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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.
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.
Abstract
The primary immunological target of COVID-19 vaccines is the SARS-CoV-2 spike (S) protein. S is exposed on the viral surface and mediates viral entry into the host cell. To identify possible antibody binding sites, we performed multi-microsecond molecular dynamics simulations of a 4.1 million atom system containing a patch of viral membrane with four full-length, fully glycosylated and palmitoylated S proteins. By mapping steric accessibility, structural rigidity, sequence conservation, and generic antibody binding signatures, we recover known epitopes on S and reveal promising epitope candidates for structure-based vaccine design. We find that the extensive and inherently flexible glycan coat shields a surface area larger than expected from static structures, highlighting the importance of structural dynamics. The protective glycan shield and the high flexibility of its hinges give the stalk overall low epitope scores. Our computational epitope-mapping procedure is general and should thus prove useful for other viral envelope proteins whose structures have been characterized.
Author summary
The SARS-CoV-2 virus has caused a global health crisis. The spike protein exposed at its surface is key for infection and the primary antibody target. However, spike is covered by highly mobile glycan molecules that could impair antibody binding. To identify accessible epitopes, we performed molecular dynamics simulations of an atomistic model of glycosylated spike embedded in a membrane. By combining extensive simulations with bioinformatics analyses, we recovered known antibody binding sites and identified several epitope candidates as targets for further vaccine development.
The severity of the COVID-19 pandemic, caused by the SARS-CoV-2 coronavirus, calls for the urgent development of a vaccine. The primary immunological target is the SARS-CoV-2 spike (S) protein. S is exposed on the viral surface to mediate viral entry into the host cell. To identify possible antibody binding sites not shielded by glycans, we performed multi-microsecond molecular dynamics simulations of a 4.1 million atom system containing a patch of viral membrane with four full-length, fully glycosylated and palmitoylated S proteins. By mapping steric accessibility, structural rigidity, sequence conservation and generic antibody binding signatures, we recover known epitopes on S and reveal promising epitope candidates for vaccine development. We find that the extensive and inherently flexible glycan coat shields a surface area larger than expected from static structures, highlighting the importance of structural dynamics in epitope mapping.
TriMem: A parallelized hybrid Monte Carlo software for efficient simulations of lipid membranes
(2022)
Lipid membranes are integral building blocks of living cells and perform a multitude of biological functions. Currently, molecular simulations of cellular-scale membrane remodeling processes at atomic resolution are extremely difficult, due to their size, complexity, and the large times-scales on which these processes occur. Instead, elastic membrane models are used to simulate membrane shapes and transitions between them and to infer their properties and functions. Unfortunately, an efficiently parallelized open-source simulation code to do so has been lacking. Here, we present TriMem, a parallel hybrid Monte Carlo simulation engine for triangulated lipid membranes. The kernels are efficiently coded in C++ and wrapped with Python for ease-of-use. The parallel implementation of the energy and gradient calculations and of Monte Carlo flip moves of edges in the triangulated membrane enable us to simulate large and highly curved membrane structures. For validation, we reproduce phase diagrams of vesicles with varying surface-to-volume ratios and area difference. We also compute the density of states to verify correct Boltzmann sampling. The software can be used to tackle a range of large-scale membrane remodeling processes as a step toward cell-scale simulations. Additionally, extensive documentation make the software accessible to the broad biophysics and computational cell biology communities.
Living cells constantly remodel the shape of their lipid membranes. In the endoplasmic reticulum (ER), the reticulon homology domain (RHD) of the reticulophagy regulator 1 (RETR1/FAM134B) forms dense autophagic puncta that are associated with membrane removal by ER-phagy. In molecular dynamics (MD) simulations, we find that FAM134B-RHD spontaneously forms clusters, driven in part by curvature-mediated attractions. At a critical size, as in a nucleation process, the FAM134B-RHD clusters induce the formation of membrane buds. The kinetics of budding depends sensitively on protein concentration and bilayer asymmetry. Our MD simulations shed light on the role of FAM134B-RHD in ER-phagy and show that membrane asymmetry can be used to modulate the kinetic barrier for membrane remodeling.
