Biology subjects

Tsumoto, K.

Publications and source records attributed to Tsumoto, K..

At least 19 recordsLinked to original sources

bioRxiv2026

Structural basis for the intestinal protocadherin-based intermicrovillar adhesion complex

The intestinal brush border (BB), composed of densely packed microvilli on enterocytes, is essential for nutrient absorption and host defense. Its organization relies on the intermicrovillar adhesion complex (IMAC), mediated by protocadherins CDHR2 and CDHR5. Despite their clinical relevance in inflammatory bowel disease and several carcinomas, structural details of IMAC assemblies have remained elusive. Herein, we report the Cryo-EM structure of the adhesive complex at 3.4 [A] resolution, revealing a heterotetrameric ensemble composed of a dimer of CDHR2 and a dimer of CDHR5. This assembly ensures uniform adhesive strength between neighboring microvilli, and facilitates hexagonal packing of microvilli. Biophysical analyses and molecular dynamics simulations revealed a kinked, Ca{superscript 2}-free linker between domains EC3 and EC4 of CDHR5 conferring the necessary flexibility to withstand the shear stress caused during intestinal peristalsis. Collectively, these findings provide a structural framework for understanding BB organization and suggest strategies for therapeutics targeting IMAC in intestinal disorders.

biophysics↗

bioRxiv2026

A bacterial thioester domain functions as a pH-responsive covalent adhesion module

Thioester domains (TEDs) represent a family of bacterial adhesin domains that mediate covalent anchoring to target ligands via an intramolecular thioester bond. Although the broad distribution of TEDs among Gram-positive bacteria suggests a critical functional role, the biological significance of this covalent mechanism remains unclear. In this study, we demonstrated that TED-mediated covalent anchoring is reversible and that its equilibrium is regulated by pH. Specifically, SfbI-TED from Group A Streptococcus binds tightly to fibrinogen at physiological pH, whereas mild acidification to pH 6.0 induces rapid dissociation of the complex. Thermodynamic analyses revealed that this pH-responsiveness arises from the intrinsic properties of the thioester bond within the TED. Similar pH-dependent behavior was observed in phylogenetically distinct TEDs, suggesting that pH-responsive adhesion is a conserved feature of the TED family across Gram-positive bacteria. TeaserA reactive thioester bond in a Gram-positive adhesin acts as a pH sensor to enable environment-responsive adhesion.

biochemistry↗

bioRxiv2025

True-atomic-resolution insights into the interactions of antibiotic rifampicin and rifamycin derivatives with orphan CYP143 of M.tuberculosis.

Analysis of drug-resistant strains of M.tuberculosis revealed a mutation in cytochrome P450 CYP143 gene located in the ESX-5 secretion cluster. The function of CYP143 is unknown. Available synteny information is insufficient to straightforwardly infer potential CYP143 substrates, because a full set of effector proteins/substrates exported by the ESX-5 secretion system has not yet been identified. Here we found that CYP143 is highly conserved among mycobacteria. We leverage the possible association of the G334A mutation in CYP143 with the development of resistance to test the frontline antitubercular drug rifampicin using purified proteins. Binding of rifampicin does not induce typical P450 spectral response, while the crystal structure at atomic resolution reveals the binding mode in the active site. The drug binds above the heme and adopts "closed" conformation of the ansa chain running almost parallel to the naphthoquinone core. In this conformation the oxygen atom bridging C12 and C29 of the macrocycle makes a hydrogen bond with a water molecule coordinating heme iron. The same binding mode was observed in crystal structures in complex with rifaximin, rifamycin S and two synthetic derivatives. In the ligand-bound state the protein retains an open conformation even in the presence of the redox partner, as evident from the crystal structure of the ternary complex. Despite binding close to the heme, no conversion was observed showing that either the rifamycins or the reduction system, or both, are insufficient to support a full catalytic cycle. These results pave the way to understand the development of rifampicin resistance/tolerance and the role of orphan CYPome of M.tuberculosis.

