Yohei Miyanoiri

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 nonrigorous stereochemical recognition is attributed to intersubdomain 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 has 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.

Self-assembly of intrinsically disordered proteins (IDPs) underlies cellular functions and disease pathogenesis. This process is mediated by two intermolecular interaction modes: point-to-point contacts described by the sticker-and-spacer framework, and surface-to-surface contacts proposed in the cross-β hypothesis. In this study, we investigated the molecular basis of these modes in the context of conformational biases, defined as sequence-encoded structural preferences of local segments. To this end, employing a five-residue model as a mechanistic framework to capture local conformational biases, we designed lag-series IDPs from the T-cell intracellular antigen-1 prion-like domain by systematically modulating conformational biases while preserving amino acid composition. The lag-series IDPs demonstrated distinct condensate properties and varying capacities for amyloid fibril formation. The structural analyses suggested that strongly biased regions preferentially adopt extended structures, including β-strands, and the spacing between these regions influences metastable β-sheet formation. Our findings suggest that local conformational biases are associated with interaction modes of IDPs, thereby linking sequence to condensate properties and amyloid fibril formation.

Monoclonal antibodies are the cornerstone biopharmaceuticals whose safety and efficacy critically depend on their higher-order structure (HOS). Nuclear magnetic resonance (NMR) spectroscopy has emerged as a promising tool for HOS evaluation, yet its application has largely relied on fingerprinting approaches without residue-level interpretation. Here, we report site-specific assignments of methyl resonances in the Fc region of human IgG1, established through amino acid-selective labeling and correlation with backbone resonances using scalar coupling and NOE connectivities, further supported by mutagenesis. These assignments allowed us to identify glycoform-dependent spectral variations, including distinct signatures of core fucosylation and terminal galactosylation, as well as an Fc-specific amino acid substitution. Importantly, these spectral probes were detectable even in antibodies at natural isotopic abundance, enabling practical applications to therapeutic products without isotopic labeling. Furthermore, dynamic filtering highlighted methyl resonances from hinge and receptor-binding residues with elevated mobility, providing localized insights into functional sites. Collectively, our results establish unlabeled methyl NMR as a robust platform for sensitive and practical HOS assessment of therapeutic antibodies. This approach is broadly applicable to monitor glycosylation heterogeneity, chemical modifications, and batch-to-batch consistency, thereby offering a valuable framework for development and quality control of both innovative biopharmaceuticals and biosimilars.

AI-designed protein variants have demonstrated remarkable resistance to heat and chemical stress, yet the molecular mechanisms underlying this stability remain unclear. Here, we present a comprehensive biophysical and nuclear magnetic resonance (NMR) analysis of thermally stable ubiquitin and its ProteinMPNN-designed variants, R4 and R10, together with a second system based on the less stable ISG15 C-terminal domain (ISG15-CTD). Both R4/R10 and ProteinMPNN-designed ISG15-CTD variants (ICVs) exhibit extraordinary thermostability beyond 120 °C, and resist extreme denaturation at pH 3.0 in 8 M urea. NMR relaxation and hydrogen-deuterium exchange, and molecular-dynamics simulations reveal a protective mesostructured hydration shell that strengthens the hydrogen bonding network between protein-bound and bulk water, thereby suppressing unfolding. Sequence and electrostatic analyses indicate that this hydration arises from charge enrichment and clustering on the protein surface. These findings identify mesostructured hydration as a general, sequence-encoded mechanism of ProteinMPNN-driven stability and provide a physical framework for designing highly resilient biomolecules.

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.

Self-assembly of intrinsically disordered proteins (IDPs) underlies cellular functions and disease pathogenesis. This process is mediated by two intermolecular interaction modes: transient point-to-point contacts described by the sticker-and-spacer framework, and persistent surface-to-surface contacts proposed in the cross-β hypothesis. We investigated the molecular basis of these modes in the context of conformational biases, defined as sequence-encoded structural preferences of local segments. Using a five-residue model, we generated lag-series IDPs from the TIA-1 prion-like domain by systematically modulating conformational biases while preserving amino acid composition. The lag-series IDPs demonstrated distinct condensate properties and varying capacities for amyloid fibril formation. Their structural analyses revealed that strongly biased regions preferentially adopt extended structures, including β-strands, and the spacing between these regions influences metastable β-sheet formation. Our findings demonstrate that local conformational biases shape interaction modes of IDPs, thereby linking sequence to condensate properties and amyloid fibril formation. Significance Statement: Proteins fold into three-dimensional structures defined by their amino acid sequences. In contrast, intrinsically disordered proteins (IDPs) lack stable structures, yet their sequences encode unique self-assembly behaviors, including phase separation and amyloid fibril formation. Can such behaviors be explained within structure-based frameworks? Using a five-residue model, we showed that IDP sequences encode local structural preferences, termed conformational biases, within short segments. These biases determine whether segments engage in transient point-to-point interactions driving phase separation or persistent surface-to-surface interactions leading to amyloid fibril formation. By bridging sequence, structure, and interaction modes, our work provides a comprehensive mechanism for self-assembly and conceptual tools for understanding IDP-related biological functions and disease mechanisms.

