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Snapshots of acetyl-CoA synthesis, the final step of CO₂ fixation in the Wood-Ljungdahl pathway
(2024)
In the ancient microbial Wood-Ljungdahl pathway, CO2 is fixed in a multi-step process 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.
Human feline leukaemia virus subgroup C receptor-related proteins 1 and 2 (FLVCR1 and 2) are major facilitator superfamily transporters from the solute carrier family 49. Dysregulation of these ubiquitous transporters has been linked to various haematological and neurological disorders. While both FLVCRs were initially proposed to hold a physiological function in heme transport, subsequent studies questioned this notion. Here, we used structural, computational and biochemical methods and conclude that these two FLVCRs function as human choline transporters. We present cryo-electron microscopy structures of FLVCRs in different inward- and outward-facing conformations, captured in the apo state or in complex with choline in their translocation pathways. Our findings provide insights into the molecular framework of choline coordination and transport, largely mediated by conserved cation-π interactions, and further illuminate the conformational dynamics of the transport cycle. Moreover, we identified a heme binding site on the protein surface of the FLVCR2 N-domain, and observed that heme actively drives the conformational transitions of the protein. This auxiliary binding site might indicate a potential regulatory role of heme in the FLVCR2 transport mechanisms. Our work resolves the contested substrate specificity of the FLVCRs, and sheds light on the process of maintaining cellular choline homeostasis at the molecular level.
Complex I couples the free energy released from quinone (Q) reduction to pump protons across the biological membrane in the respiratory chains of mitochondria and many bacteria. The Q reduction site is separated by a large distance from the proton-pumping membrane domain. To address the molecular mechanism of this long-range proton-electron coupling, we perform here full atomistic molecular dynamics simulations, free energy calculations, and continuum electrostatics calculations on complex I from Thermus thermophilus. We show that the dynamics of Q is redox-state-dependent, and that quinol, QH2, moves out of its reduction site and into a site in the Q tunnel that is occupied by a Q analog in a crystal structure of Yarrowia lipolytica. We also identify a second Q-binding site near the opening of the Q tunnel in the membrane domain, where the Q headgroup forms strong interactions with a cluster of aromatic and charged residues, while the Q tail resides in the lipid membrane. We estimate the effective diffusion coefficient of Q in the tunnel, and in turn the characteristic time for Q to reach the active site and for QH2 to escape to the membrane. Our simulations show that Q moves along the Q tunnel in a redox-state-dependent manner, with distinct binding sites formed by conserved residue clusters. The motion of Q to these binding sites is proposed to be coupled to the proton-pumping machinery in complex I.
We investigate system-size effects on the rotational diffusion of membrane proteins and other membrane-embedded molecules in molecular dynamics simulations. We find that the rotational diffusion coefficient slows down relative to the infinite-system value by a factor of one minus the ratio of protein and box areas. This correction factor follows from the hydrodynamics of rotational flows under periodic boundary conditions and is rationalized in terms of Taylor-Couette flow. For membrane proteins like transporters, channels, or receptors in typical simulation setups, the protein-covered area tends to be relatively large, requiring a significant finite-size correction. Molecular dynamics simulations of the protein adenine nucleotide translocase (ANT1) and of a carbon nanotube porin in lipid membranes show that the hydrodynamic finite-size correction for rotational diffusion is accurate in standard-use cases. The dependence of the rotational diffusion on box size can be used to determine the membrane viscosity.
Cysteine cross-linking in native membranes establishes the transmembrane architecture of Ire1
(2021)
The ER is a key organelle of membrane biogenesis and crucial for the folding of both membrane and secretory proteins. Sensors of the unfolded protein response (UPR) monitor the unfolded protein load in the ER and convey effector functions for maintaining ER homeostasis. Aberrant compositions of the ER membrane, referred to as lipid bilayer stress, are equally potent activators of the UPR. How the distinct signals from lipid bilayer stress and unfolded proteins are processed by the conserved UPR transducer Ire1 remains unknown. Here, we have generated a functional, cysteine-less variant of Ire1 and performed systematic cysteine cross-linking experiments in native membranes to establish its transmembrane architecture in signaling-active clusters. We show that the transmembrane helices of two neighboring Ire1 molecules adopt an X-shaped configuration independent of the primary cause for ER stress. This suggests that different forms of stress converge in a common, signaling-active transmembrane architecture of Ire1.
