Biology subjects

Sugahara, M.

Publications and source records attributed to Sugahara, M..

6 recordsLinked to original sources

bioRxiv2025

Structural and Thermodynamic Determinants of Anti-PGE2 mAb Specificity

We investigated the molecular determinants underlying the highly specific recognition of prostaglandin (PG) E2 and its structural analogues by an anti-PGE2 monoclonal antibody (mAb) and its Fab fragment using high-resolution X-ray crystallography coupled with detailed thermodynamic analyses, including conditions that mimic macromolecular crowding. Crystal structures of the Fab fragment in complex with PGE2, PGE1, and ONO5 were determined at 1.9 [A], 1.7 [A], and 1.7 [A] resolution, respectively, and the antigen-free Fab structure was solved at 2.0 [A]. The Fab accommodates the L-shaped PG scaffold within a deep antigen-binding crevasse formed between the {beta}-sheet frameworks of the VH and VL domains, where ligand recognition is mediated by multiple water-bridged hydrogen bonds and extensive hydrophobic contacts, including putative CH-O and CH-{pi} interactions. Comparison of antigen-free and ligand-bound structures revealed compensatory conformational rearrangements accompanied by reorganization of bound water networks. Upon PG binding, a short 310 helix forms at the tip of CDR-H1, stabilized by a cluster of hydrogen bonds and capping water molecules. This helix formation is associated with rotation of the VL domain and an increased Fab elbow angle. The CDR-H1 loop undergoes a flip-in transition in which Phe29H inserts into a hydrophobic cavity while Glu31H is extruded toward solvent, accompanied by {pi}-{pi} stacking between Tyr27H and Tyr32H. These changes collectively reduce hydrophobic surface area and promote enthalpy-driven ligand binding. The flexible -chain of PGE1, lacking the C5-C6 double bond, adopts altered conformations that disrupt C1-carboxyl interactions with three aromatic residues and lead to closure of a secondary water channel, consistent with subtle but meaningful differences in binding thermodynamics and crystallographic temperature factors relative to PGE2 and ONO5. Isothermal titration calorimetry (ITC) revealed high-affinity, enthalpy-driven PG binding to the Fab, in agreement with differential scanning calorimetry (DSC), which showed increased thermal stability upon complex formation. Under macromolecular crowding conditions induced by high bovine serum albumin (BSA) concentrations, PG binding to the intact mAb--but not to the Fab--exhibited improved discrimination between PGE2 and PGE1, suggesting that reduced entropy-enthalpy compensation under crowding amplifies subtle structural mismatches in ligand binding.

molecular biology↗

bioRxiv2025

High throughput protein serial crystallography using a grease matrix and a large-area support film

Serial femtosecond crystallography (SFX) using ultrashort pulses from X-ray free- electron lasers (XFELs) enables the determination of crystal structures at room temperature while minimizing radiation damage to the samples. This method involves irradiating numerous crystals one by one with XFEL pulses, allowing even the capture snapshots of dynamical structures in biological macromolecules. To achieve this, an efficient sample delivery system is essential for acquiring a large number of diffraction patterns. The most common approach uses a highly viscous grease matrix containing sample crystals, injected into the XFEL path from a narrow nozzle. However, the injection often suffers from clogging issues inside the injector nozzle, resulting in additional challenges such as the need for suitably sized crystals, increased sample consumption and unstable flow rates. Alternatively, a fixed-target approach, which scans a two-dimensional substrate with dispersed samples, can circumvent these issues. However, it must ensure the integrity of biological samples and provide sufficient surface area for efficient data collection. We here present an approach that utilizes a grease matrix and a large-area support film specially designed to address these requirements. This system offers a fast and reliable solution for protein SFX, enabling high-quality structure determination while significantly reducing sample consumption.

molecular biology↗

bioRxiv2022

Mapping Protein Dynamics at High-Resolution with Temperature-Jump X-ray Crystallography