Living cells constantly remodel the shape of their lipid membranes. In the endo-plasmic reticulum (ER), the reticulon homology domain (RHD) of the reticulophagy regulator 1 (RETR1/FAM134B) forms dense autophagic puncta that are associated with membrane removal by ER-phagy. In molecular dynamics (MD) simulations, we find that FAM134B-RHD spontaneously forms clusters, driven in part by curvature-mediated attraction. At a critical size, the FAM134B-RHD clusters induce the formation of membrane buds. The kinetics of budding depends sensitively on protein concentration and bilayer asymmetry. Our MD simulations shed light on the role of FAM134B-RHD in ER-phagy and show that membrane asymmetry can be used to modulate the kinetics barrier for membrane remodeling.
During infection the SARS-CoV-2 virus fuses its viral envelope with cellular membranes of its human host. The viral spike (S) protein mediates both the initial contact with the host cell and the subsequent membrane fusion. Proteolytic cleavage of S at the S2′ site exposes its fusion peptide (FP) as the new N-terminus. By binding to the host membrane, the FP anchors the virus to the host cell. The reorganization of S2 between virus and host then pulls the two membranes together. Here we use molecular dynamics (MD) simulations to study the two core functions of the SARS-CoV-2 FP: to attach quickly to cellular membranes and to form an anchor strong enough to withstand the mechanical force during membrane fusion. In eight 10 μs long MD simulations of FP in proximity to endosomal and plasma membranes, we find that FP binds spontaneously to the membranes and that binding proceeds predominantly by insertion of two short amphipathic helices into the membrane interface. Connected via a flexible linker, the two helices can bind the membrane independently, yet binding of one promotes the binding of the other by tethering it close to the target membrane. By simulating mechanical pulling forces acting on the C-terminus of the FP, we then show that the bound FP can bear forces up to 250 pN before detaching from the membrane. This detachment force is more than 10-fold higher than an estimate of the force required to pull host and viral membranes together for fusion. We identify a fully conserved disulfide bridge in the FP as a major factor for the high mechanical stability of the FP membrane anchor. We conclude, first, that the sequential binding of two short amphipathic helices allows the SARS-CoV-2 FP to insert quickly into the target membrane, before the virion is swept away after shedding the S1 domain connecting it to the host cell receptor. Second, we conclude that the double attachment and the conserved disulfide bridge establish the strong anchoring required for subsequent membrane fusion. Multiple distinct membrane-anchoring elements ensure high avidity and high mechanical strength of FP–membrane binding.
During infection the SARS-CoV-2 virus fuses its viral envelope with cellular membranes of its human host. Initial contact with the host cell and membrane fusion are both mediated by the viral spike (S) protein. Proteolytic cleavage of S at the S2′ site exposes its 40 amino acid long fusion peptide (FP). Binding of the FP to the host membrane anchors the S2 domain of S in both the viral and the host membrane. The reorganization of S2 then pulls the two membranes together. Here we use molecular dynamics (MD) simulations to study the two core functions of the SARS-CoV-2 FP: to attach quickly to cellular membranes and to form an anchor strong enough to withstand the mechanical force during membrane fusion. In eight 10 μs-long MD simulations of FP in proximity to endosomal and plasma membranes, we find that FP binds spontaneously to the membranes and that binding proceeds predominantly by insertion of two short amphipathic helices into the membrane interface. Connected via a flexible linker, the two helices can bind the membrane independently, yet binding of one promotes the binding of the other by tethering it close to the target membrane. By simulating mechanical pulling forces acting on the C-terminus of the FP we then show that the bound FP can bear forces up to 250 pN before detaching from the membrane. This detachment force is more than ten-fold higher than an estimate of the force required to pull host and viral membranes together for fusion. We identify a fully conserved disulfide bridge in the FP as a major factor for the high mechanical stability of the FP membrane anchor. We conclude, first, that the sequential binding of two short amphipathic helices allows the SARS-CoV-2 FP to insert quickly into the target membrane, before the virion is swept away after shedding the S1 domain connecting it to the host cell receptor. Second, we conclude that the double attachment and the conserved disulfide bridge establish the strong anchoring required for subsequent membrane fusion. Multiple distinct membrane-anchoring elements ensure high avidity and high mechanical strength of FP-membrane binding.
