Membrane structure and internalization dynamics of human Flower isoforms hFWE3 and hFWE4 indicate a conserved endocytic role for hFWE4.

Human Flower (hFWE) isoforms hFWE1-4 are putative transmembrane (TM) proteins that reportedly mediate fitness comparisons during cell competition through extracellular display of their C-terminal tails. Isoform topology, subcellular localization, and duration of plasma membrane presentation are essential to this function. However, disagreement persists regarding the structure of orthologous fly and mouse FWEs, and experimental evidence for hFWE isoform subcellular localization or membrane structure is lacking. Here, we used AlphaFold2 and subsequent molecular dynamics-based structural predictions to construct epitope-tagged hFWE3 and hFWE4, the most abundant human isoforms, for experimental determination of their structure and internalization dynamics. We demonstrate that hFWE3 resides in the membrane of the endoplasmic reticulum (ER), while hFWE4 partially colocalizes with Rab4-, Rab5-, and Rab11-positive vesicles as well as with the plasma membrane. An array of imaging techniques revealed that hFWE4 positions both N- and C-terminal tails and a loop between second and third TM segments within the cytosol, while small (4-12aa) loops between the first and second and the third and fourth TM segments are either exposed to the extracellular space or within the lumen of cytoplasmic vesicles. Similarly, we found hFWE3 positions both N- and C-terminal tails in the cytosol, while a short loop between TM domains extends into the ER lumen. Finally, we demonstrate that hFWE4 exists only transiently at the cell surface and is rapidly internalized in an AP-2- and dynamin-1-dependent manner. Collectively, these data are consistent with a conserved role for hFWE4 in endocytic processes.

EMC chaperone-CaV structure reveals an ion channel assembly intermediate.

Voltage-gated ion channels (VGICs) comprise multiple structural units, the assembly of which is required for function1,2. Structural understanding of how VGIC subunits assemble and whether chaperone proteins are required is lacking. High-voltage-activated calcium channels (CaVs)3,4 are paradigmatic multisubunit VGICs whose function and trafficking are powerfully shaped by interactions between pore-forming CaV1 or CaV2 CaVα1 (ref. 3), and the auxiliary CaVß5 and CaVα2δ subunits6,7. Here we present cryo-electron microscopy structures of human brain and cardiac CaV1.2 bound with CaVß3 to a chaperone-the endoplasmic reticulum membrane protein complex (EMC)8,9-and of the assembled CaV1.2-CaVß3-CaVα2δ-1 channel. These structures provide a view of an EMC-client complex and define EMC sites-the transmembrane (TM) and cytoplasmic (Cyto) docks; interaction between these sites and the client channel causes partial extraction of a pore subunit and splays open the CaVα2δ-interaction site. The structures identify the CaVα2δ-binding site for gabapentinoid anti-pain and anti-anxiety drugs6, show that EMC and CaVα2δ interactions with the channel are mutually exclusive, and indicate that EMC-to-CaVα2δ hand-off involves a divalent ion-dependent step and CaV1.2 element ordering. Disruption of the EMC-CaV complex compromises CaV function, suggesting that the EMC functions as a channel holdase that facilitates channel assembly. Together, the structures reveal a CaV assembly intermediate and EMC client-binding sites that could have wide-ranging implications for the biogenesis of VGICs and other membrane proteins.

Immunoglobulin M perception by FcµR.

Immunoglobulin M (IgM) is the first antibody to emerge during embryonic development and the humoral immune response1. IgM can exist in several distinct forms, including monomeric, membrane-bound IgM within the B cell receptor (BCR) complex, pentameric and hexameric IgM in serum and secretory IgM on the mucosal surface. FcµR, the only IgM-specific receptor in mammals, recognizes different forms of IgM to regulate diverse immune responses2-5. However, the underlying molecular mechanisms remain unknown. Here we delineate the structural basis of the FcµR-IgM interaction by crystallography and cryo-electron microscopy. We show that two FcµR molecules interact with a Fcµ-Cµ4 dimer, suggesting that FcµR can bind to membrane-bound IgM with a 2:1 stoichiometry. Further analyses reveal that FcµR-binding sites are accessible in the context of IgM BCR. By contrast, pentameric IgM can recruit four FcµR molecules to bind on the same side and thereby facilitate the formation of an FcµR oligomer. One of these FcµR molecules occupies the binding site of the secretory component. Nevertheless, four FcµR molecules bind to the other side of secretory component-containing secretory IgM, consistent with the function of FcµR in the retrotransport of secretory IgM. These results reveal intricate mechanisms of IgM perception by FcµR.

