Chao Wang

Kinesin-1 is a major microtubule-based molecular motor that transports diverse cellular cargoes, yet how assembly of its subunits is coupled to cargo recognition and motor regulation remains poorly understood. Mammalian kinesin-1 functions as a heterotetrameric holoenzyme composed of kinesin heavy chains (KIF5s) and kinesin light chains (KLCs), but the structural principles linking holoenzyme assembly to motor regulation remain unclear. Here, using KIF5C as a model system, we define the molecular mechanisms underlying kinesin-1 holoenzyme assembly and cargo-adaptor regulation. We identify a conserved coiled-coil interface between KIF5C and KLC1 that mediates their 2:2 assembly, with quantitative mutagenesis revealing key hydrophobic determinants of complex formation. We further demonstrate that the mitochondrial adaptor TRAK2 directly engages the KIF5C CC4 cargo-binding platform through a defined 2:2 interaction required for mitochondrial recruitment of KIF5C. Although KLC1 and TRAK2 bind distinct regions of KIF5C, KLC1 modulates TRAK2 association through steric and conformational effects, revealing how holoenzyme composition influences cargo-adaptor accessibility. Mechanistically, we identify a previously unrecognized intramolecular interaction between the KIF5C CC1 and CC4 domains that forms a stalk-mediated autoinhibitory latch. TRAK2 and KLC1 release this inhibitory interaction through distinct mechanisms. Together, our study establishes a molecular framework in which kinesin-1 holoenzyme assembly regulates cargo-adaptor recognition and autoinhibitory remodeling, providing mechanistic insight into how molecular motors coordinate cargo engagement with activation.

The cadherin–catenin complex governs neuronal development and synaptic signaling by linking cell adhesion to cytoskeletal dynamics. CTNND2 (δ‐catenin) is a neuron‐enriched p120 catenin whose C‐terminal PDZ domain‐binding motif (PBM) plays essential roles in dendritic spine morphogenesis and synaptic plasticity; however, its direct postsynaptic binding partner(s) and the structural determinant of binding selectivity remain unclear. Here, we systematically screened major postsynaptic PDZ proteins and identified MAGI2 as a specific, high‐affinity binder that recognizes the CTNND2 PBM exclusively via its PDZ5 domain. The crystal structure of the CTNND2 PBM–MAGI2 PDZ5 complex reveals a previously unrecognized electrostatic contact between D(−3) of the PBM and R1154 of PDZ5 that dictates specificity, explaining why other PDZ domains fail to bind CTNND2 PBM. This atypical PDZ–PBM interaction mode is conserved among p120 catenin family members ARVCF and PKP4, and extends to the classic catenin β‐catenin. Our findings define a non‐canonical PDZ recognition mechanism underlying the CTNND2–MAGI2 synaptic complex, with implications for CTNND2‐related diseases.

Kinesin-1 is a major microtubule-based molecular motor that transports diverse cellular cargoes, yet how assembly of its subunits is coupled to cargo recognition and motor regulation remains poorly understood. Mammalian kinesin-1 functions as a heterotetrameric holoenzyme composed of kinesin heavy chains (KIF5s) and kinesin light chains (KLCs), but the structural principles linking holoenzyme assembly to motor regulation remain unclear. Here, using KIF5C as a model system, we define the molecular mechanisms underlying kinesin-1 holoenzyme assembly and cargo-adaptor regulation. We identify a conserved coiled-coil interface between KIF5C and KLC1 that mediates their 2:2 assembly, with quantitative mutagenesis revealing key hydrophobic determinants of complex formation. We further demonstrate that the mitochondrial adaptor TRAK2 directly engages the KIF5C CC4 cargo-binding platform through a defined 2:2 interaction required for mitochondrial recruitment of KIF5C. Although KLC1 and TRAK2 bind distinct regions of KIF5C, KLC1 modulates TRAK2 association through steric and conformational effects, revealing how holoenzyme composition influences cargo-adaptor accessibility. Mechanistically, we identify a previously unrecognized intramolecular interaction between the KIF5C CC1 and CC4 domains that forms a stalk-mediated autoinhibitory latch. TRAK2 and KLC1 release this inhibitory interaction through distinct mechanisms. Together, our study establishes a molecular framework in which kinesin-1 holoenzyme assembly regulates cargo-adaptor recognition and autoinhibitory remodeling, providing mechanistic insight into how molecular motors coordinate cargo engagement with activation.

