Si Wu

The Parkinson’s disease-related protein α-synuclein can form solid amyloid fibrils through liquid–liquid phase separation (LLPS) and liquid-to-solid phase transition. The most deleterious familial mutation E46K has recently been shown to enhance α-synuclein LLPS and subsequent solidification; yet, the precise mechanisms remain largely unknown. Here, using molecular dynamics simulations at different spatiotemporal scales combined with biochemical experiments, we show that the E46K mutation acts as an electrostatic switch to remodel interactions between the oppositely charged N-terminal domain (NTD) and C-terminal domain (CTD) of α-synuclein. This remodeling shifts the interaction site of NTD with CTD from its N-terminus to the mutation region. Such reorganization increases the hydrophobic solvent exposure of key LLPS-promoting motifs, potentially facilitating intermolecular interactions that initiate phase separation. Phase coexistence simulations further support this hypothesis, showing that the E46K mutant exhibits enhanced LLPS and solidification propensities. This enhancement is primarily driven by intermolecular electrostatic interactions between the mutation region and the CTD, followed by the hydrophobic NAC–NAC interactions mediated by the LLPS-promoting motifs. Collectively, these results reveal that NTD–CTD electrostatic crosstalk acts as the key modulator of α-synuclein phase separation, while NAC–NAC interactions play an auxiliary role, both of which synergistically govern α-synuclein phase separation. This study offers a complete and detailed mechanistic framework for understanding α-synuclein phase separation and its enhancement induced by the E46K mutation.

The liquid‐liquid phase separation (LLPS) of nucleophosmin1 (NPM1) is essential for the assembly of pre‐ribosomal subunits. However, the interplay between the conformational transitions of the intrinsically disordered region (IDR) of NPM1 and its biologically critical phase transition remains poorly understood. Here, we uncover a salt‐concentration‐dependent reentrant phase behavior of NPM1 originating from electrostatically driven IDR plasticity. By integrating molecular dynamics (MD) simulations and single‐molecule Förster resonance energy transfer (smFRET) techniques, we demonstrate that low salt concentrations stabilize intrachain interactions, maintaining a compact conformation that suppresses intermolecular contact and thereby inhibits LLPS. At intermediate ionic strengths, weakened intrachain interactions result in conformational extension, enabling interchain electrostatic networks that facilitate LLPS. Excessive charge screening at high salt concentrations disrupts both intra‐ and intermolecular interactions, leading to condensate dissolution. Our study establishes a mechanistic framework linking conformational plasticity, intra‐ and interchain electrostatic interactions, and phase behavior, expanding our understanding of how dynamic molecular architectures of the NPM1 IDR govern biomolecular condensate assembly and its functions in ribosome biogenesis within the granular component region of the nucleolus.

Among the distinct adaptor protein (AP) complexes, AP-4 primarily functions as a non-clathrin-coated vesicle machinery essential for intracellular membrane trafficking. ARF1 is a master regulator of AP-4 membrane recruitment, but the underlying mechanism remains elusive. Here, we present the cryo-EM structures of soluble AP-4 and the AP-4/ARF1 complex. Unexpectedly, AP-4 adopts a dynamic equilibrium between closed and open conformations, caused by loose contacts between its medium subunit and central core. ARF1 binding induces only subtle changes in AP-4, which retains its conformational equilibrium. Mutations at the AP-4/ARF1 interface disrupt complex formation and impair ARF1-dependent membrane recruitment. Efficient membrane recruitment of AP-4 likely requires the synergistic engagement of ARF1 and cargoes. Disrupting the conformational flexibility of AP-4 interferes with this synergistic effect and compromises AP-4-mediated membrane trafficking. Our findings may redefine AP-4 as a conformationally dynamic complex modulated by cooperative interactions, providing insights into neurodevelopmental disorders associated with AP-4 dysfunction.