Clathrin-associated AP-1 controls termination of STING signalling.

Stimulator of interferon genes (STING) functions downstream of cyclic GMP-AMP synthase in DNA sensing or as a direct receptor for bacterial cyclic dinucleotides and small molecules to activate immunity during infection, cancer and immunotherapy1-10. Precise regulation of STING is essential to ensure balanced immune responses and prevent detrimental autoinflammation11-16. After activation, STING, a transmembrane protein, traffics from the endoplasmic reticulum to the Golgi, where its phosphorylation by the protein kinase TBK1 enables signal transduction17-20. The mechanism that ends STING signalling at the Golgi remains unknown. Here we show that adaptor protein complex 1 (AP-1) controls the termination of STING-dependent immune activation. We find that AP-1 sorts phosphorylated STING into clathrin-coated transport vesicles for delivery to the endolysosomal system, where STING is degraded21. We identify a highly conserved dileucine motif in the cytosolic C-terminal tail (CTT) of STING that, together with TBK1-dependent CTT phosphorylation, dictates the AP-1 engagement of STING. A cryo-electron microscopy structure of AP-1 in complex with phosphorylated STING explains the enhanced recognition of TBK1-activated STING. We show that suppression of AP-1 exacerbates STING-induced immune responses. Our results reveal a structural mechanism of negative regulation of STING and establish that the initiation of signalling is inextricably associated with its termination to enable transient activation of immunity.

Cryo-EM structure of an active bacterial TIR-STING filament complex.

Stimulator of interferon genes (STING) is an antiviral signalling protein that is broadly conserved in both innate immunity in animals and phage defence in prokaryotes1-4. Activation of STING requires its assembly into an oligomeric filament structure through binding of a cyclic dinucleotide4-13, but the molecular basis of STING filament assembly and extension remains unknown. Here we use cryogenic electron microscopy to determine the structure of the active Toll/interleukin-1 receptor (TIR)-STING filament complex from a Sphingobacterium faecium cyclic-oligonucleotide-based antiphage signalling system (CBASS) defence operon. Bacterial TIR-STING filament formation is driven by STING interfaces that become exposed on high-affinity recognition of the cognate cyclic dinucleotide signal c-di-GMP. Repeating dimeric STING units stack laterally head-to-head through surface interfaces, which are also essential for human STING tetramer formation and downstream immune signalling in mammals5. The active bacterial TIR-STING structure reveals further cross-filament contacts that brace the assembly and coordinate packing of the associated TIR NADase effector domains at the base of the filament to drive NAD+ hydrolysis. STING interface and cross-filament contacts are essential for cell growth arrest in vivo and reveal a stepwise mechanism of activation whereby STING filament assembly is required for subsequent effector activation. Our results define the structural basis of STING filament formation in prokaryotic antiviral signalling.

A peroxisomal ubiquitin ligase complex forms a retrotranslocation channel.

Peroxisomes are ubiquitous organelles that house various metabolic reactions and are essential for human health1-4. Luminal peroxisomal proteins are imported from the cytosol by mobile receptors, which then recycle back to the cytosol by a poorly understood process1-4. Recycling requires receptor modification by a membrane-embedded ubiquitin ligase complex comprising three RING finger domain-containing proteins (Pex2, Pex10 and Pex12)5,6. Here we report a cryo-electron microscopy structure of the ligase complex, which together with biochemical and in vivo experiments reveals its function as a retrotranslocation channel for peroxisomal import receptors. Each subunit of the complex contributes five transmembrane segments that co-assemble into an open channel. The three ring finger domains form a cytosolic tower, with ring finger 2 (RF2) positioned above the channel pore. We propose that the N terminus of a recycling receptor is inserted from the peroxisomal lumen into the pore and monoubiquitylated by RF2 to enable extraction into the cytosol. If recycling is compromised, receptors are polyubiquitylated by the concerted action of RF10 and RF12 and degraded. This polyubiquitylation pathway also maintains the homeostasis of other peroxisomal import factors. Our results clarify a crucial step during peroxisomal protein import and reveal why mutations in the ligase complex cause human disease.

