Biology subjects

Hummer, G.

Publications and source records attributed to Hummer, G..

At least 19 recordsLinked to original sources

bioRxiv2026

Structure and function of the DFCP1 membrane barrier responsible for ER microdomain organization in lysosome repair

The endoplasmic reticulum (ER) forms specialized microdomains that organize autophagy, lipid droplet formation, and the repair of damaged lysosomes. The PI3P-binding protein DFCP1/ZFYVE1 marks these microdomains, raising the question as to whether and how one protein could define such a subcompartment. Here we determine the structure of full-length human DFCP1 bound to membranes by cryo-electron tomography and single-particle cryo-electron microscopy. DFCP1 assembles into a helical coat, built from tetramers that stack around a constricted membrane tube. Its ATPase domains form the organizing core of the helix, while its ER-targeting domains insert deeply into the outer leaflet, and its FYVE domains grip PI3P. Coarse-grained molecular dynamics simulations show that the coat does not simply follow the curvature of the membrane but acts as a barrier to lipid diffusion, almost completely blocking the movement of its cognate lipid PI3P past the filament and concentrating it beneath the coat. Point mutations that break the assembly, membrane-insertion or PI3P-binding interfaces each impair lysosomal repair in cells, and the assembly is also required for the focal recruitment of the lipid transporter VPS13C. We propose that DFCP1 collars fence off a specialized ER microdomain, so that locally made lipids can reach sites of repair rather than escaping into bulk ER.

cell biology↗

bioRxiv2026

Structural mechanism of Gasdermin E-mediated mtDNA release from apoptotic mitochondria

During apoptosis, the permeabilization of the mitochondrial inner membrane (MIM) through still-unclear mechanisms releases mtDNA into the cytosol, triggering the inflammatory cGAS/STING pathway under low caspase activity. Here, we report that, in apoptosis, the active form of the pore-forming protein Gasdermin E (GSDME-N) damages mitochondria before plasma membrane disruption. We visualize GSDME-N pore-like nano-assemblies in the MIM of apoptotic cells and of isolated mitochondria, which we bridge with the cryo-EM structure of the GSDME-N pore in mitochondria-like membranes. Deep membrane insertion of the anchor domain, which acts as a determinant of GSDME-N cardiolipin binding preferences, supports a role in pore formation. Notably, GSDME depletion results in a reduction in cristae swelling, MIM extrusion and mtDNA release during apoptosis. Subsequently, this decreases STING activation and inflammatory responses under caspase inhibition. Our findings reveal that GSDME mediates MIM permeabilization and mtDNA release during apoptosis and define the mechanism of GSDME-mediated membrane damage.

molecular biology↗

bioRxiv2026

Structural mechanism of nuclear membrane sealing by LEM2-ESCRT-III

In open mitosis, re-establishing nucleocytoplasmic compartmentalization requires the LEM2-ESCRT machinery to coordinate spindle clearance with sealing of the remaining nuclear envelope pores. The structural basis of this topologically unique and fundamental membrane-remodeling process is poorly understood. Here, we combine biochemical reconstitution, cryo-electron tomography, subtomogram averaging and large-scale molecular dynamics simulations to define the structural mechanism of nuclear membrane sealing. We structurally resolve that LEM2s winged-helix domain (WH) co-polymerizes with the ESCRT-II/III protein CHMP7 to form a membrane-bound scaffold whose geometry is progressively remodeled by downstream ESCRT-III proteins as it transitions from the flat membrane surrounding the pore towards the negatively curved membrane neck. In parallel, LEM2 positions its intrinsically disordered low-complexity domain within the pore, where condensation around spindle microtubules mechanically couples the membrane-ESCRT-LEM2 scaffold to the spindle and narrows the remaining diffusion path, restoring compartmentalization before membrane closure is complete. Remarkably, the LEM2-WH domain alone forms tightly constricted membrane tubes, coating the negatively curved inner surface, revealing an intrinsic membrane-remodeling activity of the receptor itself. Together, our work establishes a structural framework for how receptor-ESCRT co-polymerization, low complexity domain-mediated sealing and receptor-driven membrane remodeling guide nuclear-envelope pores from spindle-containing openings to terminal constriction and fusion.