biochemistry↗

bioRxiv2025

Non-DNA-damaging DNA-PK activation improving hearing and prolonging life due to NAD+ and SIRT upregulation

Emerging evidence strongly supports a close relationship between age-related hearing loss and frailty, highlighting the importance of early detection and intervention. Recently, we invented a mitochondria-homing drug named mitochonic acid 5 (MA-5), that increases the adenosine triphosphate (ATP) levels, rescue mitochondrial function, and protect tissue damages. Currently, the phase I clinical trial has been finished in Japan (jRCT2031210495) and the phase 2 clinical trial has already been approved by PMDA. Here we show that MA-5 improved various types of hearing loss in mouse models. Structural chemical bioanalysis revealed that MA-5 is a mixture of equal amount of S- and R- enantiomer and both S- and R- enantiomer increase ATP by binding mitochondrial protein, mitofilin. However, S-enantiomer significantly increased the NAD+ levels by binding to the NAD+-producing key enzyme nicotinamide phosphoribosyltransferase (NAMPT). Moreover, the S-enantiomer increased the sirtuin 1 protein by suppressing polyubiquitination induced by tripartite motif containing 28 (TRIM28) phosphorylation which was triggered by DNA-dependent protein kinase (DNA-PK) activation in the absence of DNA damage. Transcriptomic signatures showed that the signature of MA-5 shows an inverse correlation with aging and mortality and is oriented in the same direction as the OSKM-related iPSCs, suggesting the modification of aging pathways. Oral administration of MA-5 to mitochondrial disease model mouse showed increased survival. Our findings suggest that, in addition to enhancing ATP levels, the coordinated regulation of NAD+ metabolism, SIRT protein expression, and DNA-PK activity-constituting a novel therapeutic triad may contribute to the amelioration of hearing impairment and mitochondrial dysfunction, thereby improving life prognosis.

cell biology↗

bioRxiv2025

De novo chemo-optogenetics through rational small-molecule design and TRAP display

Optical control of biomolecules is a powerful technique for biological research, but current tools derived from natural proteins face significant limitations. Engineering these proteins to meet researchers needs remains challenging. In this study, we present a novel approach to the de novo creation of tailored chemo-optogenetic tools. This method combines the rational design of synthetic photo-switchable molecules with in vitro selection of artificial protein binders that specifically recognize a particular conformation of these molecules. We demonstrate the utility of this approach by generating artificial proteins that bind exclusively to the cis-form of designed azobenzene derivatives. The newly created pairs enable sustained protein activation with a single light pulse and allow for precise, reversible, and repeatable switching of cellular processes using two distinct wavelengths of light in mammalian cells. This de novo technique provides tailored synthetic photoswitch-protein binder pairs, expanding the potential for optical biomolecule manipulation.

synthetic biology↗

bioRxiv2024

Generation of antagonistic biparatopic anti-CD30 antibody from an agonistic antibody by precise epitope determination and utilization of structural characteristics of CD30 molecule

CD30 is a type I membrane protein that has been successfully targeted for lymphoma therapy using Brentuximab vedotin, an antibody-drug conjugate. Recently, the potential of blocking CD30-dependent NF-{kappa}B intracellular signaling has gained attention for treating inflammatory disorders. Development of antibody-based CD30 antagonists would broaden therapeutic strategies. A challenge in developing antagonistic antibodies is that the bivalent form of natural antibody format inevitably cross-links trace amounts of CD30 molecules, leading to signal transduction. In this study, we developed a series of biparatopic antibodies with each pair of antibody variable domains (Fvs) binding to distinct epitopes on CD30, and evaluated their biological activities and binding modes. Initially, we precisely identified epitope sites of the nine antibodies precisely by assessing binding to multiple orthologous CD30 proteins and mutants. We then produced 36 biparatopic antibodies covering all possible combinations of the nine Fvs, and analyzed their biological activities. Among these, we identified both potent agonists and antagonists. Notably, a significant proportion of the biparatopic antibodies displayed reduced agonistic activities, including 1:1-binding antagonists derived from a Fv of a strong agonist previously developed for lymphoma therapy, AC10. The mechanism of signaling activity induction is discussed using epitope information, which leads to the strategies of the development of biparatopic antibodies.