We investigate the impact of glycoform alterations on the dynamic structure of the human immunoglobulin G1 (IgG1) Fc region using integrated computational and experimental approaches. Four distinct IgG1-Fc glycoforms, varying in core fucosylation and nonreducing terminal galactosylation, were generated through a combination of cell engineering and in vitro enzymatic reactions. Stable-isotope-assisted NMR spectroscopy, incorporating both glycan and protein signals, revealed that galactosylation induces chemical shift perturbations extending from the glycan–protein interface to the C _H 2–C _H 3 domain boundary. Molecular dynamics simulations demonstrated that the absence of galactose enhances the mobility of both the glycan and the C _H 2 domain, broadening the conformational landscape of the Fc quaternary structure. This increased flexibility likely contributes to a greater entropic penalty upon binding to effector molecules, which constrain the Fc in an asymmetric conformation. Conversely, the effects of fucosylation are more localized, primarily influencing the dynamics of residues involved in Fcγ receptor IIIa binding. These findings provide atomic-level insights into the distinct yet synergistic mechanisms by which galactosylation and fucosylation modulate IgG1-Fc dynamics and effector functions, offering crucial information for the optimization of therapeutic antibodies.

Cyanophages play a critical role in shaping host metabolism during infection by encoding auxiliary metabolic genes (AMGs), including enzymes involved in phycobilin biosynthesis. PebS (phycoerythrobilin synthase) is a phage-encoded ferredoxin-dependent biliverdin reductase (FDBR) that catalyses the two-step reduction of biliverdin IXα (BV) to phycoerythrobilin (PEB). Although high-resolution structures are available for both PebS and its electron donor ferredoxin (Fd), the structural details governing their interaction remain unclear. This study leverages the well-characterised structural models of PebS and a host-like ferredoxin from Thermosynechococcus elongatus (Te-Fd), whose NMR structure provides a reliable basis for probing protein– protein interactions. Using [¹⁵N]-labelled, gallium-substituted Te-Fd, we employed NMR spectroscopy to monitor chemical shift perturbations upon binding, enabling us to probe the interaction interface in solution. Based on these data, we conducted protein-protein docking with HADDOCK to predict the interaction interface between Te-Fd and PebS. PebS variants designed to disrupt this interface did indeed show corresponding alterations in enzymatic efficiency and product formation as determined by time resolved UV/Vis spectroscopy and HPLC analyses. Utilizing the Fd encoded in the cyanophage PSSM2-Fd in our assays, we could observe a significantly improved catalytic activity, suggesting an coevolution of phage enzyme and electron donor. A comparison of the two available X-ray structures of Te-Fd and PSSM2-Fd with an alphafold-model of the Fd of the natural host Prochlorococcus NATL1A (NATL1A-Fd) also supports this evolutionary adaptation and the role of both PSSM2-Fd and PebS as AMGs involved in viral infection by PSSM2.

Liquid-liquid phase separation (LLPS) of proteins can form membraneless organelles in the cell and can lead to pathological aggregation associated with neurodegenerative diseases. However, progress in controlling LLPS has been limited, and there has been no emergence of engineered de novo molecules to induce and modulate LLPS of targeted proteins. Here, we report de novo peptides that efficiently induce the LLPS of α-synuclein (αSyn), a protein involved in Parkinson's disease, discovered by the RaPID (random nonstandard peptides integrated discovery) system. These peptides primarily interact with the C-terminal region of αSyn, leading to the formation of an interaction network with αSyn and resulting in efficient droplet formation. Our study demonstrates the capacity of target-specific peptides to control LLPS and the subsequent liquid-solid phase transition (LSPT). Our novel LLPS-inducing and LSPT-modulating peptides may serve as a promising tool for fundamental investigations of LLPS and potentially for therapeutic intervention in amyloid diseases.

α‐Synuclein (αSyn) inclusions are a pathological hallmark of several neurodegenerative disorders. While cryo‐electron microscopy studies have revealed distinct fibril polymorphs across different synucleinopathies, the molecular switches controlling polymorphism remain unveiled. In this study, we found that fibril morphology is associated with the conformational state of monomeric αSyn. Through systematic evaluation of the ionic strength and temperature, we generated two distinct polymorphs: a twisted morphology at low ionic strength and temperature, and a rod‐like morphology at higher ionic strength and temperature. Using solid‐state NMR, we revealed that both polymorphs share a highly conserved core structure, with morphological differences arising probably from distinct structural arrangements at the protofilament interfaces. Furthermore, we found that a specific conformational change in the C‐terminal domain of the monomeric αSyn serves as a molecular switch for the formation of polymorphs. Interestingly, this conformational change can also be triggered by calcium binding to the C‐terminus, connecting environmental factors to specific fibril architectures. Our results reveal a conformational role for the C‐terminal domain that influences αSyn fibril morphology, providing significant insights into the fibrogenesis of αSyn.