as that of FABP3. However, FABP4 can bind with a higher affinity to ANS than FABP3. To understand the difference in their ligand specificities, a structural comparison was performed between FABP3-ANS and FABP4-ANS complexes. The result revealed that the orientation of ANS binding to FABP3 is completely opposite to that of ANS binding to FABP4, and the substitution of valine in FABP4 to leucine in FABP3 may result in greater steric hindrance between the side-chain of Leu115 and the aniline ring of ANS.

Journal of Biological Chemistry, 2005

The Journal of Biochemistry, 2013

Copper-containing nitrite reductase (CuNIR) catalyzes the reduction of nitrite (NO À 2) to nitric... more Copper-containing nitrite reductase (CuNIR) catalyzes the reduction of nitrite (NO À 2) to nitric oxide (NO) during denitrification. We determined the crystal structures of CuNIR from thermophilic gram-positive bacterium, Geobacillus thermodenitrificans (GtNIR) in chloride-and formate-bound forms of wild type at 1.15 Å resolution and the nitrite-bound form of the C135A mutant at 1.90 Å resolution. The structure of C135A with nitrite displays a unique g 1-O coordination mode of nitrite at the catalytic copper site (T2Cu), which has never been observed at the T2Cu site in known wildtype CuNIRs, because the mobility of two residues essential to catalytic activity, Asp98 and His244, are sterically restricted in GtNIR by Phe109 on a characteristic loop structure that is found above Asp98 and by an unusually short CHO hydrogen bond observed between His244 and water, respectively. A detailed comparison of the WT structure with the nitrite-bound C135A structure implies the replacement of hydrogen-bond networks around His244 and predicts the flow path of protons consumed by nitrite reduction. On the basis of these observations, the reaction mechanism of GtNIR through the g 1-O coordination manner is proposed.

Journal of Biochemistry, 2007

Orotidine 5 0-monophoshate decarboxylase (OMPDC) catalyses the decarboxylation of orotidine 5 0-m... more Orotidine 5 0-monophoshate decarboxylase (OMPDC) catalyses the decarboxylation of orotidine 5 0-monophosphate (OMP) to uridine 5 0-monophosphate (UMP). Here, we report the X-ray analysis of apo, substrate or product-complex forms of OMPDC from Plasmodium falciparum (PfOMPDC) at 2.7, 2.65 and 2.65 Å , respectively. The structural analysis provides the substrate recognition mechanism with dynamic structural changes, as well as the rearrangement of the hydrogen bond array at the active site. The structural basis of substrate or product binding to PfOMPDC will help to uncover the decarboxylation mechanism and facilitate structure-based optimization of antimalarial drugs.