MPI für Biophysik
Refine
Year of publication
Document Type
- Article (129)
- Preprint (21)
- Doctoral Thesis (4)
- Bachelor Thesis (1)
- Conference Proceeding (1)
- Review (1)
Has Fulltext
- yes (157)
Is part of the Bibliography
- no (157)
Keywords
Institute
- MPI für Biophysik (157)
- Physik (61)
- Biochemie und Chemie (46)
- Biowissenschaften (22)
- Biochemie, Chemie und Pharmazie (14)
- Buchmann Institut für Molekulare Lebenswissenschaften (BMLS) (12)
- Medizin (12)
- Exzellenzcluster Makromolekulare Komplexe (4)
- MPI für Hirnforschung (4)
- Frankfurt Institute for Advanced Studies (FIAS) (3)
Nuclear pore complexes (NPCs) constitute giant channels within the nuclear envelope that mediate nucleocytoplasmic exchange. NPC diameter is thought to be regulated by nuclear envelope tension, but how such diameter changes are physiologically linked to cell differentiation, where mechanical properties of nuclei are remodeled and nuclear mechanosensing occurs, remains unstudied. Here we used cryo-electron tomography to show that NPCs dilate during differentiation of mouse embryonic stem cells into neural progenitors. In Nup133-deficient cells, which are known to display impaired neural differentiation, NPCs however fail to dilate. By analyzing the architectures of individual NPCs with template matching, we revealed that the Nup133-deficient NPCs are structurally heterogeneous and frequently disintegrate, resulting in the formation of large nuclear envelope openings. We propose that the elasticity of the NPC scaffold mechanically safeguards the nuclear envelope. Our studies provide a molecular explanation for how genetic perturbation of scaffolding components of macromolecular complexes causes tissue-specific phenotypes.
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.
The majority of bacterial membrane-bound NiFe-hydrogenases and formate dehydrogenases have homologous membrane-integral cytochrome b subunits. The prototypic NiFe-hydrogenase of Wolinella succinogenes (HydABC complex) catalyzes H2 oxidation by menaquinone during anaerobic respiration and contains a membrane-integral cytochrome b subunit (HydC) that carries the menaquinone reduction site. Using the crystal structure of the homologous FdnI subunit of Escherichia coli formate dehydrogenase-N as a model, the HydC protein was modified to examine residues thought to be involved in menaquinone binding. Variant HydABC complexes were produced in W. succinogenes, and several conserved HydC residues were identified that are essential for growth with H2 as electron donor and for quinone reduction by H2. Modification of HydC with a C-terminal Strep-tag II enabled one-step purification of the HydABC complex by Strep-Tactin affinity chromatography. The tagged HydC, separated from HydAB by isoelectric focusing, was shown to contain 1.9 mol of heme b/mol of HydC demonstrating that HydC ligates both heme b groups. The four histidine residues predicted as axial heme b ligands were individually replaced by alanine in Strep-tagged HydC. Replacement of either histidine ligand of the heme b group proximal to HydAB led to HydABC preparations that contained only one heme b group. This remaining heme b could be completely reduced by quinone supporting the view that the menaquinone reduction site is located near the distal heme b group. The results indicate that both heme b groups are involved in electron transport and that the architecture of the menaquinone reduction site near the cytoplasmic side of the membrane is similar to that proposed for E. coli FdnI.
The cell division cycle protein 37 (Cdc37) and the 90-kDa heat shock protein (Hsp90) are molecular chaperones, which are crucial elements in the protein signaling pathway. The largest class of client proteins for Cdc37 and Hsp90 are protein kinases. The catalytic domains of these kinases are stabilized by Cdc37, and their proper folding and functioning is dependent on Hsp90. Here, we present the x-ray crystal structure of the 16-kDa middle domain of human Cdc37 at 1.88 angstroms resolution and the structure of this domain in complex with the 23-kDa N-terminal domain of human Hsp90 based on heteronuclear solution state NMR data and docking. Our results demonstrate that the middle domain of Cdc37 exists as a monomer. NMR and mutagenesis experiments reveal Leu-205 in Cdc37 as a key residue enabling complex formation. These findings can be very useful in the development of small molecule inhibitors against cancer.
Membrane-bound complex I (NADH:ubiquinone oxidoreductase) of the respiratory chain is considered the main site of mitochondrial radical formation and plays a major role in many mitochondrial pathologies. Structural information is scarce for complex I, and its molecular mechanism is not known. Recently, the 49-kDa subunit has been identified as part of the "catalytic core" conferring ubiquinone reduction by complex I. We found that the position of the 49-kDa subunit is clearly separated from the membrane part of complex I, suggesting an indirect mechanism of proton translocation. This contradicts all hypothetical mechanisms discussed in the field that link proton translocation directly to redox events and suggests an indirect mechanism of proton pumping by redox-driven conformational energy transfer.
