Biology subjects

Matsuura, H.

Publications and source records attributed to Matsuura, H..

11 recordsLinked to original sources

bioRxiv2026

Structural basis for monobody OP-4 binding to open-form adenylate kinase

Monobodies are fibronectin type-III-based binding proteins that specifically bind target proteins and regulate their functions. We previously identified monobodies that selectively recognize either the OPEN or CLOSED conformation of adenylate kinase (Adk), revealing that monobodies can discriminate distinct conformational states of a target protein. However, the molecular basis of OPEN-form recognition has remained unclear because the structure of the complex between an OPEN-form-specific monobody and Adk had not been determined. To address this issue, we determined the crystal structure of the complex between Adk and the OPEN-form-specific monobody OP-4, employing hierarchical clustering analysis of X-ray diffraction datasets. The structure revealed that OP-4 binds to the surface formed by the expanded LID and CORE domains of Adk. Mutations in the interface residues reduced the OP- 4-binding affinity, indicating that the crystallographically identified interface is also relevant in solution. In particular, R123 mutations markedly impaired OP-4 binding. Molecular dynamics simulations further suggested that the R123-D159-R156 hydrogen-bond network is retained in solution and may contribute to efficient complex formation. These findings establish the structural basis for monobody OP-4 binding to open-form Adk and identify the R123-centered interaction network as a key determinant of complex formation. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=153 SRC="FIGDIR/small/742914v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@67570forg.highwire.dtl.DTLVardef@84b9caorg.highwire.dtl.DTLVardef@1c1b782org.highwire.dtl.DTLVardef@f6ed05_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIThe crystal structure of the OPEN-form adenylate kinase/monobody OP-4 complex was successfully determined. C_LIO_LIThe crystal structure revealed that OP-4 binds to surface formed by the expanded LID and CORE domains in adenylate kinase. C_LIO_LIIsothermal titration calorimetry measurements for adenylate kinase mutants confirmed that the binding modes observed in solution are consistent with those observed in the crystal structure. C_LIO_LIMolecular dynamics simulations suggested that the R123-D159-R156 hydrogen bond network prior to OP-4 binding is important for complex formation. C_LI

biochemistry↗

bioRxiv2025

Two phytohormones synergistically induce parasitic weeds seed germination via KAI2d receptors

Root parasitic plants can severely decrease global crop production. The germination of their seeds is induced by host root-derived strigolactones (SLs). Considering this unique germination system, a "suicidal germination" method has been proposed to control root parasitic plants. However, this method requires the cost-effective production of germination inducers. In this study, we determined that jasmonates and SLs can synergistically induce the seed germination of root parasitic plants (Orobanche minor and Striga hermonthica). Biochemical analyses indicated that jasmonates and SLs cooperatively activate multiple divergent KARRIKIN INSENSITIVE 2 receptors. Our findings have elucidated the host recognition systems associated with these highly duplicated promiscuous receptors, with potential implications for developing a new strategy to protect crops against root parasitic plants.

plant biology↗

bioRxiv2025

Structural basis of stereochemical promiscuity by an umami taste receptor ortholog, Tas1r1/Tas1r3 from pufferfish

Taste receptor type 1 (TAS1R), which consists of sweet and umami receptors in humans, senses nutrients (such as sugars and amino acids) with substrate specificity that is often broad and varies among animals and subtypes. However, the structural basis for achieving diverse specificities remains largely elusive. Here, we present the crystal structure of the ligand-binding domain (LBD) of Tas1r1/Tas1r3 heterodimer from pufferfish, an ortholog of the human umami taste receptor. The overall structure of Tas1r1/Tas1r3LBD resembles previously reported TAS1R structures, indicating a conserved core architecture within the family. Nevertheless, pufferfish Tas1r1/Tas1r3 was found to bind and respond to both L- and D-amino acids, even though TAS1Rs are considered to respond to either enantiomer. Structural and mutational analyses revealed that this non-rigorous stereochemical recognition is attributed to inter- subdomain interactions that latch the cleft containing the amino acid-binding site. These interactions prevent the cleft from fully opening, thereby stabilizing the active conformation, even if the ligand shares a different chirality. These results suggest that different substrate specificities in TAS1Rs can be acquired not only by the gain or loss of direct interactions with the substrate but also by the gain of intramolecular interactions, which alter the conformational equilibrium of the receptor.

