Refine
Document Type
- Preprint (26) (remove)
Language
- English (26)
Has Fulltext
- yes (26)
Is part of the Bibliography
- no (26)
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.
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.
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.
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.
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 structures at atomic resolution are nearly impossible, due to their size, complexity, and the large times-scales required. Instead, elastic membrane models are used to simulate membrane topologies and transitions between them, and to infer their properties and functions. Unfortunately, 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 also large and highly curved sub-cellular structures. For validation, we reproduce phase diagrams of vesicles with varying surface-to-volume ratios and area difference. The software can tackle a range of membrane remodelling processes on sub-cellular and cellular scales. 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 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. 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.
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-π 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.
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.
Nuclear pore complexes (NPCs) mediate nucleocytoplasmic transport. Their intricate 120 MDa architecture remains incompletely understood. Here, we report a near-complete structural model of the human NPC scaffold with explicit membrane and in multiple conformational states. We combined AI-based structure prediction with in situ and in cellulo cryo-electron tomography and integrative modeling. We show that linker Nups spatially organize the scaffold within and across subcomplexes to establish the higher-order structure. Microsecond-long molecular dynamics simulations suggest that the scaffold is not required to stabilize the inner and outer nuclear membrane fusion, but rather widens the central pore. Our work exemplifies how AI-based modeling can be integrated with in situ structural biology to understand subcellular architecture across spatial organization levels.
Binding of the spike protein of SARS-CoV-2 to the human angiotensin converting enzyme 2 (ACE2) receptor triggers translocation of the virus into cells. Both the ACE2 receptor and the spike protein are heavily glycosylated, including at sites near their binding interface. We built fully glycosylated models of the ACE2 receptor bound to the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Using atomistic molecular dynamics (MD) simulations, we found that the glycosylation of the human ACE2 receptor contributes substantially to the binding of the virus. Interestingly, the glycans at two glycosylation sites, N90 and N322, have opposite effects on spike protein binding. The glycan at the N90 site partly covers the binding interface of the spike RBD. Therefore, this glycan can interfere with the binding of the spike protein and protect against docking of the virus to the cell. By contrast, the glycan at the N322 site interacts tightly with the RBD of the ACE2-bound spike protein and strengthens the complex. Remarkably, the N322 glycan binds into a conserved region of the spike protein identified previously as a cryptic epitope for a neutralizing antibody. By mapping the glycan binding sites, our MD simulations aid in the targeted development of neutralizing antibodies and SARS-CoV-2 fusion inhibitors.
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.
The interaction between the Heat Shock Proteins 70 and 40 is at the core of the ATPase regulation of the chaperone machinery that maintains protein homeostasis. However, the structural details of this fundamental interaction are still elusive and contrasting models have been proposed for the transient Hsp70/Hsp40 complexes. Here we combine molecular simulations based on both coarsegrained and atomistic models with co-evolutionary sequence analysis to shed light on this problem by focusing on the bacterial DnaK/DnaJ system. The integration of these complementary approaches resulted into a novel structural model that rationalizes previous experimental observations. We identify an evolutionary-conserved interaction surface formed by helix II of the DnaJ J-domain and a groove on lobe IIA of the DnaK nucleotide binding domain, involving the inter-domain linker.
Transport of lipids across membranes is fundamental for diverse biological pathways in cells. Multiple ion-coupled transporters participate in lipid translocation, but their mechanisms remain largely unknown. Major facilitator superfamily (MFS) lipid transporters play central roles in cell wall synthesis, brain development and function, lipids recycling, and cell signaling. Recent structures of MFS lipid transporters revealed overlapping architectural features pointing towards a common mechanism. Here we used cysteine disulfide trapping, molecular dynamics simulations, mutagenesis analysis, and transport assays in vitro and in vivo, to investigate the mechanism of LtaA, a proton-dependent MFS lipid transporter essential for lipoteichoic acids synthesis in the pathogen Staphylococcus aureus. We reveal that LtaA displays asymmetric lateral openings with distinct functional relevance and that cycling through outward- and inward-facing conformations is essential for transport activity. We demonstrate that while the entire amphipathic central cavity of LtaA contributes to lipid binding, its hydrophilic pocket dictates substrate specificity. We propose that LtaA catalyzes lipid translocation by a ‘trap-and-flip’ mechanism that might be shared among MFS lipid transporters.
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.
A key event in cellular physiology is the decision between membrane biogenesis and fat storage. Phosphatidic acid (PA) is an important lipid intermediate and signaling lipid at the branch point of these pathways and constantly monitored by the transcriptional repressor Opi1 to orchestrate lipid metabolism. Here, we report on the mechanism of membrane recognition by Opi1 and identify an amphipathic helix (AH) for the selective binding to membranes containing PA over phosphatidylserine (PS). The insertion of the AH into the hydrophobic core of the membrane renders Opi1 sensitive to the lipid acyl chain composition as an important factor contributing to the regulation of membrane biogenesis. Based on these findings, we rationally designed the membrane binding properties of Opi1 to control its responsiveness in the physiological context. Using extensive molecular dynamics (MD) simulations, we identified two PA-selective three-finger grips that tightly bind the phosphate headgroup, while interacting less intimately and more transiently with PS. This work establishes lipid headgroup selectivity as a new feature in the family of AH-containing membrane property sensors.
Autophagy is a highly conserved catabolic process through which defective or otherwise harmful cellular components are targeted for degradation via the lysosomal route. Regulatory pathways, involving post-translational modifications such as phosphorylation, play a critical role in controlling this tightly orchestrated process. Here, we demonstrate that TBK1 regulates autophagy by phosphorylating autophagy modifiers LC3C and GABARAP-L2 on surface-exposed serine residues (LC3C S93 and S96; GABARAP-L2 S87 and S88). This phosphorylation event impedes their binding to the processing enzyme ATG4 by destabilizing the complex. Phosphorylated LC3C/GABARAP-L2 cannot be removed from liposomes by ATG4 and are thus protected from ATG4-mediated premature removal from nascent autoph-agosomes. This ensures a steady coat of lipidated LC3C/GABARAP-L2 throughout the early steps in autophagosome formation and aids in maintaining a unidirectional flow of the autophagosome to the lysosome. Taken together, we present a new regulatory mechanism of autophagy, which influences the conjugation and de-conjugation of LC3C and GABARAP-L2 to autophagosomes by TBK1-mediated phosphorylation.
Intrinsically disordered regions (IDRs) are essential for membrane receptor regulation but often remain unresolved in structural studies. TRPV4, a member of the TRP vanilloid channel family involved in thermo- and osmosensation, has a large N-terminal IDR of approximately 150 amino acids. With an integrated structural biology approach, we analyze the structural ensemble of the TRPV4 IDR and identify a network of regulatory elements that modulate channel activity in a hierarchical lipid-dependent manner through transient long-range interactions. A highly conserved autoinhibitory patch acts as a master regulator by competing with PIP2 binding to attenuate channel activity. Molecular dynamics simulations show that loss of the interaction between the PIP2-binding site and the membrane reduces the force exerted by the IDR on the structured core of TRPV4. This work demonstrates that IDR structural dynamics are coupled to TRPV4 activity and highlights the importance of IDRs for TRP channel function and regulation.