biochemistry↗

bioRxiv2024

Malaria parasites require a divergent heme oxygenase for apicoplast gene expression and biogenesis

Malaria parasites have evolved unusual metabolic adaptations that specialize them for growth within heme-rich human erythrocytes. During blood-stage infection, Plasmodium falciparum parasites internalize and digest abundant host hemoglobin within the digestive vacuole. This massive catabolic process generates copious free heme, most of which is biomineralized into inert hemozoin. Parasites also express a divergent heme oxygenase (HO)-like protein (PfHO) that lacks key active-site residues and has lost canonical HO activity. The cellular role of this unusual protein that underpins its retention by parasites has been unknown. To unravel PfHO function, we first determined a 2.8 [A]-resolution X-ray structure that revealed a highly -helical fold indicative of distant HO homology. Localization studies unveiled PfHO targeting to the apicoplast organelle, where it is imported and undergoes N-terminal processing but retains most of the electropositive transit peptide. We observed that conditional knockdown of PfHO was lethal to parasites, which died from defective apicoplast biogenesis and impaired isoprenoid-precursor synthesis. Complementation and molecular-interaction studies revealed an essential role for the electropositive N-terminus of PfHO, which selectively associates with the apicoplast genome and enzymes involved in nucleic acid metabolism and gene expression. PfHO knockdown resulted in a specific deficiency in levels of apicoplast-encoded RNA but not DNA. These studies reveal an essential function for PfHO in apicoplast maintenance and suggest that Plasmodium repurposed the conserved HO scaffold from its canonical heme-degrading function in the ancestral chloroplast to fulfill a critical adaptive role in organelle gene expression.

biochemistry↗

bioRxiv2023

Mitochondrial protein FKBP8 captures PDZD8 to form mitochondria-ER contacts

Mitochondria-ER membrane contact sites (MERCS) represent a fundamental ultrastructural feature underlying unique biochemistry and physiology in eukaryotic cells. The ER protein PDZD8 is required for the formation of MERCS in many cell types, however, its tethering partner on the outer mitochondrial membrane (OMM) is currently unknown. Here we identified the OMM protein FKBP8 as the tethering partner of PDZD8 using a combination of unbiased proximity proteomics, CRISPR-Cas9 endogenous protein tagging, Cryo-Electron Microscopy (Cryo-EM) tomography, and correlative light-EM (CLEM). Single molecule tracking revealed highly dynamic diffusion properties of PDZD8 along the ER membrane with significant pauses and capture at MERCS. Overexpression of FKBP8 was sufficient to narrow the ER-OMM distance, whereas independent versus combined deletions of these two proteins demonstrated their interdependence for MERCS formation. Furthermore, PDZD8 enhances mitochondrial complexity in a FKBP8-dependent manner. Our results identify a novel ER-mitochondria tethering complex that regulates mitochondrial morphology in mammalian cells.

cell biology↗

bioRxiv2023

Conformational features and interaction mechanisms of VHH antibodies with β-hairpin-like CDR-H3: A case of Nb8-HigB2 interaction.

