Biology subjects

Hummer, G.

Publications and source records attributed to Hummer, G..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

AlphaFold modeling of polyubiquitin complexes and covalently linked proteins

AbstractCells use the covalent attachment of Ubiquitin (Ub) chains to mark proteins for degradation, alter their cellular localization or drive their association. Protein fate is encoded in the distinct poly-Ub linkages, exploiting the vast combinatorial space of linear and branched Ub modifications. AlphaFold has emerged as a powerful tool to predict the structure of protein-protein complexes. However, standard AlphaFold does not consider linkages between individual protein chains, limiting its applicability to Ub chains. The near complete conservation of the ubiquitin sequence and the large number of binding partners suppresses coevolutionary signals, further challenging the prediction of poly-Ub complex structures. We address this challenge, first, by introducing correlated cysteine mutations to induce linkage-specific proximity of Ubs in complex with interacting proteins. Second, we introduce short covalent linker groups in AlphaFold 3 calculations that mimic the isopeptide bonds between linked lysines and Ub C-terminal carboxylates. These two approaches enable the robust structural modeling of complexes involving poly-Ub chains with AlphaFold. The linker approach is general and can be used for other covalent inter-chain connections and to enforce distance restraints for integrative structural modeling.

bioinformatics↗

bioRxiv2025

Mechanical forces drive mitochondrial matrix extrusion and apoptotic pore growth

Apoptotic pore opening by BAX and BAK at the mitochondrial outer membrane is a key step in the cell commitment to death. The subsequent inner membrane extrusion and permeabilization releases mitochondrial DNA into the cytosol, which can trigger inflammatory signaling. However, the underlying mechanisms have not been elucidated. Here we developed CLOSE microscopy, a multi-correlative approach that enabled the simultaneous analysis of BAK stoichiometry and nanoscale organization in individual apoptotic pore complexes in relation to mitochondrial ultrastructure. We find that the outer membrane opening at the apoptotic pore defines the spatial arrangement of BAK nanoassemblies. We identify mechanical stress as a driver of inner membrane extrusion, which can be perturbed by osmoregulation. The extruded inner membrane in turn exerts forces on the outer membrane that promote apoptotic pore growth, in line with membrane dynamics simulations. Our study reveals a tight interplay between the inner and outer membranes during mitochondrial permeabilization in apoptosis and establishes a biophysical mechanism for inner membrane extrusion that defines the structural organization of the apoptotic pore.

cell biology↗

bioRxiv2025

Molecular architecture of heterochromatin at the nuclear periphery of primary human cells

In eukaryotes, meters of DNA are packaged into micrometer scale nuclei. Nucleosomes, as the major organizational unit, have been extensively studied in vitro, yet the elaborate 3D structure of chromatin inside cells and its distinct multi-nucleosome arrangements remain poorly resolved. Here, we combine cryo-electron tomography with template matching, subtomogram averaging and molecular simulations to visualize nucleosomes and chromatin structure inside human cells. We confidently assign individual nucleosomes and report their in-situ structure at secondary structure resolution. By tracing linker DNA, we identify multi-nucleosome arrangements and uncover higher-order chromatin structures in situ, including a 37-nm wide, elongated but non-fibrous arrangement. In situ structural biology thus reveals the molecular chromatin organization inside cells and sets the stage for 3D genomics.

biophysics↗

bioRxiv2025

STIM1 transmembrane helix dimerization captured by AI-guided transition path sampling

