Plasma membrane preassociation drives ß-arrestin coupling to receptors and activation.

ß-arrestin plays a key role in G protein-coupled receptor (GPCR) signaling and desensitization. Despite recent structural advances, the mechanisms that govern receptor-ß-arrestin interactions at the plasma membrane of living cells remain elusive. Here, we combine single-molecule microscopy with molecular dynamics simulations to dissect the complex sequence of events involved in ß-arrestin interactions with both receptors and the lipid bilayer. Unexpectedly, our results reveal that ß-arrestin spontaneously inserts into the lipid bilayer and transiently interacts with receptors via lateral diffusion on the plasma membrane. Moreover, they indicate that, following receptor interaction, the plasma membrane stabilizes ß-arrestin in a longer-lived, membrane-bound state, allowing it to diffuse to clathrin-coated pits separately from the activating receptor. These results expand our current understanding of ß-arrestin function at the plasma membrane, revealing a critical role for ß-arrestin preassociation with the lipid bilayer in facilitating its interactions with receptors and subsequent activation.

Delineating the conformational landscape of the adenosine A2A receptor during G protein coupling.

G-protein-coupled receptors (GPCRs) represent a ubiquitous membrane protein family and are important drug targets. Their diverse signaling pathways are driven by complex pharmacology arising from a conformational ensemble rarely captured by structural methods. Here, fluorine nuclear magnetic resonance spectroscopy (19F NMR) is used to delineate key functional states of the adenosine A2A receptor (A2AR) complexed with heterotrimeric G protein (Gαsß1γ2) in a phospholipid membrane milieu. Analysis of A2AR spectra as a function of ligand, G protein, and nucleotide identifies an ensemble represented by inactive states, a G-protein-bound activation intermediate, and distinct nucleotide-free states associated with either partial- or full-agonist-driven activation. The Gßγ subunit is found to be critical in facilitating ligand-dependent allosteric transmission, as shown by 19F NMR, biochemical, and computational studies. The results provide a mechanistic basis for understanding basal signaling, efficacy, precoupling, and allostery in GPCRs.

Molecular mechanism of prestin electromotive signal amplification.

Hearing involves two fundamental processes: mechano-electrical transduction and signal amplification. Despite decades of studies, the molecular bases for both remain elusive. Here, we show how prestin, the electromotive molecule of outer hair cells (OHCs) that senses both voltage and membrane tension, mediates signal amplification by coupling conformational changes to alterations in membrane surface area. Cryoelectron microscopy (cryo-EM) structures of human prestin bound with chloride or salicylate at a common "anion site" adopt contracted or expanded states, respectively. Prestin is ensconced within a perimeter of well-ordered lipids, through which it induces dramatic deformation in the membrane and couples protein conformational changes to the bulk membrane. Together with computational studies, we illustrate how the anion site is allosterically coupled to changes in the transmembrane domain cross-sectional area and the surrounding membrane. These studies provide insight into OHC electromotility by providing a structure-based mechanism of the membrane motor prestin.

The ZAR1 resistosome is a calcium-permeable channel triggering plant immune signaling.

Nucleotide-binding, leucine-rich repeat receptors (NLRs) are major immune receptors in plants and animals. Upon activation, the Arabidopsis NLR protein ZAR1 forms a pentameric resistosome in vitro and triggers immune responses and cell death in plants. In this study, we employed single-molecule imaging to show that the activated ZAR1 protein can form pentameric complexes in the plasma membrane. The ZAR1 resistosome displayed ion channel activity in Xenopus oocytes in a manner dependent on a conserved acidic residue Glu11 situated in the channel pore. Pre-assembled ZAR1 resistosome was readily incorporated into planar lipid-bilayers and displayed calcium-permeable cation-selective channel activity. Furthermore, we show that activation of ZAR1 in the plant cell led to Glu11-dependent Ca2+ influx, perturbation of subcellular structures, production of reactive oxygen species, and cell death. The results thus support that the ZAR1 resistosome acts as a calcium-permeable cation channel to trigger immunity and cell death.

PspA adopts an ESCRT-III-like fold and remodels bacterial membranes.