{beta}-hairpin conformation is regarded as an important basic motif to form and regulate protein-protein interactions. Single-domain VHH antibodies are potential therapeutic and diagnostic tools, and the third complementarity-determining regions of the heavy chains (CDR-H3s) of these antibodies are critical for antigen recognition. Although the sequences and conformations of the CDR-H3s are diverse, CDR-H3s sometimes adopt {beta}-hairpin-like conformations. However, characteristic features and interaction mechanisms of {beta}-hairpin-like CDR-H3s remain to be fully elucidated. In this study, we investigated the molecular recognition of the anti-HigB2 VHH antibody Nb8, which has a CDR-H3 that forms a {beta}-hairpin-like conformation. The interaction was analyzed by evaluation of alanine-scanning mutants, molecular dynamics simulations, and hydrogen/deuterium exchange mass spectrometry. These experiments demonstrated that positions 93 and 94 (Chothia numbering) in framework region 3, which is right outside CDR-H3 by definition, play pivotal roles in maintaining structural stability and binding properties of Nb8. These findings will facilitate design and optimization of single-domain antibodies.

biochemistry↗

bioRxiv2022

Development of a 1:1-binding biparatopic anti-TNFR2 antagonist by epitope selection

Conventional bivalent antibodies against cell surface receptors often initiate unwanted signal transduction by crosslinking two antigen molecules. Biparatopic antibodies (BpAbs) bind to two different epitopes on the same antigen, thus altering crosslinking ability. In this study, we developed BpAbs against tumor necrosis factor receptor 2 (TNFR2), which is an attractive immune checkpoint target. Using different pairs of variable regions specific to topographically distinct TNFR2 epitopes, we successfully regulated the size of BpAb-TNFR2 immunocomplexes to result in controlled agonistic activities. One particular antagonist BpAb bound TNFR2 in 1:1 ratio without unwanted signal transduction, with its structural basis revealed by cryo-electron microscopy. This antagonist suppressed the proliferation of regulatory T cells expressing TNFR2. Therefore, the BpAb format would be useful in designing specific and distinct antibody functions.

biochemistry↗

bioRxiv2022

Structural insights into 3Fe-4S ferredoxins diversity in M.tuberculosis highlighted by a first redox complex with P450.

Ferredoxins are small iron-sulfur proteins and key players in essential metabolic pathways. Among all types, 3Fe-4S ferredoxins are less studied mostly due to anaerobic requirements. Their complexes with cytochrome P450 redox partners have not been structurally characterized. In the present work, we solved the structures of both 3Fe-4S ferredoxins from M. tuberculosis - Fdx alone and the fusion FdxE-CYP143. Our SPR analysis demonstrated a high affinity binding of FdxE to CYP143. According to SAXS data, the same complex is present in solution. The structure reveals extended multipoint interactions and the shape/charge complementarity of redox partners. Furthermore, FdxE binding induced conformational changes in CYP143 as evident from the solved CYP143 structure alone. The comparison of FdxE-CYP143 and modeled Fdx-CYP51 complexes further revealed the specificity of ferredoxins. Our results illuminate the diversity of electron transfer complexes for the production of different secondary metabolites.

biochemistry↗

bioRxiv2022

Molecular mechanism underlying the increased risk of colorectal cancer metastasis caused by single nucleotide polymorphisms in LI-cadherin gene

LI-cadherin is a member of the cadherin superfamily. LI-cadherin mediates Ca2+-dependent cell-cell adhesion by forming a homodimer. A previous study reported two single nucleotide polymorphisms (SNPs) in the LI-cadherin-coding gene (CDH17). These SNPs correspond to the amino acid changes of Lys115 to Glu and Glu739 to Ala. Patients with colorectal cancer carrying these SNPs are reported to have a higher risk of lymph node metastasis than patients without the SNPs. Although proteins associated with metastasis have been identified, the molecular mechanisms underlying the functions of these proteins remain unclear, making it difficult to develop effective strategies to prevent metastasis. In this study, we employed biochemical assays and molecular dynamics (MD) simulations to elucidate the molecular mechanisms by which the amino acid changes caused by SNPs in the LI-cadherin-coding gene increase the risk of cancer metastasis. Cell aggregation assays showed that the amino acid changes weakened the LI-cadherin-dependent cell-cell adhesion. In vitro assays demonstrated a decrease in homodimerization tendency due to the mutation of Lys115, and MD simulations suggested an alteration in the intramolecular hydrogen bond network due to the amino acid change. Taken together, our results indicate that the increased risk of lymph node metastasis is due to weakened cell-cell adhesion caused by the decrease in homodimerization tendency.