The m ⁶ A methyltransferase complex (“writer”) regulates mRNA stability and translation, but how its assembly is orchestrated remains incompletely understood. Wilms’ tumor 1-associating protein (WTAP) is a conserved regulatory subunit essential for m ⁶ A deposition and cell proliferation, yet its structural organization and mechanistic contributions remain elusive. Here, we report that WTAP dimerizes and further assembles into a stable tetramer through its middle coiled-coil domain, as revealed by high-resolution crystal structures. Disruption of this tetrameric interface abolishes WTAP’s interaction with METTL3, METTL14, and ZC3H13, impairs m ⁶ A deposition, and fails to rescue proliferation defects in WTAP-depleted cells. Live-cell imaging demonstrates that WTAP is required for accurate chromosome segregation, and MeRIP-seq analysis identifies WTAP-dependent m ⁶ A regulation as a critical determinant sustaining the expression of mitotic regulators, including KIF20A. Together, our study defines a tetrameric scaffold function for WTAP that is essential for writer complex integrity and highlights its pivotal role in linking m ⁶ A methylation to cell cycle progression.

MORC2 is a chromatin-associated ATPase essential for transcriptional silencing and genome stability, yet the biophysical principles governing its regulatory activity remain elusive. Here, we demonstrate that full-length MORC2 undergoes biomolecular condensation to form dynamic nuclear assemblies, a process fundamentally required for its repressor function. Endogenous MORC2 forms discrete, dynamic condensates in neurons from Morc2a ^EGFP chimeric mice, supporting the physiological relevance of these assemblies in vivo. Mechanistically, a 3.1 Å crystal structure of coiled-coil 3 (CC3) identifies a dimeric scaffold that serves as a structural hub, while multivalent ‘sticker’ interactions between an intrinsically disordered region (IDR) and a newly defined IDR-binding domain (IBD) drive condensation. We show that DNA acts as a molecular scaffold that triggers MORC2 condensation, which in turn allosterically stimulates its ATPase activity. Critically, by employing a ‘killswitch’ strategy to decouple assembly from internal fluidity, we reveal that only dynamic MORC2 condensates, not static aggregates or condensation-deficient mutants, can restore transcriptional regulation in MORC2 -knockout cells. Furthermore, pathogenic variants linked to CMT2Z and SMA differentially perturb these material properties and enzymatic turnover, providing a mechanistic link between condensate dysregulation and human neuropathies. Together, our findings establish a DNA-templated condensation mechanism for MORC2 and provide a molecular framework for understanding how the material state of chromatin-associated machinery dictates gene regulation and disease pathogenesis.

Several orthoebolaviruses have been established, including Orthoebolavirus zairense [for Ebola virus (EBOV)], Orthoebolavirus sudanense [for Sudan virus (SUDV)], and Orthoebolavirus bundibugyoense [for Bundibugyo virus (BDBV)]. Orthoebolaviruses are highly virulent pathogens that cause severe disease in humans. However, most existing vaccines primarily target EBOV, and fail to provide robust cross-protection against other lethal orthoebolaviruses. The development of a broad-spectrum vaccine has the potential to efficiently mitigate outbreaks caused by multiple orthoebolaviruses. In this study, we developed and evaluated a broad-spectrum mRNA vaccine, designated [GPs+NP]@LNP, formulated with a single lipid nanoparticle (LNP) platform to encapsulate a mixed mRNA payload encoding the glycoproteins (GPs) of EBOV, BDBV, and SUDV, along with the nucleoprotein (NP) of EBOV. This rational antigenic combination was designed to engage both humoral and cellular arms of the immune system. Consistent with this design, the compatibility of humoral immunity-driving GPs and cellular immune-dominating NPs supports complementary immune responses, which together are associated with broad and durable protection against EBOV, BDBV, and SUDV in animal models. Antigenic coordination within a single LNP formulation may therefore represent an effective strategy to optimize vaccine immunogenicity, protective effect, durability, and safety. Collectively, these findings highlight [GPs+NP]@LNP as a promising approach for the development of next-generation vaccines targeting multiple pathogenic orthoebolaviruses.