biochemistry↗

bioRxiv2025

(3R, 7S)-11-hydroxy-jasmonic acid is a major oxidative shunt product of jasmonate catabolism in Arabidopsis thaliana

Jasmonoyl-L-isoleucine (JA-Ile) is a pivotal oxylipin plant hormone that regulates numerous physiological processes and stress responses. Because JA-Ile is synthesized from jasmonic acid (JA), the cellular level of JA is tightly controlled. Although the oxidative catabolism of JA to 12-hydroxy-JA (12-OH-JA) is well-characterized, the biosynthesis, accumulation dynamics, and biological functions of 11-hydroxy-JA (11-OH-JA) remain poorly understood. Here, we conducted a comprehensive investigation of 11-OH-JA in Arabidopsis thaliana, encompassing its chemical synthesis, accumulation patterns, biological activity, and biosynthetic pathways. Using chemically synthesized 11-OH-JA of naturally occurring (3R,7S)-stereochemistry, we identified 11-OH-JA as the predominant hydroxylated JA derivative that accumulates following wounding. Enzymatic assays and in silico docking studies revealed that JOX1/2/3/4 exclusively catalyze the conversion of JA to 11-OH-JA, whereas 12-OH-JA is produced by distinct pathways. Notably, the quadruple mutant joxQ completely abolished 11-OH-JA accumulation without affecting 12-OH-JA levels. Furthermore, 11-OH-JA exhibited no binding affinity for the COI1-JAZ co-receptor, indicating that it acts as a biologically inactive shunt product of JA catabolism. Our findings establish JOX-mediated 11-hydroxylation of JA as a primary inactivation pathway, separate from 12-hydroxylation. Parallel, non-redundant inactivation routes deliver layered signal termination of costly defense signals. This study provides new insights into JA catabolism, highlighting 11-OH-JA as a crucial factor in the attenuation of jasmonate signaling, and thereby redefining the framework of JA turnover in A. thaliana.

plant biology↗

bioRxiv2025

Deciphering the altered conformational states of bifunctional thaumarchaeal crotonyl-CoA hydratase and 3-hydroxypropionyl-CoA dehydratase from Nitrosopumilus maritimus

The thaumarchaeal 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) cycle represents one of the most efficient mechanisms for CO2 fixation discovered to date. Within this cycle, the enzyme encoded by Nmar_1308 from Nitrosopumilus maritimus SCM1 plays a crucial role due to its dual functionality as both a crotonyl-CoA hydratase (CCAH) and a 3-hydroxypropionyl-CoA dehydratase (3HPD). Although the importance of a bifunctional enzyme for lowering the cost of biosynthesis, the details of structural dynamics are still missing. Here, in addition to our cryogenic temperature structures, we determined the first ambient temperature structures of the Nmar_1308 protein by Serial Femtosecond X-ray Crystallography (SFX). The determined structures capture previously unobserved conformational dynamics of the Nmar_1308 protein, providing invaluable information for future synthetic biology applications.

biochemistry↗

bioRxiv2025

Solubility, Hydration, and Sulphate Coordination in Cubic Insulin Crystals from Esrapid™ Monomers Stabilized in Divalent Anionic Form of Citric Acid