STIM1 is a Ca2+-sensing protein in the endoplasmic reticulum (ER) membrane. The depletion of ER Ca2+ stores induces a large conformational transition of the cytosolic STIM1 C-terminus, initiated by the dimerization of the transmembrane (TM) domain. We use the AI-guided transition path sampling algorithm aimmd to extensively sample the dimerization of STIM1-TM helices in an ER-mimicking lipid bilayer. In nearly 0.5 milliseconds of all-atom molecular dynamics simulations without bias potentials, we harvest over 170 transition paths, each about 1.2 {micro}s long on average. We find that STIM1 dimerizes into three distinct and coexisting configurations, which reconciles conflicting results from earlier crosslinking studies. The dominant X-shaped bound state centers around contacts supported by the SxxxG TM interfacial motif. Mutating residues in this contact interface allows us to tune the STIM1-dimerization propensity in fluorescence experiments. From the trained model of the committor probability of dimerization, we identify the transition state ensemble for TM-helix dimerization. At the transition state, interhelical contacts in the luminal halves of the two monomers dominate, which likely enables the luminal Ca2+-sensing domain in STIM1 to condition the dimerization of the TM helices. Our work demonstrates the unique power of AI-guided simulations to sample rare and slow molecular transitions, and to produce detailed atomistic insight into the mechanism of STIM1 TM-helix dimerization as a key step in ER Ca2+-sensing. SignificanceSTIM1 is a protein sensor that signals drops in the Ca2+ concentration inside the endoplasmic reticulum (ER) to the cytosol. As Ca2+ levels decrease, the STIM1 transmembrane (TM) helices dimerize. We sample this TM helix dimerization in AI-guided atomistic molecular simulations, revealing two distinct pathways of dimerization. We reconcile conflicting results of earlier crosslinking studies by showing that both reported dimer configurations coexist as the end points of distinct association pathways. The dominance of interhelical contacts on the luminal side at the transition state enables the luminal Ca2+-sensing domain to condition helix dimerization. AI-guided path sampling made it possible to sample rare helix dimerization events far outside the current reach of regular molecular simulations.

biophysics↗

bioRxiv2024

cryoTIGER: Deep-Learning Based Tilt Interpolation Generator for Enhanced Reconstruction in Cryo Electron Tomography

Cryo-electron tomography enables the visualization of macromolecular complexes within native cellular environments, but is limited by incomplete angular sampling and the maximal electron dose that biological specimen can be exposed to. Here, we developed cryoTIGER (Tilt Interpolation Generator for Enhanced Reconstruction), a computational workflow leveraging a deep learning-based frame interpolation to generate intermediate tilt images. By interpolating between tilt series projections, cryoTIGER improves angular sampling, leading to enhanced 3D reconstructions, more refined particle localization, and improved segmentation of cellular structures. We evaluated our interpolation workflow on diverse datasets and compared its performance against non-interpolated data. Our results demonstrate that deep learning-based interpolation improves image quality and structural recovery. The presented cryoTIGER framework offers a computational alternative to denser sampling during tilt series acquisition, paving the way for enhanced cryo-ET workflows and advancing structural biology research.

molecular biology↗

bioRxiv2024

AlphaFold2 SLiM screen for LC3-LIR interactions in autophagy

In selective autophagy, cargo recruitment is mediated by LC3-interacting regions (LIRs) / Atg8-interacting motifs (AIMs) in the cargo or cargo receptor proteins. The binding of these motifs to LC3/Atg8 proteins at the phagophore membrane is often modulated by post-translational modifications, especially phosphorylation. As a challenge for computational LIR predictions, sequences may contain the short canonical (W/F/Y)XX(L/I/V) motif without being functional. Conversely, LIRs may be formed by non-canonical but functional sequence motifs. AlphaFold2 has proven to be useful for LIR predictions, even if some LIRs are missed and proteins with thousands of residues reach the limits of computational feasibility. We present a fragment-based approach to address these limitations. We find that fragment length and phosphomimetic mutations modulate the interactions predicted by AlphaFold2. Systematic fragment screening for a range of target proteins yields structural models for interactions that AlphaFold2 and AlphaFold3 fail to predict for full-length targets. We provide guidance on fragment choice, sequence tuning, and LC3 isoform effects for optimal LIR screens. Finally, we also test the transferability of this general framework to SUMO-SIM interactions, another type of protein-protein interaction involving short linear motifs (SLiMs).

bioinformatics↗

bioRxiv2024

Reconstitution of BNIP3/NIX-mediated autophagy reveals two pathways and hierarchical flexibility of the initiation machinery

Selective autophagy is a lysosomal degradation pathway that is critical for maintaining cellular homeostasis by disposing of harmful cellular material. While the mechanisms by which soluble cargo receptors recruit the autophagy machinery are becoming increasingly clear, the principles governing how organelle-localized transmembrane cargo receptors initiate selective autophagy remain poorly understood. Here, we demonstrate that transmembrane cargo receptors can initiate autophagosome biogenesis not only by recruiting the upstream FIP200/ULK1 complex but also via a WIPI-ATG13 complex. This latter pathway is employed by the BNIP3/NIX receptors to trigger mitophagy. Additionally, other transmembrane mitophagy receptors, including FUNDC1 and BCL2L13, exclusively use the FIP200/ULK1 complex, while FKBP8 and the ER-phagy receptor TEX264 are capable of utilizing both pathways to initiate autophagy. Our study defines the molecular rules for initiation by transmembrane cargo receptors, revealing remarkable flexibility in the assembly and activation of the autophagy machinery, with significant implications for therapeutic interventions.