Emerging evidence indicates that liquid‐liquid phase separation of α‐synuclein occurs during the nucleation step of its aggregation, a pivotal step in the onset of Parkinson's disease. Elucidating the molecular determinants governing this process is essential for understanding the pathological mechanisms of diseases and developing therapeutic strategies that target early‐stage aggregation. While previous studies have identified residues critical for α‐synuclein amyloid formation, the key residues and molecular drivers of its phase separation remain largely unexplored. Herein, multiscale simulations and experimental approaches are employed to uncover the molecular determinants dictating α‐synuclein phase separation and the pre‐solidification of its condensates. Seven motifs are identified that exhibit high β‐sheet propensity in the monomeric state of α‐synuclein and progressively increase in β‐sheet content during condensation. Notably, two C‐terminal motifs engage in a percolated network of intermolecular interactions through transient hydrogen bonds, contributing to the phase boundary properties. Deletion of these motifs reduces the phase separation ability of α‐synuclein, underscoring their essential roles in this process. Together, the findings reveal crucial phase separation hotspots and shed light on the molecular mechanism underlying α‐synuclein phase separation, offering significant insights and novel potential therapeutic targets for Parkinson's disease.

Liquid–liquid phase separation (LLPS) is now recognized as one of the key mechanisms underlying the formation of membraneless organelles. Typically, condensates formed through LLPS are dynamic and play a crucial role in the spatiotemporal regulation of essential cellular processes. In some cases, however, condensates can undergo an aberrant liquid-to-solid transition, which is now recognized as being related to the onset of cancers and neurodegeneration. The microtubule-associated protein Tau, the aberrant aggregation of which is implicated in neurodegenerative disorders like Alzheimer’s and Parkinson’s, has been found to undergo LLPS. The Tau condensates formed through LLPS are considered to be intermediate on-pathway precursors of amyloid aggregates. Unlike other known phase-separating proteins (e.g., FUS or TDP-43) that have low-complexity domains (LCDs), Tau is intrinsically disordered. Thus, Tau exhibits a highly flexible structure that can be modulated by changes in environmental changes. The intricate relationship between different conformations of full-length Tau and its phase behavior remains poorly understood. To bridge this gap, here, by employing a combination of single-molecule FRET and molecular dynamics simulations, we demonstrate that Tau undergoes conformational transitions from compact to extended states during LLPS, irrespective of diverse driving forces. Moreover, we show that intramolecular interactions responsible for stabilizing the compact conformations of monomeric Tau correlate with the intermolecular interactions driving the LLPS of Tau, thereby facilitating the formation of dynamic networks. These findings provide crucial mechanistic insights into how the conformational state of Tau governs its propensity for phase separation, shedding light on sequence-encoded structural processes that ultimately drive biological phase separation.

Macromolecular crowding compacts the disordered 4.1G-CTD while preserving its disorder, enhances its affinity and association kinetics for NuMA (with crowder-specific effects), thereby fine-tuning IDP functionality in cellular environments.

The 70-kDa heat shock protein, Hsp70, is a key chaperone involved in cellular protein homeostasis. The structure of the Hsp70 protein family is highly conserved, including a nucleotide-binding domain (NBD) and a substrate-binding domain (SBD). ATP binding and hydrolysis in the NBD of Hsp70 regulates the binding and release of substrates in the SBD via interdomain allosteric communication. Growing evidence shows that the conformational dynamics of Hsp70 are crucial for its function, which are difficult to probe by traditional bulk-based methods. Single-molecule techniques are emerging as powerful tools to explore the dynamics of proteins that are obscured in bulk measurements. In this review, we summarize recent progress in the study of the molecular dynamics of Hsp70 and its interactions with cochaperones and substrates using single-molecule fluorescence spectroscopy and single-molecule force spectroscopy. We discuss how the application of single-molecule techniques facilitates a deeper understanding of the mechanistic details of the chaperone functions of Hsp70.