Insulins structural adaptations have been extensively studied at neutral and basic pH; however, the effects of water and anion coordination on allosteric regions under various acidic conditions remain unexplored. Given the critical role of polar interactions in allosteric modulation, investigating structured water movements across extended pH intervals is essential for understanding solvent-mediated stabilization mechanisms in the monomer form of insulin. Structures of acid-stable cubic insulin crystals were determined in the divalent anionic form of citric acid solutions over a pH range of 2 to 6 to investigate the effects of water and anion coordination, along with charge distribution, on protein conformation. Synchrotron X-ray diffraction data were collected at resolutions ranging from 1.4 to 1.76 [A], with refined models exhibiting R-factors between 0.19 and 0.21. While the spatial arrangement of most proteins is highly conserved, [~]90% of the water coordination network and polar interactions alter local residue motion and intrinsic dynamics of the structures as the pH is changed. This is in line with previously determined structures at pH 7-11, allowing for a comprehensive structural analysis in the pH range of pH 2-11. Three key observations emerged: (i) water coordinations undergo a prominent shift toward the isoelectric point of insulin (pH 5-7), (ii) water molecules and anions function as allosteric modulators to stabilize the T-state of insulin at the pH range 2 to 6, and (iii) at extreme pH values 2 and 11, increased solubility correlates with the structural adoption of insulins most active form, wherein hydration within the allosteric pocket supports monomer stabilization in the T-state. Combined with the computational analyses, pH-dependent electrostatic redistributions primarily affect side-chain dynamics and local protein motion. This solvent-coupled allosteric regulation provides a mechanistic framework for solvent-mediated protein stabilization, offering a novel insight into the rational design of insulin formulations through controlled protonation and hydration strategies.

biophysics↗

bioRxiv2024

Experimental and Computational Insights into the Structural Dynamics of the Fc Fragment of IgG1 Subtype from Biosimilar VEGF-Trap

The constant fragment (Fc) of the Immunoglobulin G1 (IgG1) subtype is increasingly recognized as a crucial scaffold in the development of advanced therapeutics due to its enhanced specificity, efficacy, and extended half-life. A prime example is VEGF-Trap (Aflibercept), a recombinant fusion protein that merges the Fc region of the IgG1 subtype with the binding domains of vascular endothelial growth factor receptors (VEGFR)-1 and VEGFR-2. The Fc regions role in N-glycosylation is particularly important, as it significantly influences protein stability. In this study, we present the first near-physiological temperature structures of the N-glycan-bound Fc fragment of IgG1 subtype from a biosimilar VEGF-Trap, determined using the SPring-8 Angstrom Compact free electron LAser (SACLA) and the Turkish Light Source (Turkish DeLight). Comparative analysis with cryogenic structures, including our own data, reveals alternate conformations within the glycan-binding pocket. Furthermore, molecular dynamics (MD) simulations highlight an unexpected degree of structural plasticity. These findings offer new insights into the molecular basis of Fc-mediated functions and provide valuable information for the design of next-generation therapeutics.

molecular biology↗

bioRxiv2022

Elucidating polymorphs of crystal structures with intensity-based hierarchical clustering analysis on multiple diffraction datasets

In macromolecular structure determination using X-ray diffraction from multiple crystals, the presence of different structures (structural polymorphs) necessitates the classification of diffraction data for appropriate structural analysis. Hierarchical clustering analysis (HCA) is a promising technique that has so far been used to extract isomorphous data, mainly for single structure determination. Although in principle the use of HCA can be extended to detect polymorphs, the absence of a reference for defining a threshold used for grouping the isomorphous datasets ( isomorphic threshold) poses a challenge. Here, we have applied unit cell-based and intensity-based HCAs to the datasets of apo-trypsin and inhibitor-bound trypsin that were mixed post-data acquisition to investigate how effective HCA is in classifying polymorphous datasets. Single-step intensity-based HCA successfully classified polymorphs with a certain isomorphic threshold. In datasets of several samples containing an unknown degree of structural heterogeneity, polymorphs could be identified by intensity-based HCA using the suggested isomorphic threshold. Polymorphs were also detected in single crystals using the data collected by the continuous helical scheme. These findings are expected to facilitate the determination of multiple structural snapshots by exploiting automated data collection and analysis. SynopsisSingle-step intensity-based hierarchical clustering is demonstrated to allow the detection of structural polymorphs in the diffraction datasets obtained from multiple crystals. By splitting the datasets collected by continuous helical scheme into several chunks, both inter and intra-crystal polymorphs can be successfully analyzed.

molecular biology↗

bioRxiv2022

The bHLH transcription factor MpHYPNOS is a critical regulator of gemma dormancy in the liverwort Marchantia polymorpha.