Amyloid fibrils in FTLD-TDP are composed of TMEM106B and not TDP-43.

Frontotemporal lobar degeneration (FTLD) is the third most common neurodegenerative condition after Alzheimer's and Parkinson's diseases1. FTLD typically presents in 45 to 64 year olds with behavioural changes or progressive decline of language skills2. The subtype FTLD-TDP is characterized by certain clinical symptoms and pathological neuronal inclusions with TAR DNA-binding protein (TDP-43) immunoreactivity3. Here we extracted amyloid fibrils from brains of four patients representing four of the five FTLD-TDP subclasses, and determined their structures by cryo-electron microscopy. Unexpectedly, all amyloid fibrils examined were composed of a 135-residue carboxy-terminal fragment of transmembrane protein 106B (TMEM106B), a lysosomal membrane protein previously implicated as a genetic risk factor for FTLD-TDP4. In addition to TMEM106B fibrils, we detected abundant non-fibrillar aggregated TDP-43 by immunogold labelling. Our observations confirm that FTLD-TDP is associated with amyloid fibrils, and that the fibrils are formed by TMEM106B rather than TDP-43.

FliL ring enhances the function of periplasmic flagella.

SignificanceHow flagella sense complex environments and control bacterial motility remain fascinating questions. Here, we deploy cryo-electron tomography to determine in situ structures of the flagellar motor in wild-type and mutant cells of Borrelia burgdorferi, revealing that three flagellar proteins (FliL, MotA, and MotB) form a unique supramolecular complex in situ. Importantly, FliL not only enhances motor function by forming a ring around the stator complex MotA/MotB in its extended, active conformation but also facilitates assembly of the stator complex around the motor. Our in situ data provide insights into how cooperative remodeling of the FliL-stator supramolecular complex helps regulate the collective ion flux and establishes the optimal function of the flagellar motor to guide bacterial motility in various environments.

Liquid-phase scanning electron microscopy for single membrane protein imaging.

Liquid-phase electron microscopy is highly desirable for observing biological samples in their native liquid state at high resolution. We developed liquid imaging approaches for biological cells using scanning electron microscopy. Novel approaches included scanning transmission electron imaging using a liquid-cell apparatus (LC-STEM), as well as correlative cathodoluminescence and electron microscopy (CCLEM) imaging. LC-STEM enabled imaging at a ∼2 nm resolution and excellent contrast for the precise recognition of localization, distribution, and configuration of individually labeled membrane proteins on the native cells in solution. CCLEM improved the resolution of fluorescent images down to 10 nm. Liquid SEM technologies will bring unique and wide applications to the study of the structure and function of cells and membrane proteins in their near-native states at the monomolecular level.

Shedding light on membrane rafts structure and dynamics in living cells.

Cellular membranes are fundamental building blocks regulating an extensive repertoire of biological functions. These structures contain lipids and membrane proteins that are known to laterally self-aggregate in the plane of the membrane, forming defined membrane nanoscale domains essential for protein activity. Membrane rafts are described as heterogeneous, dynamic, and short-lived cholesterol- and sphingolipid-enriched membrane nanodomains (10-200 nm) induced by lipid-protein and lipid-lipid interactions. Those membrane nanodomains have been extensively characterized using model membranes and in silico methods. However, despite the development of advanced fluorescence microscopy techniques, undoubted nanoscale visualization by imaging techniques of membrane rafts in the membrane of unperturbed living cells is still uncompleted, increasing the skepticism about their existence. Here, we broadly review recent biochemical and microscopy techniques used to investigate membrane rafts in living cells and we enumerate persistent open questions to answer before unlocking the mystery of membrane rafts in living cells.