Gasdermin-D (GSDMD) is the ultimate effector of pyroptosis, a form of programmed cell death associated with pathogen invasion and inflammation. After proteolytic cleavage by caspases activated by the inflammasome, the GSDMD N-terminal domain (GSDMDNT) assembles on the inner leaflet of the plasma membrane and induces the formation of large membrane pores. We use atomistic molecular dynamics simulations to study GSDMDNT monomers, oligomers, and rings in an asymmetric plasma membrane mimetic. We identify distinct interaction motifs of GSDMDNT with phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) and phosphatidylserine (PS) head-groups and describe differential lipid binding between the pore and prepore conformations. Oligomers are stabilized by shared lipid binding sites between neighboring monomers acting akin to double-sided tape. We show that already small GSDMDNT oligomers form stable, water-filled and ion-conducting membrane pores bounded by curled beta-sheets. In large-scale simulations, we resolve the process of pore formation by lipid detachment from GSDMDNT arcs and lipid efflux from partial rings. We find that that high-order GSDMDNT oligomers can crack under the line tension of 86 pN created by an open membrane edge to form the slit pores or closed GSDMDNT rings seen in experiment. Our simulations provide a detailed view of key steps in GSDMDNT-induced plasma membrane pore formation, including sublytic pores that explain nonselective ion flux during early pyroptosis.
Gasdermin-D (GSDMD) is the ultimate effector of pyroptosis, a form of programmed cell death associated with pathogen invasion and inflammation. After proteolytic cleavage by caspases, the GSDMD N-terminal domain (GSDMDNT) assembles on the inner leaflet of the plasma membrane and induces the formation of membrane pores. We use atomistic molecular dynamics simulations to study GSDMDNT monomers, oligomers, and rings in an asymmetric plasma membrane mimetic. We identify distinct interaction motifs of GSDMDNT with phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) and phosphatidylserine (PS) headgroups and describe their conformational dependence. Oligomers are stabilized by shared lipid binding sites between neighboring monomers acting akin to double-sided tape. We show that already small GSDMDNT oligomers support stable, water-filled, and ion-conducting membrane pores bounded by curled beta-sheets. In large-scale simulations, we resolve the process of pore formation from GSDMDNT arcs and lipid efflux from partial rings. We find that high-order GSDMDNT oligomers can crack under the line tension of 86 pN created by an open membrane edge to form the slit pores or closed GSDMDNT rings seen in atomic force microscopy experiments. Our simulations provide a detailed view of key steps in GSDMDNT-induced plasma membrane pore formation, including sublytic pores that explain nonselective ion flux during early pyroptosis.
New drugs are urgently needed to combat the global TB epidemic. Targeting simultaneously multiple respiratory enzyme complexes of Mycobacterium tuberculosis is regarded as one of the most effective treatment options to shorten drug administration regimes, and reduce the opportunity for the emergence of drug resistance. During infection and proliferation, the cytochrome bd oxidase plays a crucial role for mycobacterial pathophysiology by maintaining aerobic respiration at limited oxygen concentrations. Here, we present the cryo-EM structure of the cytochrome bd oxidase from M. tuberculosis at 2.5 Å. In conjunction with atomistic molecular dynamics (MD) simulation studies we discovered a previously unknown MK-9-binding site, as well as a unique disulfide bond within the Q-loop domain that defines an inactive conformation of the canonical quinol oxidation site in Actinobacteria. Our detailed insights into the long-sought atomic framework of the cytochrome bd oxidase from M. tuberculosis will form the basis for the design of highly specific drugs to act on this enzyme.
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.
Molecular mechanisms of inorganic-phosphate release from the core and barbed end of actin filaments
(2023)
The release of inorganic phosphate (Pi) from actin filaments constitutes a key step in their regulated turnover, which is fundamental to many cellular functions. However, the molecular mechanisms underlying Pi release from both the core and barbed end of actin filaments remain unclear. Here, we combine cryo-EM with molecular dynamics simulations and in vitro reconstitution to demonstrate how actin releases Pi through a ‘molecular backdoor’. While constantly open at the barbed end, the backdoor is predominantly closed in filament-core subunits and only opens transiently through concerted backbone movements and rotameric rearrangements of residues close to the nucleotide binding pocket. This mechanism explains why Pi escapes rapidly from the filament end and yet slowly from internal actin subunits. In an actin variant associated with nemaline myopathy, the backdoor is predominantly open in filament-core subunits, resulting in greatly accelerated Pi release after polymerization and filaments with drastically shortened ADP-Pi caps. This demonstrates that the Pi release rate from F-actin is controlled by steric hindrance through the backdoor rather than by the disruption of the ionic bond between Pi and Mg2+ at the nucleotide-binding site. Our results provide the molecular basis for Pi release from actin and exemplify how a single, disease-linked point mutation distorts the nucleotide state distribution and atomic structure of the actin filament.