The carnitine transporter CaiT from Escherichia coli belongs to the betaine, choline, and carnitine transporter family of secondary transporters. It acts as an L-carnitine/gamma-butyrobetaine exchanger and is predicted to span the membrane 12 times. Unlike the other members of this transporter family, it does not require an ion gradient and does not respond to osmotic stress (Jung, H., Buchholz, M., Clausen, J., Nietschke, M., Revermann, A., Schmid, R., and Jung, K. (2002) J. Biol. Chem. 277, 39251-39258). The structure and oligomeric state of the protein was examined in detergent and in lipid bilayers. Blue native gel electrophoresis indicated that CaiT was a trimer in detergent solution. This result was further supported by gel filtration and cross-linking studies. Electron microscopy and single particle analysis of the protein showed a triangular structure of three masses or two parallel elongated densities. Reconstitution of CaiT into lipid bilayers yielded two-dimensional crystals that indicated that CaiT was a trimer in the membrane, similar to its homologue BetP. The implications of the trimeric structure on the function of CaiT are discussed.
IHMCIF: an extension of the PDBx/mmCIF data standard for integrative structure determination methods
(2024)
IHMCIF (github.com/ihmwg/IHMCIF) is a data information framework that supports archiving and disseminating macromolecular structures determined by integrative or hybrid modeling (IHM), and making them Findable, Accessible, Interoperable, and Reusable (FAIR). IHMCIF is an extension of the Protein Data Bank Exchange/macromolecular Crystallographic Information Framework (PDBx/mmCIF) that serves as the framework for the Protein Data Bank (PDB) to archive experimentally determined atomic structures of biological macromolecules and their complexes with one another and small molecule ligands (e.g., enzyme cofactors and drugs). IHMCIF serves as the foundational data standard for the PDB-Dev prototype system, developed for archiving and disseminating integrative structures. It utilizes a flexible data representation to describe integrative structures that span multiple spatiotemporal scales and structural states with definitions for restraints from a variety of experimental methods contributing to integrative structural biology. The IHMCIF extension was created with the benefit of considerable community input and recommendations gathered by the Worldwide Protein Data Bank (wwPDB) Task Force for Integrative or Hybrid Methods (wwpdb.org/task/hybrid). Herein, we describe the development of IHMCIF to support evolving methodologies and ongoing advancements in integrative structural biology. Ultimately, IHMCIF will facilitate the unification of PDB-Dev data and tools with the PDB archive so that integrative structures can be archived and disseminated through PDB.
The 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 field’s methods to determine a robotic arm’s 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.
Highlights
• Sampling the large conformational space of disordered proteins requires extensive molecular dynamics (MD) simulations.
• Fragment assembly complements MD simulations to produce extensive ensembles of disordered proteins with atomic detail.
• Hierarchical chain growth (HCG) ensembles capture key experimental descriptors “out of the box”.
• HCG has revealed local structural characteristics associated with protein dysfunction in neurodegeneration.
Abstract
Disordered proteins and nucleic acids play key roles in cellular function and disease. Here, we review recent advances in the computational exploration of the conformational dynamics of flexible biomolecules. While atomistic molecular dynamics (MD) simulation has seen a lot of improvement in recent years, large-scale computing resources and careful validation are required to simulate full-length disordered biopolymers in solution. As a computationally efficient alternative, hierarchical chain growth (HCG) combines pre-sampled chain fragments in a statistically reproducible manner into ensembles of full-length atomically detailed biomolecular structures. Experimental data can be integrated during and after chain assembly. Applications to the neurodegeneration-linked proteins α-synuclein, tau, and TDP-43, including as condensate, illustrate the use of HCG. We conclude by highlighting the emerging connections to AI-based structural modeling including AlphaFold2.
Cryo-electron tomography combined with subtomogram averaging (StA) has yielded high-resolution structures of macromolecules in their native context. However, high-resolution StA is not commonplace due to beam-induced sample drift, images with poor signal-to-noise ratios (SNR), challenges in CTF correction, and limited particle number. Here we address these issues by collecting tilt series with a higher electron dose at the zero-degree tilt. Particles of interest are then located within reconstructed tomograms, processed by conventional StA, and then re-extracted from the high-dose images in 2D. Single particle analysis tools are then applied to refine the 2D particle alignment and generate a reconstruction. Use of our hybrid StA (hStA) workflow improved the resolution for tobacco mosaic virus from 7.2 to 4.4 Å and for the ion channel RyR1 in crowded native membranes from 12.9 to 9.1 Å. These resolution gains make hStA a promising approach for other StA projects aimed at achieving subnanometer resolution.