Cryo-EM structures of the endoplasmic reticulum membrane complex.

The transmembrane α-helices of membrane proteins are in general highly hydrophobic, and they enter the lipid bilayer through a lateral gate in the Sec61 translocon. However, some transmembrane α-helices are less hydrophobic and form membrane channels or substrate-binding pockets. Insertion of these amphipathic transmembrane α-helices into the membrane requires the specific membrane-embedded insertase called the endoplasmic reticulum membrane complex (EMC), which is a multi-subunit chaperone distinct from the GET insertase complex. Four recent cryo-electron microscopy studies on the eukaryotic EMC have revealed their remarkable architectural conservation from yeast to humans; a general consensus on the substrate transmembrane helix-binding pocket; and the evolutionary link to the prokaryotic insertases of the tail-anchored membrane proteins. These structures provide a solid framework for future mechanistic investigation.

Dynamics properties of membrane proteins in native cell membranes revealed by solid-state NMR spectroscopy.

Cell membranes provide an environment that is essential to the functions of membrane proteins. Cell membranes are mainly composed of proteins and highly diverse phospholipids. The influence of diverse lipid compositions of native cell membranes on the dynamics of the embedded membrane proteins has not been examined. Here we employ solid-state NMR to investigate the dynamics of E. coli Aquaporin Z (AqpZ) in its native inner cell membranes, and reveal the influence of diverse lipid compositions on the dynamics of AqpZ by comparing it in native cell membranes to that in POPC/POPG bilayers. We demonstrate that the dynamic rigidity of AqpZ generally conserves in both native cell membranes and POPC/POPG bilayers, due to its tightly packed tetrameric structure. In the gel and the liquid crystal phases of lipids, our experimental results show that AqpZ is more dynamic in native cell membranes than that in POPC/POPG bilayers. In addition, we observe that phase transitions of lipids in native membranes are less sensitive to temperature variations compared with that in POPC/POPG bilayers, which results in that the dynamics of AqpZ is less affected by the phase transitions of lipids in native cell membranes than that in POPC/POPG bilayers. This study provides new insights into the dynamics of membrane proteins in native cell membranes.

A structurally conserved site in AUP1 binds the E2 enzyme UBE2G2 and is essential for ER-associated degradation.

Endoplasmic reticulum-associated degradation (ERAD) is a protein quality control pathway of fundamental importance to cellular homeostasis. Although multiple ERAD pathways exist for targeting topologically distinct substrates, all pathways require substrate ubiquitination. Here, we characterize a key role for the UBE2G2 Binding Region (G2BR) of the ERAD accessory protein ancient ubiquitous protein 1 (AUP1) in ERAD pathways. This 27-amino acid (aa) region of AUP1 binds with high specificity and low nanomolar affinity to the backside of the ERAD ubiquitin-conjugating enzyme (E2) UBE2G2. The structure of the AUP1 G2BR (G2BRAUP1) in complex with UBE2G2 reveals an interface that includes a network of salt bridges, hydrogen bonds, and hydrophobic interactions essential for AUP1 function in cells. The G2BRAUP1 shares significant structural conservation with the G2BR found in the E3 ubiquitin ligase gp78 and in vitro can similarly allosterically activate ubiquitination in conjunction with ERAD E3s. In cells, AUP1 is uniquely required to maintain normal levels of UBE2G2; this is due to G2BRAUP1 binding to the E2 and preventing its rapid degradation. In addition, the G2BRAUP1 is required for both ER membrane recruitment of UBE2G2 and for its activation at the ER membrane. Thus, by binding to the backside of a critical ERAD E2, G2BRAUP1 plays multiple critical roles in ERAD.

Structure, mechanism, and inhibition of Hedgehog acyltransferase.