cell biology↗

bioRxiv2024

Refining Martini Force Field Interactions for Accurate Glycoprotein Modeling

Covalently attached sugar molecules play important roles as mediators of biomolecular interactions. Molecular dynamics simulations are an indispensable tool to explore these interactions at the molecular level. The large time and length scales involved frequently necessitate the use of coarse-grained representations, which heavily depend on the parameterization of sugar-protein interactions. Here, we adjust the sugar-protein interactions in the widely used Martini 2.2 force field to reproduce the experimental second virial coefficients between sugars and proteins. In simulations of two model proteins in glucose solutions with adjusted force field parameters, we observe weak protein-sugar interaction. The sugar molecules are thus acting mainly as crowding agents, in agreement with experimental measurements. The procedure to fine-tune sugar-protein interactions is generally applicable and could prove useful also for atomistic force fields.

biophysics↗

bioRxiv2024

Snapshots of acetyl-CoA synthesis, the final step of CO2 fixation in the Wood-Ljungdahl pathway

In the ancient microbial Wood-Ljungdahl pathway, CO2 is fixed in a multi-step process ending with acetyl-CoA synthesis at the bifunctional carbon monoxide dehydrogenase/acetyl-CoA synthase complex (CODH/ACS). Here, we present catalytic snapshots of the CODH/ACS from the gas-converting acetogen Clostridium autoethanogenum, characterizing the molecular choreography of the overall reaction including electron transfer to the CODH for CO2 reduction, methyl transfer from the corrinoid iron-sulfur protein (CoFeSP) partner to the ACS active site and acetyl-CoA production. Unlike CODH, the multidomain ACS undergoes large conformational changes to form an internal connection to the CODH active site, accommodate the CoFeSP for methyl transfer and protect the reaction intermediates. Altogether, the structures allow us to draw a detailed reaction mechanism of this enzyme crucial for CO2 fixation in anaerobic organisms. One-Sentence Summary: Structural description of key states of CO2 fixation by the carbon monoxide dehydrogenase/acetyl-CoA synthase complex.

biophysics↗

bioRxiv2024

Passage of the HIV capsid cracks the nuclear pore

Upon infection, human immunodeficiency virus (HIV-1) releases its cone-shaped capsid into the cytoplasm of infected T-cells and macrophages. As its largest known cargo, the capsid enters the nuclear pore complex (NPC), driven by interactions with numerous FG-repeat nucleoporins (FG-Nups). Whether NPCs structurally adapt to capsid passage and whether capsids are modified during passage remains unknown, however. Here, we combined super-resolution and correlative microscopy with cryo electron tomography and molecular simulations to study nuclear entry of HIV-1 capsids in primary human macrophages. We found that cytosolically bound cyclophilin A is stripped off capsids entering the NPC, and the capsid hexagonal lattice remains largely intact inside and beyond the central channel. Strikingly, the NPC scaffold rings frequently crack during capsid passage, consistent with computer simulations indicating the need for NPC widening. The unique cone shape of the HIV-1 capsid facilitates its entry into NPCs and helps to crack their rings.

biophysics↗

bioRxiv2024

Robotic-inspired approach to multi-domain membrane receptor conformation space: theory and SARS-CoV-2 spike protein case study

1The spike protein of SARS-CoV-2 is a highly flexible membrane receptor that triggers the translocation of the virus into cells by attaching to the human receptors. Like other type I membrane receptors, this protein has several extracellular domains connected by flexible hinges. The presence of these hinges results in high flexibility, which consequently results in challenges in defining the conformation of the protein. Here, We developed a new method to define the conformational space based on a few variables inspired by the robotic fields methods to determine a robotic arms forward kinematics. Using newly performed atomistic molecular dynamics (MD) simulations and publicly available data, we found that the Denavit-Hartenberg (DH) parameters can reliably show the changes in the local conformation. Furthermore, the rotational and translational components of the homogenous transformation matrix constructed based on the DH parameters can identify the changes in the global conformation of the spike and also differentiate between the conformation with a similar position of the spike head, which other types of parameters, such as spherical coordinates, fail to distinguish between such conformations. Finally, the new method will be beneficial for looking at the conformational heterogeneity in all other type I membrane receptors.

