Biology subjects

Toma-Fukai, S.

Publications and source records attributed to Toma-Fukai, S..

3 recordsLinked to original sources

bioRxiv2026

Metastable Dimeric Intermediate Drives Native-like Spidroin Fibrillogenesis, while Dehydration Promotes Cross-β Transition

The exceptional mechanical properties of spider dragline silk originate from hierarchical spidroin self-assembly, yet mimicking this process to generate native-like structures remains challenging. Here, we report a metastable dimeric intermediate as an essential molecular factor for fibrillogenesis. Small-angle X-ray scattering (SAXS) and atomic force microscopy (AFM) revealed a compact core stabilized by solvent-excluded "dry interfaces" surrounded by extended random coil regions. Within the core, a subset of the polyalanine blocks is held by side-chain packing rather than by main-chain {beta}-sheets; the majority remain exposed in the surrounding coil regions. Upon lowering the denaturant concentration, these exposed polyalanine segments pair with their counterparts at a fibril end, forming intermolecular {beta}-sheets that act as linkers and drive the stepwise end-to-end polymerization of the dimeric particles. In solution, the resulting fibrils exhibit an ordered {beta}-structure in which the polypeptide chains run parallel to the fibril axis, in contrast to amyloid-like cross-{beta}, in which the strands lie perpendicular to it; this matches key features of native silk nanofibril architecture. However, X-ray diffraction (XRD) analysis demonstrates that these native-like nanofibrils undergo an effectively irreversible transition toward amyloid-like cross-{beta} structures upon drying. Native-like fibrils are thus achievable by self-assembly, but structural control is lost during the final dehydration step. Accordingly, we propose a general principle for decoupling self-assembly from dehydration: native-like nanofibrils are formed first, then dried under conditions that restrict molecular-chain mobility (for example, mechanical constraint or immobilization on a surface), which we expect to suppress thermodynamic relaxation into cross-{beta} structures.

biophysics↗

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↗

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↗