molecular biology↗

bioRxiv2026

Identification of lipid specificity in membrane organizing protein complexes

Organelle membrane identity is encoded by protein and lipid composition1. Human cells produce thousands of distinct lipid species for this purpose2,3. Yet, the precise molecular functions of most lipids remain unknown, owing to limited methods for in situ investigations of individual lipid species. Here, we use minimally modified bifunctional lipid probes to detect and functionally characterize lipid-protein interactions across time and subcellular compartments. We leverage this approach to map time-resolved protein interactomes of individual lipids representing major membrane lipid classes as they are transported through the organelle system of human cells. We identify hundreds of unique lipid-protein interaction candidates, measure lipid-induced protein abundance changes, and distinguish between directly and indirectly interacting subunits of associated protein complexes. Informed by this multimodal dataset, we interrogate the lipid specificity of membrane organizing complexes, in particular the selective co-association of phosphatidylethanolamine with the ancestral subunits of the nuclear pore (NDC1) and MICOS (MIC60) complexes. Using super-resolution microscopy and molecular dynamics simulations, we demonstrate that phosphatidylethanolamine stabilizes both NDC1 and MIC60 in highly curved membrane nanodomains, indicating that major membrane organizing complexes are positioned by selective lipid-protein interactions. Altogether, our experimental strategy provides a blueprint for discovering and characterizing cellular functions of individual lipids.

cell biology↗

bioRxiv2026

Effects of PTMs on Tau Protein Aggregation: Insights from HCG and Atomistic MD Simulations

Post-translational modifications (PTMs) of the tau protein are increasingly recognized as pivotal regulators in the onset and progression of tauopathies, such as Alzheimers disease (AD). To systematically evaluate the structural and functional consequences of specific PTMs, we generated and analyzed seven distinctly modified variants of the tau-K32 construct. These included phosphorylation at Ser202/Thr205, phosphorylation at Ser258/Ser262/Ser356, full phosphorylation at all reported Ser/Thr sites, acetylation at Lys274/Lys281, acetylation at Lys280, full acetylation at all sites, and an unmodified control. Selection of PTM sites was guided by prior experimental literature. By incorporating fully modified tau models, we assessed the global impact of widespread modifications on structural properties and aggregation behavior. Our findings establish a comparative framework for understanding how discrete and cumulative PTMs modulate tau aggregation and provide mechanistic insight into PTM-induced tau dysfunction relevant to neurodegenerative diseases.

biochemistry↗

bioRxiv2026

Chemically tunable permeability of engineered alpha-Hemolysin in synthetic cells

Controlling molecular transport across membranes is a defining feature of living cells, yet replicating this functionality in synthetic systems remains a major challenge. Self-inserting protein nanopores, such as -hemolysin (HL), offer a promising route towards programmable membrane permeability due to their robust assembly, compatibility with diverse membrane systems, and intrinsic permeability for diverse biomolecules. Here, we explored the use of chemically functionalized HL nanopores as tunable transport modules. To quantify translocation of peptide substrates across HL-containing membranes, we developed a high-throughput luminescence-based breakage-controlled assay using large unilamellar vesicles. With this assay we introduce a one-pot nanopore modification and strategy, compatible with scalable workflows. Electrophysiology and molecular simulations demonstrate that the introduction of cysteine residues at defined pore locations, combined with targeted chemical modification, enables controlled tuning of HL-nanopore selectivity based on peptide structure and charge. Together, these findings position engineered protein nanopores as versatile and responsive components for controlling membrane transport in synthetic biology.

synthetic biology↗

bioRxiv2026

Does the sequence of a disordered protein encode small molecule binding paths?

Ligand binding to intrinsically disordered proteins resists description in terms of conventional binding pockets, yet it can be analysed as a dynamic process in which ligands move across transient surface interaction sites. Here we characterise a pathway-based representation in which ligand binding is described as a sequence of transitions between residue-defined microstates, enabling ligand-specific effects to be distinguished from intrinsic properties of the peptide conformational ensemble. Using all-atom molecular dynamics simulations of A{beta}42 and the C-terminal region of -synuclein in complex with chemically diverse small molecules, we construct transition matrices that encode ligand movement across the peptide surface and use Markov state models to identify dominant binding pathways and relative binding propensities. Pairwise enrichment-factor and AUC analyses reveal strong conservation of the highest-ranked pathways across chemically diverse ligands, with enrichment factors of 15-45 for the top-ranked states and AUC values typically [≥]0.75, well above random expectation. These dominant pathways are also preserved across changes in pH and temperature, whereas a urea control, included as a non-specific binder, shows reduced enrichment, indicating that ligands primarily modulate pathway weights rather than define the underlying network topology. Ensemble docking across chemically diverse libraries further supports the presence of recurrent ligand-accessible hotspots within the peptide conformational ensemble. Building on this framework, we apply a prospective screening pipeline to A{beta}42, combining MSM-derived hotspots with sequence-based Ligand-Transformer scoring and Gnina docking across 1.66 million compounds, to nominate 19 candidates for prospective experimental evaluation. Together, these results indicate that disordered protein sequences give rise to conformational ensembles that exhibit characteristic binding pathways for small molecules.