biophysics↗

bioRxiv2024

Intrinsically disordered region amplifies membrane remodeling to augment selective ER-phagy

Intrinsically disordered regions (IDRs) play a pivotal role in organellar remodeling. They transduce signals across membranes, scaffold signaling complexes, and mediate vesicular traffic. Their functions are regulated by constraining conformational ensembles through specific intra- and inter-molecular interactions, physical tethering, and post-translational modifications. The ER-phagy receptor FAM134B/RETREG1, known for its Reticulon homology domain (RHD), includes a substantial C-terminal intrinsically disorder region (IDR) housing the LC3 interacting (LIR) motif. Beyond engaging the autophagic machinery, the function of the FAM134B-IDR is unclear. Here, we investigate the characteristics of the FAM134B-IDR by extensive modeling and molecular dynamics (MD) simulations. We present detailed structural models for the IDR, mapping its conformational landscape in solution and membrane-anchored configurations. Our analysis reveals that depending on the membrane anchor, the IDRs collapse onto the membrane and induce positive membrane curvature to varying degrees. The charge patterns underlying this Janus-like behavior are conserved across other ER-phagy receptors. We found that IDRs alone are sufficient to sense curvature. When combined with RHDs, they intensify membrane remodeling and drive efficient protein clustering, leading to faster budding, thereby amplifying RHD remodeling functions. Our simulations provide a new perspective on IDRs of FAM134B, their Janus-like membrane interactions, and the resulting modulatory functions during large-scale ER remodeling.

biophysics↗

bioRxiv2024

Nuclear pores as conduits for fluid flow during osmotic stress

Changing environmental conditions necessitate an immediate cellular adaptation to ensure survival. Dictyostelium discoideum, a bacteriovore slime mold present in the soil of most terrestrial ecosystems, is known for its ability to tolerate drastic changes in osmolarity. How the cells cope with the resulting mechanical stress remains understudied. Here we show that D. discoideum has extraordinarily elaborate and resilient nuclear pores that serve as conduits for massive fluid exchange between cytosol and nucleus. We capitalize on the unique properties of D. discoideum cells to quantify flow across the nuclear envelope that is necessitated by changing nuclear size in response to osmotic stress. Based on mathematical concepts adapted from hydrodynamics, we conceptualize this phenomenon as porous flow across nuclear pores. This type of fluid flow is distinct from the canonically characterized modes of nucleocytoplasmic transport, i.e. passive diffusion and active nuclear transport, because of its dependence on pressure. Our insights are relevant in any biological condition that necessitates rapid nuclear size changes, which includes metastasizing cancer cells squeezing through constrictions, migrating cells and differentiating tissues.

cell biology↗

bioRxiv2023

Structure and regulation of GSDMD pores at the plasma membrane of pyroptotic cells

Gasdermin D (GSDMD) executes inflammatory cell death pyroptosis by permeabilizing the plasma membrane (PM). We introduce polymer-supported PM (PSPM) to gain access to the cytoplasmic side of the PM with imaging techniques while preserving the native PM complexity and lipid microenvironment. By combining PSPM with DNA-PAINT super-resolution microscopy we visualized, for the first time, GSDMD nanostructures directly at the PM of pyroptotic cells. We resolved diverse macromolecular architectures with ring-and arc-shaped GSDMD oligomers that enable PM permeabilization. The pyroptotically-inactive mutant GSDMD-C192A (human C191A) still interacts with the PM however fails to form pores. GSDMD expression levels affect pore density but not permeabilization ability. Finally, we identified the local PI(3,4,5)P3 concentration as a key regulatory element of PM permeabilization. Increase in PI(3,4,5)P3 levels in the PM during pyroptosis facilitates growth into large ring-shaped pores. Using molecular dynamics (MD) simulations, we identified the mechanism by which PI(3,4,5)P3 stabilizes the GSDMD assembly.

biophysics↗

bioRxiv2023

Mechanism and cellular function of direct membrane binding by the ESCRT and ERES-associated Ca2+-sensor ALG-2