Understanding and controlling protein motion at atomic resolution is a hallmark challenge for structural biologists and protein engineers because conformational dynamics are essential for complex functions such as enzyme catalysis and allosteric regulation. Time-resolved crystallography offers a window into protein motions, yet without a universal perturbation to initiate conformational changes the method has been limited in scope. Here we couple a solvent-based temperature jump with time-resolved crystallography to visualize structural motions in lysozyme, a dynamic enzyme. We observed widespread atomic vibrations on the nanosecond timescale, which evolve on the sub-millisecond timescale into localized structural fluctuations that are coupled to the active site. An orthogonal perturbation to the enzyme, inhibitor binding, altered these dynamics by blocking key motions that allow energy to dissipate from vibrations into functional movements linked to the catalytic cycle. Because temperature-jump is a universal method for perturbing molecular motion, the method demonstrated here is broadly applicable for studying protein dynamics.

biophysics↗

bioRxiv2021

Unidirectional ion transport mechanism of a light-driven chloride pump revealed using X-ray free electron lasers

Light-driven chloride-pumping rhodopsins actively transport anions, including various halide ions, across cell membranes. Recent studies using time-resolved serial femtosecond crystallography (TR-SFX) have uncovered the structural changes and ion transfer mechanisms in light-driven cation-pumping rhodopsins. However, the mechanism by which the conformational changes pump an anion to achieve unidirectional ion transport, from the extracellular side to the cytoplasmic side, in anion-pumping rhodopsins remains enigmatic. We have collected TR-SFX data of Nonlabens marinus rhodopsin-3 (NM-R3), derived from a marine flavobacterium, at 10 s and 1 ms time-points after photoexcitation. Our structural analysis reveals the conformational alterations during ion transfer and after ion release. Movements of the retinal chromophore initially displace a conserved tryptophan to the cytoplasmic side of NM-R3, accompanied with a slight shift of the halide ion bound to the retinal. After ion release, the inward movements of helix C and helix G and the lateral displacements of the retinal block access to the extracellular side of NM-R3. Anomalous signal data have also been obtained from NM-R3 crystals containing iodide ions. The anomalous density maps provide insight into the halide binding site for ion transfer in NM-R3. SignificanceLight-driven chloride pumps have been identified in various species, including archaea and marine flavobacteria. The function of ion transportation controllable by light is utilized for optogenetics tools in neuroscience. Chloride pumps differ among species, in terms of amino acid homology and structural similarity. Our time-resolved crystallographic studies using X-ray free electron lasers reveal the molecular mechanism of halide ion transfer in a light-driven chloride pump from a marine flavobacterium. Our data indicate a common mechanism in chloride pumping rhodopsins, as compared to previous low temperature trapping studies of chloride pumps. These findings are significant not only for further improvements of optogenetic tools but also for a general understanding of the ion pumping mechanisms of microbial rhodopsins.

molecular biology↗

bioRxiv2019

Comparing serial X-ray crystallography and microcrystal electron diffraction (MicroED) as methods for routine structure determination from small macromolecular crystals

Innovative new crystallographic methods are facilitating structural studies from ever smaller crystals of biological macromolecules. In particular, serial X-ray crystallography and microcrystal electron diffraction (MicroED) have emerged as useful methods for obtaining structural information from crystals on the nanometer to micron scale. Despite the utility of these methods, their implementation can often be difficult, as they present many challenges not encountered in traditional macromolecular crystallography experiments. Here, we describe XFEL serial crystallography experiments and MicroED experiments using batch-grown microcrystals of the enzyme cyclophilin A (CypA). Our results provide a roadmap for researchers hoping to design macromolecular microcrystallography experiments, and they highlight the strengths and weaknesses of the two methods. Specifically, we focus on how the different physical conditions imposed by the sample preparation and delivery methods required for each type of experiment effect the crystal structure of the enzyme.

biophysics↗

bioRxiv2017

Atomic resolution structure of serine protease proteinase K at ambient temperature

Atomic resolution structures (beyond 1.20 [A]) at ambient temperature, which is usually hampered by the radiation damage in synchrotron X-ray crystallography (SRX), will add to our understanding of the structure-function relationships of enzymes. Serial femtosecond crystallography (SFX) has attracted surging interest by providing a route to bypass such challenges. Yet the progress on atomic resolution analysis with SFX has been rather slow. In this report, we describe the 1.20 [A] resolution structure of proteinase K using 13 keV photon energy. Hydrogen atoms, water molecules, and a number of alternative side-chain conformations have been resolved. The increase in the value of B-factor in SFX suggests that the residues and water molecules adjacent to active sites were flexible and exhibited dynamic motions at specific substrate-recognition sites.

biochemistry↗