biophysics↗

bioRxiv2026

Activation of the bridge-like lipid transfer protein Atg2 by Atg1-mediated phosphorylation

Bridge-like lipid transfer proteins (BLTPs) have emerged as major contributors to lipid flux and thus cellular organization. BLTPs harbor a hydrophobic channel connecting two membranes to enable the bulk flow of lipids between them. The regulation of BLTPs is incompletely understood. Employing in vitro reconstitution, molecular dynamics simulations, and cell biology, we discovered that the BLTP Atg2, which mediates lipid transfer during autophagosome biogenesis, is activated by the Atg1 kinase. Atg1 phosphorylates two serines in the N-terminal region of Atg2. This triggers membrane binding and opening of the channel to enable lipid transfer. The Atg1 kinase complex is localized to the endoplasmic reticulum through binding of its Atg13 subunit to the VAP protein Scs2. Phosphorylated Atg2 can subsequently establish contacts with Atg9 vesicles. Our study delineates a pathway for the Atg1 kinase mediated activation of the BLTP Atg2 to establish membrane contact site formation and lipid flux.

cell biology↗

bioRxiv2026

Coenzyme A is bound to tafazzin - a paradigm change for transacylation

Cardiolipin (CL) is the signature phospholipid of mitochondria. In an obligatory remodeling process, the mitochondrial transacylase tafazzin exchanges its acyl chains to create the highly unsaturated, mature form of CL. Tafazzin dysfunction causes Barth syndrome, a severe multisystem disorder. We determined the structure of tafazzin at a resolution of 2.2 [A] using cryo-electron microscopy (cryo-EM). Until now, the tafazzin reaction has been thought to be independent of coenzyme A (CoA). However, our structure clearly shows an acyl-CoA molecule bound to tafazzin. To decipher how substrates bind to the active site, we combine cryo-EM with structure predictions and molecular dynamics simulations, giving us detailed insights into a transacylation mechanism mediated by CoA. By providing molecular explanations of tafazzin dysfunction caused by pathogenic mutations, we gain a molecular understanding of Barth syndrome.

biochemistry↗

bioRxiv2026

Cathepsin-dependent amyloid formation drives mechanical rupture of lysosomal membranes

Lysosomal membrane integrity is essential for cellular homeostasis, and its failure drives lysosomal storage disorders (LSD) and neurodegeneration. The dipeptide L-leucyl-L-leucine methyl ester (LLOMe) is widely used to model lysosomal damage, yet its mechanism remains poorly understood. The prevailing view holds that LLOMe polymerizes into membrane-permeabilizing peptide chains within the lysosomal lumen. Using cryo-electron tomography in cultured cells and primary neurons, we visualized the structural basis of LLOMe-induced lysosomal damage. We reveal that LLOMe forms amyloid structures within lysosomes that directly interact with and rupture the limiting membrane through mechanical stress. In vitro reconstitution confirms this amyloid-mediated mechanism. These findings establish a structural paradigm for lysosomal membrane disruption and provide insights into how disease-relevant protein aggregates, implicated in neurodegeneration and LSD, may compromise lysosomal integrity.

cell biology↗

bioRxiv2026

Biomolecular condensate architecture of an autophagic cargo at molecular resolution in situ

Biomolecular condensates organise cellular biochemistry, yet their molecular architecture in situ remains poorly understood. During selective autophagy, macromolecules frequently accumulate into biomolecular condensates, forming discrete entities for autophagic engulfment and degradation - ideal systems for structural analysis. We employed in situ cryo-electron tomography to determine the near-atomic resolution structure of Aminopeptidase 1 condensates within cells. These condensates form densely packed, spherical assemblies with amorphous organisation and liquid-like properties, elucidating the requirements of a selective autophagic cargo for exclusive targeting. Structural analysis and multiscale simulations reveal that the short, transient -helical structures in the disordered N-terminus of Aminopeptidase 1 enable site-specific, coiled-coil-like interactions required for condensate formation and properties. A single point mutation that increases -helical propensity directly modulates condensate viscosity and dynamics from a liquid-like to a glass-like state, while preserving local molecular packing. Our results demonstrate that disordered regions encode both specificity and material properties through transiently structured motifs, linking sequence specificity to phase behaviour in cells and expanding the molecular logic of phase separation in cells.