The Sonic Hedgehog (SHH) morphogen pathway is fundamental for embryonic development and stem cell maintenance and is implicated in various cancers. A key step in signaling is transfer of a palmitate group to the SHH N terminus, catalyzed by the multi-pass transmembrane enzyme Hedgehog acyltransferase (HHAT). We present the high-resolution cryo-EM structure of HHAT bound to substrate analog palmityl-coenzyme A and a SHH-mimetic megabody, revealing a heme group bound to HHAT that is essential for HHAT function. A structure of HHAT bound to potent small-molecule inhibitor IMP-1575 revealed conformational changes in the active site that occlude substrate binding. Our multidisciplinary analysis provides a detailed view of the mechanism by which HHAT adapts the membrane environment to transfer an acyl chain across the endoplasmic reticulum membrane. This structure of a membrane-bound O-acyltransferase (MBOAT) superfamily member provides a blueprint for other protein-substrate MBOATs and a template for future drug discovery.

Enzymatic Synthesis of 3'-5', 3'-5' Cyclic Dinucleotides, Their Binding Properties to the Stimulator of Interferon Genes Adaptor Protein, and Structure/Activity Correlations.

The 3'-5', 3'-5' cyclic dinucleotides (3'3'CDNs) are bacterial second messengers that can also bind to the stimulator of interferon genes (STING) adaptor protein in vertebrates and activate the host innate immunity. Here, we profiled the substrate specificity of four bacterial dinucleotide synthases from Vibrio cholerae (DncV), Bacillus thuringiensis (btDisA), Escherichia coli (dgcZ), and Thermotoga maritima (tDGC) using a library of 33 nucleoside-5'-triphosphate analogues and then employed these enzymes to synthesize 24 3'3'CDNs. The STING affinity of CDNs was evaluated in cell-based and biochemical assays, and their ability to induce cytokines was determined by employing human peripheral blood mononuclear cells. Interestingly, the prepared heterodimeric 3'3'CDNs bound to the STING much better than their homodimeric counterparts and showed similar or better potency than bacterial 3'3'CDNs. We also rationalized the experimental findings by in-depth STING-CDN structure-activity correlations by dissecting computed interaction free energies into a set of well-defined and intuitive terms. To this aim, we employed state-of-the-art methods of computational chemistry, such as quantum mechanics/molecular mechanics (QM/MM) calculations, and complemented the computed results with the {STING:3'3'c-di-ara-AMP} X-ray crystallographic structure. QM/MM identified three outliers (mostly homodimers) for which we have no clear explanation of their impaired binding with respect to their heterodimeric counterparts, whereas the R2 = 0.7 correlation between the computed ΔG'int_rel and experimental ΔTm's for the remaining ligands has been very encouraging.

Dispatched uses Na+ flux to power release of lipid-modified Hedgehog.

The Dispatched protein, which is related to the NPC1 and PTCH1 cholesterol transporters1,2 and to H+-driven transporters of the RND family3,4, enables tissue-patterning activity of the lipid-modified Hedgehog protein by releasing it from tightly -localized sites of embryonic expression5-10. Here we determine a cryo-electron microscopy structure of the mouse protein Dispatched homologue 1 (DISP1), revealing three Na+ ions coordinated within a channel that traverses its transmembrane domain. We find that the rate of Hedgehog export is dependent on the Na+ gradient across the plasma membrane. The transmembrane channel and Na+ binding are disrupted in DISP1-NNN, a variant with asparagine substitutions for three intramembrane aspartate residues that each coordinate and neutralize the charge of one of the three Na+ ions. DISP1-NNN and variants that disrupt single Na+ sites retain binding to, but are impaired in export of the lipid-modified Hedgehog protein to the SCUBE2 acceptor. Interaction of the amino-terminal signalling domain of the Sonic hedgehog protein (ShhN) with DISP1 occurs via an extensive buried surface area and contacts with an extended furin-cleaved DISP1 arm. Variability analysis reveals that ShhN binding is restricted to one extreme of a continuous series of DISP1 conformations. The bound and unbound DISP1 conformations display distinct Na+-site occupancies, which suggests a mechanism by which transmembrane Na+ flux may power extraction of the lipid-linked Hedgehog signal from the membrane. Na+-coordinating residues in DISP1 are conserved in PTCH1 and other metazoan RND family members, suggesting that Na+ flux powers their conformationally driven activities.

Structural Protein Analysis of Driver Gene Mutations in Conjunctival Melanoma.