Huntington disease (HD) is associated with aggregation of huntingtin (HTT) protein containing over 35 continuous Q residues within the N‐terminal exon 1 encoded region. The C‐terminal of the HTT protein consists mainly of HEAT repeat structure which serves as a scaffold for multiple cellular activities. Structural and biochemical analysis of the intact HTT protein has been hampered by its huge size (~300 kDa) and most in vitro studies to date have focused on the properties of the exon 1 region. To explore the interaction between HTT exon 1 and the HEAT repeat structure, we constructed chimeric proteins containing the N‐terminal HTT exon 1 region and the HEAT repeat protein PR65/A. The results indicate that HTT exon 1 slightly destabilizes the downstream HEAT repeat structure and endows the HEAT repeat structure with more conformational flexibility. Wild‐type and pathological lengths of polyQ did not show differences in the interaction between HTT exon 1 and the HEAT repeats. With the C‐terminal fusion of PR65/A, HTT exon 1 containing pathological lengths of polyQ could still form amyloid fibrils, but the higher‐order architecture of fibrils and kinetics of fibril formation were affected by the C‐terminal fusion of HEAT repeats. This indicates that interaction between HTT exon 1 and HEAT repeat structure is compatible with both normal function of HTT protein and the pathogenesis of HD, and this study provides a potential model for further exploration.

Preventing tau aggregation is a potential therapeutic strategy in Alzheimer’s disease and other tauopathies. Recently, liquid–liquid phase separation has been found to facilitate the formation of pathogenic tau conformations and fibrillar aggregates, although many aspects of the conformational transitions of tau during the phase transition process remain unknown. Here, we demonstrate that the tau aggregation inhibitor methylene blue promotes tau liquid–liquid phase separation and accelerates the liquid-to-gel transition of tau droplets independent of the redox activity of methylene blue. We further show that methylene blue inhibits the conversion of tau droplets into fibrils and reduces the cytotoxicity of tau aggregates. Although gelation slows down the mobility of tau and tubulin, it does not impair microtubule assembly within tau droplets. These findings suggest that methylene blue inhibits tau amyloid fibrillization and accelerates tau droplet gelation via distinct mechanisms, thus providing insights into the activity of tau aggregation inhibitors in the context of phase transition.

Dynamic interdomain interactions within the Hsp70 protein is the basis for the allosteric and functional properties of Hsp70s. While Hsp70s are generally conserved in terms of structure, allosteric behavior, and some overlapping functions, Hsp70s also contain variable sequence regions which are related to distinct functions. In the Hsp70 sequence, the part with the greatest sequence variation is the C‐terminal α‐helical lid subdomain of substrate‐binding domain (SBDα) together with the intrinsically disordered region. Dynamic interactions between the SBDα and β‐sandwich substrate‐binding subdomain (SBDβ) contribute to the chaperone functions of Hsp70s by tuning kinetics of substrate binding. To investigate how the C‐terminal region of Hsp70 has evolved from prokaryotic to eukaryotic organisms, we tested whether this region can be exchanged among different Hsp70 members to support basic chaperone functions. We found that this region from eukaryotic Hsp70 members cannot substitute for the same region in Escherichia coli DnaK to facilitate normal chaperone activity of DnaK. In contrast, this region from E. coli DnaK and Saccharomyces cerevisiae Hsp70 (Ssa1 and Ssa4) can partially support some roles of human stress inducible Hsp70 (hHsp70) and human cognate Hsp70 (hHsc70). Our results indicate that the C‐terminal region from eukaryotic Hsp70 members cannot properly support SBDα–SBDβ interactions in DnaK, but this region from DnaK/Ssa1/Ssa4 can still form some SBDα–SBDβ interactions in hHsp70 or hHsc70, which suggests that the mode for SBDα–SBDβ interactions is different in prokaryotic and eukaryotic Hsp70 members. This study provides new insight in the divergency among different Hsp70 homologs and the evolution of Hsp70s.