cell biology↗

bioRxiv2025

The vault associates with membranes in situ

The eukaryotic vault particle is a giant ribonucleoprotein complex that assembles into an iconic barrel-like cage. Its cellular function has remained elusive despite extensive characterization. Using cryo-electron tomography of Dictyostelium discoideum cells, we define the distribution, structural states and interaction landscape of vault particles in situ. Surprisingly, we detect a subpopulation of vault particles associated with the endoplasmic reticulum (ER) and nuclear envelope membranes. This association occurs at a defined barrel height of the vault particle and at patches of reduced membrane bilayer thickness and altered curvature. We further find that a substantial fraction of vaults encloses 80S ribosomes in highly ordered orientations. These structural findings are further corroborated by proximity labeling experiments which identify ER-resident proteins and numerous ribosomal components as vault particle interactors. The membrane-bound and ribosome-encapsulating vault populations that we uncover will direct future studies towards revealing vault function.

cell biology↗

bioRxiv2025

O-GlcNAcylation and an importin-β radial gradient keep the FG barrier liquid in live-cell nuclear pores

Summary ParagraphThe nuclear pore complex (NPC) regulates the molecular traffic between nucleus and cytoplasm.1,2 Its permeability barrier is formed by intrinsically disordered proteins (IDPs) known as FG-nucleoporins (FG-NUPs), whose physical state has long been debated.3-13 Deciphering how FG-NUPs behave inside living cells is crucial for understanding how the NPC achieves selective and rapid transport. Here, by combining site-specific labelling with picosecond time-resolved fluorescence anisotropy, we reveal that FG domains exhibit nanosecond-scale, liquid-like mobility in live cells, yet undergo a liquid-to-solid transition in vitro. Experiments and coarse-grained molecular dynamics simulations further show that importin-{beta}, a major nuclear transport receptor, and O-linked {beta}-N-acetylglucosamine (O-GlcNAc), a key post-translational modification of FG-NUPs together stabilise the dynamic FG network. Finely balanced FG-FG interactions modulated by O-GlcNAcylation, along with FG-importin-{beta} interactions, maintain liquidity and enrich importin-{beta} near the NPC periphery, while extended FG domains remain in the central channel to form the transport barrier. These findings reconcile conflicting models of FG-NUP organisation4,6,8,10,14,15 and explain the recent observations in high-resolution MINFLUX studies of importin-{beta} depletion from the pore centre in mammalian cells.16 Beyond resolving debates over FG-NUP behaviour, our study underscores the importance of studying IDPs in their cellular context, with broader implications for understanding IDP-related diseases, including viral infections, cancer, and neurodegenerative disorders.

biophysics↗

bioRxiv2025

Binding Paths: Describing Small Molecule Interactionswith Disordered Proteins via Markov State Models

Disordered proteins are challenging targets for drug discovery because they lack well-defined binding pockets. Although small molecules can form relatively stable complexes with disordered proteins, the highly dynamic nature of these proteins complicates the understanding of their binding mechanism. To address this problem, we analyze the binding of the small molecule 10074-G5 to A{beta}42, which results in the formation of a disordered complex. We describe the binding mechanism in terms of binding paths, which are stochastic trajectories along which small molecule diffuse across disordered protein surfaces, forming transient contacts with overlapping groups of residues. To identify these binding paths, we define them as realizations of a stochastic process defined by a Markov State Model (MSM). The MSM is built from distinct states of the disordered complex and their corresponding transition probabilities, enabling both the dynamic mapping of binding hotspots and targetable regions where static pockets cannot be defined-extending the notion of binding pockets to disordered systems - and the quantitative calculation of binding affinities. The visualization of the MSM via knowledge graphs provides an intuitive representation of the binding paths. We further validated this approach across four additional systems, comprising C-terminal -synuclein with three distinct small-molecule binders, and the full-length peptide (140 residues) with binder fasudil. By generalizing the concept of static binding pockets to dynamic binding paths, our approach rationalizes small-molecule recognition by disordered proteins and establishes a framework for identifying druggable regions on systems that were previously considered intractable, providing insights for future drug design programs.

