Takashi S. Kodama

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.

Liquid–liquid phase separation (LLPS) underlies the formation of membraneless cellular compartments, yet experimental strategies that directly connect quantitative LLPS behavior with residue-level structural information remain limited. Here, we present an integrated protocol that combines quantitative LLPS assays with nuclear magnetic resonance (NMR) spectroscopy and structure-guided mutagenesis to regulate protein phase separation. Using the VAPB MSP domain as a representative example, this workflow links residue-specific structural features to macroscopic LLPS behavior and enables suppression or enhancement of phase separation through targeted amino acid substitutions. This protocol provides a generalizable framework for systematic, residue-level regulation of protein LLPS.

Liquid-liquid phase separation (LLPS) is critical to the formation of membraneless organelles within cells. Monitoring the size and dynamics of LLPS droplets is essential to understanding their function. However, conventional methods, such as microscopy and dynamic light scattering (DLS), have limitations, including low throughput and complex setups. Here, we present a simple, label-free, high-throughput method for estimating droplet size using ultraviolet-visible (UV–Vis) spectra obtained using a standard microplate reader. This method is theoretically supported by Mie scattering theory. By analyzing the spectral shape and comparing it to that of glass beads of known sizes, our method enables the quantitative estimation of LLPS droplet size in a minimally invasive manner. We validated our approach using peptide droplets of known sizes, then applied it to monitor the growth of protein condensates formed by vesicle-associated membrane protein-associated protein B (VAPB). This technique offers a convenient, scalable alternative for screening LLPS dynamics in various biomolecular systems.

T-cell intracellular antigen-1 (TIA-1) is a key component of stress granules and contains an intrinsically disordered region called the prion-like domain (PLD). TIA-1 PLD can form condensates via phase separation that subsequently convert into amyloid fibrils. However, the structural basis underlying TIA-1 PLD amyloid fibril formation remains unclear, hindering clinical understanding and therapeutic intervention at the molecular level. Using cryo-electron microscopy, we identified structural features that are consistent with a labile architecture, including a kinked backbone conformation, a polar zipper, and a proline-mediated cross-β structure. We also determined the fibril structure containing a G355R missense mutation and found that this mutation disrupts the tight conformation around G355 in the wild-type structure, leading to delayed fibril formation. The amyloid fibril structures of TIA-1 PLD provide an atomic-level framework for mechanistic studies of mutation-driven dysfunction.

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.

The membranes surrounding the eukaryotic cell and its organelles are continuously invaginating, budding, and undergoing membrane fusion–fission events, which enable them to perform functions not found in prokaryotic cells. In addition, organelles come into close contact with each other at membrane contact sites (MCSs), which involve many types of proteins, and which regulate the signaling and transport of various molecules. Vesicle-associated membrane protein (VAMP)-associated protein (VAP) is an important factor involved in the tethering and contact of various organelles at MCSs in almost all eukaryotes and has attracted attention for its association with various diseases, mainly neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). However, the detailed mechanism of its functional expression remains unclear. In this review, we quantitatively discuss the structural dynamics of the entire molecule, including intrinsically disordered regions and intramolecular and intermolecular interactions, focusing on the vertebrate VAP paralogs VAPA and VAPB. Molecular phylogenetic and biophysical considerations are the basis of the work.

T-cell intracellular antigen-1 (TIA-1) is a key component of stress granules with an intrinsically disordered region called the prion-like domain (PLD). TIA-1 PLD forms phase-separated droplets that subsequently transition into amyloid fibrils. However, the structural basis of TIA-1 PLD amyloid fibril formation has not been elucidated. We investigated the amyloid fibril structure of TIA-1 PLD using cryo-electron microscopy and found structural features that ensure the reversibility of the fibrils, including a kinked backbone conformation, a polar zipper, and a proline-mediated cross-b structure. We also determined the amyloid fibril structure with the amyotrophic lateral sclerosis (ALS)-associated G355R mutation and found that G355R disrupts the tight conformation surrounding G355 in the wild-type fibril structure, resulting in destabilized and delayed amyloid fibril formation. The structural disturbance of amyloid fibril formation by G355R may contribute to the pathogenesis of ALS.

T cell intracellular antigen-1 (TIA-1) plays a central role in stress granule (SG) formation by self-assembly via the prion-like domain (PLD). In the TIA-1 PLD, amino acid mutations associated with neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) or Welander distal myopathy (WDM), have been identified. However, how these mutations affect PLD self-assembly properties has remained elusive. In this study, we uncovered the implicit pathogenic structures caused by the mutations. NMR analysis indicated that the dynamic structures of the PLD are synergistically determined by the physicochemical properties of amino acids in units of five residues. Molecular dynamics simulations and three-dimensional electron crystallography, together with biochemical assays, revealed that the WDM mutation E384K attenuated the sticky properties, whereas the ALS mutations P362L and A381T enhanced the self-assembly by inducing β-sheet interactions and highly condensed assembly, respectively. These results suggest that the P362L and A381T mutations increase the likelihood of irreversible amyloid fibrillization after phase-separated droplet formation, and this process may lead to pathogenicity.

The catalytic mechanism of hammerhead ribozymes (HHRzs) attracted great attention in relation to the chemical origin of life. However, the basicity (pK_a) of the catalytic sites of HHRzs has not been studied so far. As a result, the investigation of the currently assumed mechanism from an experimentally derived pK_a value has been impossible. In HHRzs, there exists a highly functionalized structural unit (A9-G10.1 site) with a catalytic residue (G12) for the nucleophile activation and metal ion-binding residue (G10.1). As inferred from this fact, there might be a possibility that HHRzs may utilize specific functions of the A9-G10.1 motif for the catalytic reaction. Therefore, here we studied the basicity of G12/G10.1-corresponding residues using RNA duplexes including the A9-G10.1 motif without other conserved residues of HHRzs. From the pH-titration experiments with NMR spectra, we have obtained the intrinsic basicity of the G12/G10.1-corresponding residues in the motif, with pK_a > 11.5 (N1 of G12) and pK_a 4.5 (N7 of G10.1) for the first time. Based on the derived irregular basicity, their correlation with a catalytic activity and a metal affinity were investigated. In total, the derived thermodynamic properties are an intrinsic nature of the exclusive catalytic unit of HHRzs, which will be an outstanding pivot point for the mechanistic analyses.