O_LIDormancy is a key process employed by land plants to adapt to harsh terrestrial environments. The liverwort Marchantia polymorpha produces dormant propagules called gemmae for asexual reproduction. The plant hormone abscisic acid (ABA) plays important roles in regulating dormancy in both the seeds of flowering plants and the gemmae of M. polymorpha. C_LIO_LIBased on previous transcriptome analysis, we identified the basic helix-loop-helix transcription factor MpHYPNOS (MpHYP) as a key regulator of gemma dormancy. C_LIO_LIKnock-out mutants of MpHYP showed much higher germination rates of gemmae in gemma cups than ABA-related mutants, while the growth and development of these mutants resembled that of the wild type. Transient induction of MpHYP caused irreversible growth arrest of gemmae and thalli. Transcriptome and RT-qPCR analyses revealed that MpHYP represses the expression of cell cycle-related genes and induces ABA biosynthesis and ABA-responsive genes. Indeed, ABA levels increased in MpHYP overexpression lines and decreased in Mphyp knock-out lines. However, the growth arrest caused by MpHYP overexpression was not suppressed by a mutation in an ABA receptor gene. C_LIO_LIThese findings suggest that MpHYP regulates gemma dormancy and thallus growth mainly through the ABA-independent pathway, providing clues about ABA-dependent and independent regulation of dormancy in land plants. C_LI

plant biology↗

bioRxiv2022

Mechanistic insights into intramembrane proteolysis by E. coli site-2 protease homolog RseP

Site-2 proteases are a conserved family of intramembrane proteases that cleave transmembrane substrates to regulate signal transduction and maintain proteostasis. Here, we elucidated crystal structures of inhibitor-bound forms of bacterial site-2 proteases including E. coli RseP. Our observations are consistent with a rearrangement of the RseP domains surrounding the active center to expose the substrate-binding site where a conserved electrostatic linkage between the transmembrane and membrane-associated domains mediates the conformational changes, suggesting that RseP has a gating mechanism to regulate substrate entry. Mutational analysis also supports that the substrate transmembrane helix is unwound by strand addition to the intramembrane {beta} sheet and is clamped at the active center for efficient cleavage. Furthermore, this substrate accommodation mechanism appears to be common across distinct intramembrane proteases.

biochemistry↗

bioRxiv2021

A ubiquitously expressed UDP-glucosyltransferase, UGT74J1, controls basal salicylic acid levels in rice

Salicylic acid (SA) is a phytohormone that regulates a variety of physiological and developmental processes, including disease resistance. SA is a key signaling component in the immune response of many plant species. However, the mechanism underlying SA-mediated immunity is obscure in rice (Oryza sativa). Prior analysis revealed a correlation between basal SA level and blast resistance in a range of rice varieties. This suggested that resistance might be improved by increasing basal SA level. Here, we identified a novel UDP-glucosyltransferase gene, UGT74J1, which is expressed ubiquitously throughout plant development. Mutants of UGT74J1 generated by genome editing accumulated high levels of SA under non-stressed conditions, indicating that UGT74J1 is a key enzyme for SA homeostasis in rice. Microarray analysis revealed that the ugt74j1 mutants constitutively overexpressed a set of pathogenesis-related (PR) genes. An inoculation assay demonstrated that these mutants had increased resistance against rice blast, but they also exhibited stunted growth phenotypes. To our knowledge, this is the first report of a rice mutant displaying SA overaccumulation.

plant biology↗