Abstract
The endoplasmic reticulum (ER) is a key organelle of membrane biogenesis and crucial for the folding of both membrane and secretory proteins. Sensors of the unfolded protein response (UPR) monitor the unfolded protein load in the ER and convey effector functions for maintaining ER homeostasis. Aberrant compositions of the ER membrane, referred to as lipid bilayer stress, are equally potent activators of the UPR. How the distinct signals from lipid bilayer stress and unfolded proteins are processed by the conserved UPR transducer Ire1 remains unknown. Here, we have generated a functional, cysteine-less variant of Ire1 and performed systematic cysteine crosslinking experiments in native membranes to establish its transmembrane architecture in signaling-active clusters. We show that the transmembrane helices of two neighboring Ire1 molecules adopt an X-shaped configuration independent of the primary cause for ER stress. This suggests that different forms of stress converge in a common, signaling-active transmembrane architecture of Ire1.
Summary
The endoplasmic reticulum (ER) is a hotspot of lipid biosynthesis and crucial for the folding of membrane and secretory proteins. The unfolded protein response (UPR) controls the size and folding capacity of the ER. The conserved UPR transducer Ire1 senses both unfolded proteins and aberrant lipid compositions to mount adaptive responses. Using a biochemical assay to study Ire1 in signaling-active clusters, Väth et al. provide evidence that the neighboring transmembrane helices of clustered Ire1 form an ‘X’ irrespectively of the primary cause of ER stress. Hence, different forms of ER stress converge in a common, signaling-active transmembrane architecture of Ire1.
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 (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is required for cell entry and is the major focus for vaccine development. We combine cryo electron tomography, subtomogram averaging and molecular dynamics simulations to structurally analyze S in situ. Compared to recombinant S, the viral S is more heavily glycosylated and occurs predominantly in a closed pre-fusion conformation. We show that the stalk domain of S contains three hinges that give the globular domain unexpected orientational freedom. We propose that the hinges allow S to scan the host cell surface, shielded from antibodies by an extensive glycan coat. The structure of native S contributes to our understanding of SARS-CoV-2 infection and the development of safe vaccines. The large scale tomography data set of SARS-CoV-2 used for this study is therefore sufficient to resolve structural features to below 5 Ångstrom, and is publicly available at EMPIAR-10453.
The spike protein (S) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is required for cell entry and is the primary focus for vaccine development. In this study, we combined cryo–electron tomography, subtomogram averaging, and molecular dynamics simulations to structurally analyze S in situ. Compared with the recombinant S, the viral S was more heavily glycosylated and occurred mostly in the closed prefusion conformation. We show that the stalk domain of S contains three hinges, giving the head unexpected orientational freedom. We propose that the hinges allow S to scan the host cell surface, shielded from antibodies by an extensive glycan coat. The structure of native S contributes to our understanding of SARS-CoV-2 infection and potentially to the development of safe vaccines.
Cryo-electron tomography (cryo-ET) is a powerful method to elucidate subcellular architecture and to structurally analyse biomolecules in situ by subtomogram averaging (STA). Specimen thickness is a key factor affecting cryo-ET data quality. Cells that are too thick for transmission imaging can be thinned by cryo-focused-ion-beam (cryo-FIB) milling. However, optimal specimen thickness for cryo-ET on lamellae has not been systematically investigated. Furthermore, the ions used to ablate material can cause damage in the lamellae, thereby reducing STA resolution. Here, we systematically benchmark the resolution depending on lamella thickness and the depth of the particles within the sample. Up to ca. 180 nm, lamella thickness does not negatively impact resolution. This shows that there is no need to generate very thin lamellae and thickness can be chosen such that it captures major cellular features. Furthermore, we show that gallium-ion-induced damage extends to depths of up to 30 nm from either lamella surface.