MORC2 is a chromatin-associated ATPase essential for transcriptional silencing and genome stability, yet the biophysical principles governing its regulatory activity remain elusive. Here, we demonstrate that full-length MORC2 undergoes biomolecular condensation to form dynamic nuclear assemblies, a process fundamentally required for its repressor function. Endogenous MORC2 forms discrete, dynamic condensates in neurons from EGFP-MORC2 knock-in mice, supporting the physiological relevance of these assemblies in vivo. Mechanistically, a 3.1 Å crystal structure of coiled-coil 3 (CC3) identifies a dimeric scaffold that serves as a structural hub, while multivalent ‘sticker’ interactions between an intrinsically disordered region (IDR) and a newly defined IDR-binding domain (IBD) drive condensation. We show that DNA acts as a molecular scaffold that triggers MORC2 condensation, which in turn allosterically stimulates its ATPase activity. Critically, by employing a “killswitch” strategy to decouple assembly from internal fluidity, we reveal that only dynamic MORC2 condensates—not static aggregates or condensation-deficient mutants—can restore transcriptional repression in MORC2-knockout cells. Furthermore, pathogenic variants linked to CMT2Z and SMA differentially perturb these material properties and enzymatic turnover, providing a mechanistic link between condensate dysregulation and human neuropathies. Together, our findings establish a DNA-templated condensation mechanism for MORC2 and provide a molecular framework for understanding how the material state of chromatin-associated machinery dictates gene regulation and disease pathogenesis

GLT25D1 O-galactosylates hydroxylysine residues in collagen and is essential for collagen maturation and function. Dysfunctions of GLT25D1 cause various tissue disorders. Despite its biological significance, the action mechanism of GLT25D1 remains enigmatic. Here we report the cryo-EM structures of human GLT25D1 and its ternary complex with UDP and hydroxylated acceptor substrates, revealing a bi-lobe architecture for the GLT25D1 monomer that organizes into dimeric and hexameric oligomers. The N-lobe of GLT25D1 contains a high-affinity UDP-galactose binding site, and the C-lobe is the catalytic domain of the enzyme. The structures together with biochemical analyses unravel the key recognition of the consensus “Hyl-Gly” motif from collagen acceptor substrates and associated catalytic mechanism. We further demonstrate that GLT25D1 mutations linked to cerebral small vessel disease and musculoskeletal defects adversely affect its function via distinct mechanisms. Our findings elucidate the molecular mechanism underlying collagen glycosylation and provide a molecular framework for understanding GLT25D1-related diseases.

AAA+ ATPases convert the chemical energy of ATP hydrolysis into mechanical work to remodel diverse cellular assemblies, driving essential biological processes. Canonically, these molecular motors are understood to function as stable oligomeric rings in which nucleotide turnover orchestrates coordinated conformational changes among subunits to act on pore-threaded substrates. However, how the meiotic-clade AAA+ ATPases, with their transient and compositionally fluid assemblies, generate sustained mechanical output remains unclear. Here we elucidate the structural and mechanistic basis of Msp1, a membrane-anchored meiotic-clade AAA+ dislocase that removes mistargeted tail-anchored proteins from organellar membranes. Using cryo-EM, we determined high-resolution structures of substrate-engaged, full-length Msp1 across distinct oligomeric states, revealing that ATP hydrolysis powers a continuous protomer flux along a membrane-anchored spiral. This dynamic assembly encodes intrinsic directionality, coupling tight substrate grip with rapid catalytic turnover to drive stepwise extraction. Collectively, our findings define a dynamic relay mechanism, demonstrating that AAA+ motors can drive processive work through architectural self-renewal rather than cyclic movements within a fixed ring.

ARMS (ankyrin repeat-rich membrane spanning) is a scaffold protein essential for neurotrophic signaling, synaptic development, and cytoskeletal remodeling. Despite its central role in neuronal function, how ARMS is regulated at the molecular level remains poorly understood. Here, we identify GABARAP, an Atg8-family autophagy adaptor, as a novel ARMS-binding protein that directly interacts with its N-terminal ankyrin repeats. We present the crystal structure of the ARMS-GABARAP complex, revealing an atypical interaction mode distinct from canonical LIR-dependent Atg8 interactions. Remarkably, ARMS specifically binds to the GABARAP subfamily of Atg8 proteins, setting it apart from the LC3 subfamily. Functional analysis demonstrates that GABARAP negatively regulates ARMS-mediated dendritic spine development and maturation in hippocampal neurons. Additionally, disrupting the ARMS-GABARAP complex using ankyrin-derived peptides alters ARMS subcellular localization, increasing its accumulation in the soma of neurons. Collectively, our findings uncover a novel ARMS-GABARAP interaction mechanism, establish the regulatory role of this complex in neuronal protein homeostasis, and suggest potential therapeutic strategies for targeting scaffold protein interactions in neurodevelopmental and neurodegenerative disorders.