biochemistry↗

bioRxiv2022

Biophysical insight into protein-protein interactions in the Interleukin-11/Interleukin-11Rα/glycoprotein 130 signaling complex

Interleukin-11 (IL-11) is a member of the interleukin-6 (IL-6) family of cytokines. IL-11 is a regulator of multiple events in hematopoiesis, and IL-11-mediated signaling is implicated in inflammatory disease, cancer, and fibrosis. All IL-6 family cytokines signal through the signal-transducing receptor, glycoprotein 130 (gp130), but these cytokines have distinct as well as overlapping biological functions. To understand IL-11 signaling at the molecular level, we performed a comprehensive interaction analysis of the IL-11 signaling complex, comparing it with the IL-6 complex, one of the best-characterized cytokine complexes. Our thermodynamic analysis revealed a clear difference between IL-11 and IL-6. Surface plasmon resonance analysis showed that the interaction between IL-11 and IL-11 receptor (IL-11R) is entropy driven, whereas that between IL-6 and IL-6 receptor (IL-6R) is enthalpy driven. Our analysis using isothermal titration calorimetry revealed that the binding of gp130 to the IL-11/IL-11R complex results in entropy loss, but that the interaction of gp130 with the IL-6/IL-6R complex results in entropy gain. Our hydrogen-deuterium exchange mass spectrometry experiments suggested that the D2 domain of gp130 was not involved in n IL-6-like interactions in the IL-11/IL-11R complex. It has been reported that IL-6 interaction with gp130 in the signaling complex was characterized through the hydrophobic interface located in its D2 domain of gp130. Our findings suggest that unique interactions of the IL-11 signaling complex with gp130 are responsible for the distinct biological activities of IL-11 compared to IL-6.

biochemistry↗

bioRxiv2021

Lipid nanodiscs facilitate the identification of a fragment compound inhibiting the enzymatic activity of the bacterial membrane protein MsbA.

Membrane proteins are critical elements of numerous therapeutic approaches ranging from cancer to bacterial infections. MsbA is a bacterial membrane protein that has received increasing attention as an antibacterial target for its role in the processing of Lipid A, a key precursor of lipopolysaccharide that is essential for bacterial growth. When employing nanodiscs it is possible to stabilize MsbA by providing a membrane-like environment that enhances its enzymatic activity. Taking advantage of this property we have carried out a fragment screening using the biophysical method of surface plasmon resonance. This approach identified several compounds that bind specifically to MsbA. In particular, one of these fragment molecules not only binds to the target, but also inhibits the ATPase activity of the MsbA protein. The similarity of this fragment to the adenine moiety of ATP points at a route to generate stronger and more potent inhibitors for MsbA and even other proteins of its family of ABC transporters. Collectively, our study reveals biophysical approaches that facilitate the identification of fragment candidates inhibiting the activity of membrane proteins.

biophysics↗

bioRxiv2021

Binding mechanism underlying FIH-1 suppression caused by the N-terminal disordered region of Mint3

Mint3 is known to enhance aerobic ATP production, known as the Warburg effect, by binding to FIH-1. Since this effect is considered to be beneficial for cancer cells, the interaction is a promising target for cancer therapy. However, previous research has suggested that the interacting region of Mint3 with FIH-1 is intrinsically disordered, which makes investigation of this interaction challenging. Therefore, we adopted a physicochemical approach that combined thermodynamic studies with structural analyses in solution, to clarify the binding mechanism. First, using a combination of CD, NMR, and hydrogen/deuterium exchange mass spectrometry (HDX-MS), we confirmed that the N-terminal half, which is the interacting part of Mint3, is mostly disordered. Next, using isothermal titration calorimetry (ITC), we revealed that the interaction of Mint3 and FIH-1 produces an enormous change in enthalpy and entropy. The profile is consistent with the model that the flexibility of disordered Mint3 is drastically reduced upon binding to FIH-1. Moreover, we performed a series of ITC experiments with several types of truncated Mint3s, an effective approach since the interacting part of Mint3 is disordered, and identified 78-88 as a novel core site for binding to FIH-1. The truncation study of Mint3 also revealed the thermodynamic contribution of each part to the interaction, where one contributes to the affinity ({Delta}G), while the other only affects enthalpy ({Delta}H), by forming non-covalent bonds. This insight can serve as a foothold for further investigation of IDRs and drug development for cancer therapy.