In recent years, there has been tremendous enthusiasm with respect to detailing the genetic basis of many neoplasms, including conjunctival melanoma (CM). We aim to analyze five proteins associated with CM, namely BRAF, NRAS, c-KIT, NF1, and PTEN. We evaluated each protein for its intrinsically disordered protein regions (IDPRs) and its protein-protein interactions (PPI) with the Predictor of Natural Disordered Protein Regions (PONDR®) and the Search Tool for the Retrieval of Interacting Genes (STRING®). Our PONDR® analysis found high levels of IDPRs in all five proteins with mutations linked to CM. The highest levels of IDPRs were in BRAF (45.95%), followed by PTEN (31.76%), NF1 (22.19%), c-KIT (21.82%), and NRAS (14.81%). Our STRING analysis found that each of these five proteins had more predicted interactions then expected (p-value < 1.0 × 10-16). Our analysis demonstrates that the mutations linked to CM likely affected IDPRs and possibly altered their highly complex PPIs. Quantifying IDPRs in BRAF, NRAS, c-KIT, NF1, and PTEN and understanding these protein regions are important processes as IDPRs can be possible drug targets for novel targeted therapies for treating CM.

Kainate receptor modulation by NETO2.

Glutamate-gated kainate receptors are ubiquitous in the central nervous system of vertebrates, mediate synaptic transmission at the postsynapse and modulate transmitter release at the presynapse1-7. In the brain, the trafficking, gating kinetics and pharmacology of kainate receptors are tightly regulated by neuropilin and tolloid-like (NETO) proteins8-11. Here we report cryo-electron microscopy structures of homotetrameric GluK2 in complex with NETO2 at inhibited and desensitized states, illustrating variable stoichiometry of GluK2-NETO2 complexes, with one or two NETO2 subunits associating with GluK2. We find that NETO2 accesses only two broad faces of kainate receptors, intermolecularly crosslinking the lower lobe of ATDA/C, the upper lobe of LBDB/D and the lower lobe of LBDA/C, illustrating how NETO2 regulates receptor-gating kinetics. The transmembrane helix of NETO2 is positioned proximal to the selectivity filter and competes with the amphiphilic H1 helix after M4 for interaction with an intracellular cap domain formed by the M1-M2 linkers of the receptor, revealing how rectification is regulated by NETO2.

Effect of charge on protein preferred orientation at the air-water interface in cryo-electron microscopy.

The air-water interface (AWI) tends to adsorb proteins and frequently causes preferred orientation problems in cryo-electron microscopy (cryo-EM). Here, we examined cryo-EM data from protein samples frozen with different detergents and found that both anionic and cationic detergents promoted binding of proteins to the AWI. By contrast, some of the nonionic and zwitterionic detergents tended to prevent proteins from attaching to the AWI. The protein orientation distributions with different anionic detergents were similar and resembled that obtained without detergent. By contrast, cationic detergents gave distinct orientation distributions. Our results indicate that proteins adsorb to charged interface and the negative charge of the AWI plays an important role in adsorbing proteins in the conventional cryo-EM sample preparation. According to these findings, a new method was developed by adding anionic detergent at a concentration between 0.002% and 0.005%. Using this method, the protein particles exhibited a more evenly distributed orientations and still adsorbed to the AWI enabling them embedding in a thin layer of ice with high concentration, which will benefit the cryo-EM structural determination.

Cryo-EM structures of the TTYH family reveal a novel architecture for lipid interactions.

The Tweety homologs (TTYHs) are members of a conserved family of eukaryotic membrane proteins that are abundant in the brain. The three human paralogs were assigned to function as anion channels that are either activated by Ca2+ or cell swelling. To uncover their unknown architecture and its relationship to function, we have determined the structures of human TTYH1-3 by cryo-electron microscopy. All structures display equivalent features of a dimeric membrane protein that contains five transmembrane segments and an extended extracellular domain. As none of the proteins shows attributes reminiscent of an anion channel, we revisited functional experiments and did not find any indication of ion conduction. Instead, we find density in an extended hydrophobic pocket contained in the extracellular domain that emerges from the lipid bilayer, which suggests a role of TTYH proteins in the interaction with lipid-like compounds residing in the membrane.