Apoptosis Linked Gene-2 (ALG-2) is a multifunctional intracellular Ca2+ sensor and the archetypal member of the penta-EF hand protein family. ALG-2 functions in the repair of damage to both the plasma and lysosome membranes and in COPII-dependent budding at endoplasmic reticulum exit sites (ERES). In the presence of Ca2+, ALG-2 binds to ESCRT-I and ALIX in membrane repair and to SEC31A at ERES. ALG-2 also binds directly to acidic membranes in the presence of Ca2+ by a combination of electrostatic and hydrophobic interactions. By combining GUV-based experiments and molecular dynamics simulations, we show that charge-reversed mutants of ALG-2 at these locations disrupt membrane recruitment. ALG-2 membrane binding mutants have reduced or abrogated ERES localization in response to Thapsigargin-induced Ca2+ release but still localize to lysosomes following lysosomal Ca2+ release. In vitro reconstitution shows that the ALG-2 membrane-binding defect can be rescued by binding to ESCRT-I. These data thus reveal the nature of direct Ca2+-dependent membrane binding and its interplay with Ca2+-dependent protein binding in the cellular functions of ALG-2.

biophysics↗

bioRxiv2023

Structural pathway for class III PI 3-kinase activation by the myristoylated GTP-binding pseudokinase VPS15

The class III phosphatidylinositol (PI) 3-kinase complexes I and II (PI3KC3-C1 and -C2) are central to the initiation of macroautophagy and endosomal maturation, respectively. Through three-dimensional classification of a large cryo-EM dataset of human PI3KC3-C1 bound to the small GTPase RAB1A, we were able to map the structural pathway of enzyme activation. The inactive conformation is stabilized by an N-myristoyl modification of the pseudokinase (PK) subunit VPS15. The N-myristate is sequestered in the N-lobe of the VPS15 PK domain, which stabilizes a series of interactions whereby VPS15 sequesters and blocks the catalytic and membrane binding units of the VPS34 lipid kinase. In the activated conformation, the N-myristate and the VPS34 lipid kinase domain are liberated to interact with membranes and catalyze PI3P formation. The VPS15 PK domain contains a unique Arg at the gatekeeper position and binds tightly to GTP. GTP binding structurally stabilizes the N-myristate "in" conformation, which promotes the inactive conformation. This pathway provides a general mechanism for PI3KC3 activation in autophagy and endosome biogenesis and a roadmap for their pharmacological upregulation.

biochemistry↗

bioRxiv2023

Structural and mechanistic insights into human choline transport

Human feline leukemia virus subgroup C receptor-related proteins 1 and 2 (FLVCR1 and 2) are members of the major facilitator superfamily1. Their dysfunction is linked to several clinical disorders, including PCARP, HSAN, and Fowler syndrome2-7. Earlier studies concluded that FLVCR1 may function as a putative heme exporter8-12, while FLVCR2 was suggested to act as a heme importer13, yet conclusive biochemical and detailed molecular evidence remained elusive for the function of both transporters14-17. Here, we show that FLVCR1 and FLVCR2 facilitate the transport of choline and ethanolamine across human plasma membranes, utilizing a concentration-driven substrate translocation process. Through structural and computational analyses, we have identified distinct conformational states of FLVCRs and unraveled the coordination chemistry underlying their substrate interactions. Within the binding pocket of both transporters, we identify fully conserved tryptophan and tyrosine residues holding a central role in the formation of cation-{pi} interactions, essential for choline and ethanolamine selectivity. Our findings not only clarify the mechanisms of choline and ethanolamine transport by FLVCR1 and FLVCR2, enhancing our comprehension of disease-associated mutations that interfere with these vital processes, but also shed light on the conformational dynamics of these MFS-type proteins during the transport cycle.

biophysics↗

bioRxiv2023

High-confidence 3D template matching for cryo-electron tomography

Cryo-electron tomography (CryoET) resolves individual macromolecules inside living cells. However, the complex composition and high density of cells challenge the faithful identification of features in tomograms. Here, we capitalize on recent advances in electron tomography and demonstrate that 3D template matching (TM) localizes a wide range of structures inside crowded eukaryotic cells with confidence 10 to 100-fold above the noise level. We establish a TM pipeline with systematically tuned parameters for automated, objective and comprehensive feature identification. High-fidelity and high-confidence localizations of nuclear pore complexes, vaults, ribosomes, proteasomes, lipid membranes and microtubules, and individual subunits, demonstrate that TM is generic. We resolve [~]100-kDa proteins, connect the functional states of complexes to their cellular localization, and capture vaults carrying ribosomal cargo in situ. By capturing individual molecular events inside living cells with defined statistical confidence, high-confidence TM greatly speeds up the CryoET workflow and sets the stage for visual proteomics.

molecular biology↗