biophysics↗

bioRxiv2025

Pathway and gates for ATG2A-mediated lipid transport in autophagy

Autophagy is a complex process in which eukaryotic cells degrade cytosolic components by delivering them to lysosomes via double-membrane autophagosomes. The lipid transfer protein ATG2A plays a crucial role in autophagosome formation by tethering the phagophore membrane to the ER and delivering a significant fraction of the required lipids. The mechanism by which ATG2A shuttles lipids from one membrane to the other, however, remains elusive. Here, we combine structural predictions, molecular dynamics simulations and in vitro lipid transfer assays to gain mechanistic insights into ATG2A-mediated lipid transport. Using this integrative approach, we characterize the contact sites of the protein with donor and acceptor membranes. Our simulations capture multiple events of lipid uptake and delivery from and to the bound membrane. Conformational rearrangements of N-terminal amphipathic helices emerge as a critical factor for facile lipid transport. With this insight, we designed an ATG2A mutant that transfers lipids three times faster than the wild type in vitro. In complex with ATG9A, ATG2A forms a bridge between two parallel membranes at 12 nm separation. Overall, our findings suggest that ATG2A is a lipid transporter gated at the N-terminus by blocking helices that, upon release, act as additional membrane tethers.

biophysics↗

bioRxiv2025

Reconstitution of multistep recruitment of ULK1 to membranes in autophagy

The Human ULK1 autophagy-initiating complex consists of ULK1, FIP200, and the HORMA domain heterodimer ATG13:ATG101. PI3P is essential to recruit ULK1C to membranes for ULK1, but ULK1C subunits do not contain PI3P-binding domains. Here we show that the ATG13:ATG101 dimer forms a complex with the PI3P-binding protein WIPI3, as well as WIPI2. Bound to WIPI2-3, ATG13:ATG101 inserts its Trp-Phe (WF) finger into the membrane. MD simulations show that WIPIs and the WF finger cooperatively stabilizes the complex on membranes. Biochemical reconstitution and cell-based assays show that WIPI3:ATG13 engagement promotes ATG16L1 phosphorylation, autophagy and mitophagy. A kinase domain (KD)-proximal PVP motif in the ULK1 IDR docks onto the ATG13:ATG101 HORMA dimer brings the ULK1 KD close to the membrane. The PVP motif is essential for in vitro ULK1 phosphorylation of ATG16L1 and important for autophagy and mitophagy. These data establish a stepwise pathway for recruitment of the ULK1 KD to the vicinity of the membrane surface.

cell biology↗

bioRxiv2025

Structural flexibility of the human vault protein revealed by high-resolution cryo-EM and molecular dynamics simulations

Vaults are massive ribonucleoprotein complexes, highly conserved and abundant in eukaryotic cells, yet with unclear function. Their thin-walled barrel-shape architecture is composed of two symmetrical, antiparallel half-shells, each containing 39 copies of the major vault protein (MVP). The spacious lumen of the vault suggests a role in cellular transport. To facilitate cargo encapsulation and release, the vault is thought to open into two halves, yet the molecular mechanism governing vault opening remains elusive. Here, we combine cryogenic electron microscopy (cryo-EM) and multi-scale molecular dynamics (MD) simulations to reveal the structural factors giving flexibility to the human vault protein. Using cryo-EM, we identified two alternative conformational states of the human vault, along with the half-vault shell. MD simulations of these structures show extensive, breathing-like motions, porous solvent-exposed surfaces, and distinct structural variability between conformational states. The stable intermediates and the flexibility at the interface of the half vaults together suggest a possible mechanism for the dynamic assembly and disassembly of the vault.

biochemistry↗

bioRxiv2025

SND3 is the membrane insertase within a fungal multipass translocon

During the biogenesis of most eukaryotic integral membrane proteins (IMPs), transmembrane domains are inserted into the endoplasmic reticulum membrane by a dedicated insertase or the SEC61 translocon. The SRP-independent (SND) pathway is the least understood route into the membrane, despite catering for a broad range of IMP types. Here, we show that Chaetomium thermophilum SND3 is a membrane insertase of novel fold. We further present a cryo-electron microscopy structure of a ribosome-associated SND3 translocon complex involved in co-translational IMP insertion. The structure reveals that the SND3 translocon additionally comprises the complete SEC61 translocon, CCDC47 and TRAP[a]. Here, the SEC61{beta} N-terminus works together with CCDC47 to prevent substrate access to the translocon. Instead, molecular dynamics simulations show that SND3 disrupts the lipid bilayer to promote IMP insertion via its membrane-embedded hydrophilic groove. Structural and sequence comparisons indicate that the SND3 translocon is a distinct multipass translocon for the insertion of multipass IMPs in fungi, euglenozoan parasites and other eukaryotic taxa.

biophysics↗