biochemistry↗

bioRxiv2020

A new twist of rubredoxin function in M. tuberculosis

Electron transfer mediated by metalloproteins drives many biological processes. Rubredoxins are ubiquitous iron-containing electron carriers that play important roles in bacterial adaptation to changing environmental conditions. In Mycobacterium tuberculosis, oxidative and acidic stresses as well as iron starvation induce rubredoxin expression. However, their functions during M. tuberculosis infection is unknown. In the present work, we show that rubredoxin B (RubB) supports catalytic activity of mycobacterial cytochrome P450s, CYP124, CYP125, and CYP142, which are important for bacterial viability and pathogenicity. We solved the crystal structure of RubB and characterized the interaction between RubB and CYPs using site-directed mutagenesis. Mutations that neutralized single charge on the surface of RubB did not dramatically decrease activity of studied CYPs, and isothermal calorimetry (ITC) experiments indicated that interactions are transient and not highly specific. Our findings suggest that a switch from ferredoxins to rubredoxins support CYP activity in M. tuberculosis-infected macrophages. Our electrochemical experiments suggest potential applications of RubB in biotechnology.

biochemistry↗

bioRxiv2020

Dimerization mechanism and structural features of human LI-cadherin

LI-cadherin is a member of the cadherin superfamily, which encompasses a group of Ca2+-dependent cell adhesion proteins. The expression of LI-cadherin is observed on various types of cells in the human body, such as normal small intestine and colon cells, and gastric cancer cells. Because its expression is not observed on normal gastric cells, LI-cadherin is a promising target for gastric cancer imaging. However, since the cell adhesion mechanism of LI-cadherin has remained unknown, rational design of therapeutic molecules targeting this cadherin has been hampered. Here, we have studied the homodimerization mechanism of LI-cadherin. We report the crystal structure of the LI-cadherin EC1-4 homodimer. The EC1-4 homodimer exhibited a unique architecture different from that of other cadherins reported so far. The crystal structure also revealed that LI-cadherin possesses a noncanonical calcium ion-free linker between EC2 and EC3. Various biochemical techniques and molecular dynamics (MD) simulations were employed to elucidate the mechanism of homodimerization. We also showed that the formation of the homodimer observed by the crystal structure is necessary for LI-cadherin-dependent cell adhesion by performing cell aggregation assay.

biochemistry↗

bioRxiv2020

Structural basis for binding mechanism of human serum albumin complexed with cyclic peptide dalbavancin

Cyclic peptides, with unique structural features, have emerged as new candidates for drug discovery; their association with human serum albumin (HSA; long blood half-life), is crucial to improve drug delivery and avoid renal clearance. Here, we present the crystal structure of HSA complexed with dalbavancin, a clinically used cyclic peptide. SAXS and ITC experiments showed that the HSA-dalbavancin complex exists in a monomeric state; dalbavancin is only bound to the subdomain IA of HSA in solution. Structural analysis and MD simulation revealed that the swing of Phe70 and movement of the helix near dalbavancin were necessary for binding. The flip of Leu251 promoted the formation of the binding pocket with an induced-fit mechanism; moreover, the movement of the loop region including Glu60 increased the number of non-covalent interactions with HSA. These findings may support the development of new cyclic peptides for clinical use, particularly the elucidation of their binding mechanism to HSA.

biochemistry↗