PspA is the main effector of the phage shock protein (Psp) system and preserves the bacterial inner membrane integrity and function. Here, we present the 3.6 Å resolution cryoelectron microscopy (cryo-EM) structure of PspA assembled in helical rods. PspA monomers adopt a canonical ESCRT-III fold in an extended open conformation. PspA rods are capable of enclosing lipids and generating positive membrane curvature. Using cryo-EM, we visualized how PspA remodels membrane vesicles into µm-sized structures and how it mediates the formation of internalized vesicular structures. Hotspots of these activities are zones derived from PspA assemblies, serving as lipid transfer platforms and linking previously separated lipid structures. These membrane fusion and fission activities are in line with the described functional properties of bacterial PspA/IM30/LiaH proteins. Our structural and functional analyses reveal that bacterial PspA belongs to the evolutionary ancestry of ESCRT-III proteins involved in membrane remodeling.

Regulation of phospholipid distribution in the lipid bilayer by flippases and scramblases.

Cellular membranes function as permeability barriers that separate cells from the external environment or partition cells into distinct compartments. These membranes are lipid bilayers composed of glycerophospholipids, sphingolipids and cholesterol, in which proteins are embedded. Glycerophospholipids and sphingolipids freely move laterally, whereas transverse movement between lipid bilayers is limited. Phospholipids are asymmetrically distributed between membrane leaflets but change their location in biological processes, serving as signalling molecules or enzyme activators. Designated proteins - flippases and scramblases - mediate this lipid movement between the bilayers. Flippases mediate the confined localization of specific phospholipids (phosphatidylserine (PtdSer) and phosphatidylethanolamine) to the cytoplasmic leaflet. Scramblases randomly scramble phospholipids between leaflets and facilitate the exposure of PtdSer on the cell surface, which serves as an important signalling molecule and as an 'eat me' signal for phagocytes. Defects in flippases and scramblases cause various human diseases. We herein review the recent research on the structure of flippases and scramblases and their physiological roles. Although still poorly understood, we address the mechanisms by which they translocate phospholipids between lipid bilayers and how defects cause human diseases.

The Lateral Organization and Mobility of Plasma Membrane Components.

Over the last several decades, an impressive array of advanced microscopic and analytical tools, such as single-particle tracking and nanoscopic fluorescence correlation spectroscopy, has been applied to characterize the lateral organization and mobility of components in the plasma membrane. Such analysis can tell researchers about the local dynamic composition and structure of membranes and is important for predicting the outcome of membrane-based reactions. However, owing to the unresolved complexity of the membrane and the structures peripheral to it, identification of the detailed molecular origin of the interactions that regulate the organization and mobility of the membrane has not proceeded quickly. This Perspective presents an overview of how cell-surface structure may give rise to the types of lateral mobility that are observed and some potentially fruitful future directions to elucidate the architecture of these structures in more molecular detail.

LRRC8 Proteins Form Volume-Regulated Anion Channels that Sense Ionic Strength.

The volume-regulated anion channel (VRAC) is activated when a cell swells, and it plays a central role in maintaining cell volume in response to osmotic challenges. SWELL1 (LRRC8A) was recently identified as an essential component of VRAC. However, the identity of the pore-forming subunits of VRAC and how the channel is gated by cell swelling are unknown. Here, we show that SWELL1 and up to four other LRRC8 subunits assemble into heterogeneous complexes of ∼800 kDa. When reconstituted into bilayers, LRRC8 complexes are sufficient to form anion channels activated by osmolality gradients. In bilayers, as well as in cells, the single-channel conductance of the complexes depends on the LRRC8 composition. Finally, low ionic strength (Γ) in the absence of an osmotic gradient activates the complexes in bilayers. These data demonstrate that LRRC8 proteins together constitute the VRAC pore and that hypotonic stress can activate VRAC through a decrease in cytoplasmic Γ.

The Structure of the Polycystic Kidney Disease Channel PKD2 in Lipid Nanodiscs.

The Polycystic Kidney Disease 2 (Pkd2) gene is mutated in autosomal dominant polycystic kidney disease (ADPKD), one of the most common human monogenic disorders. Here, we present the cryo-EM structure of PKD2 in lipid bilayers at 3.0 Å resolution, which establishes PKD2 as a homotetrameric ion channel and provides insight into potential mechanisms for its activation. The PKD2 voltage-sensor domain retains two of four gating charges commonly found in those of voltage-gated ion channels. The PKD2 ion permeation pathway is constricted at the selectivity filter and near the cytoplasmic end of S6, suggesting that two gates regulate ion conduction. The extracellular domain of PKD2, a hotspot for ADPKD pathogenic mutations, contributes to channel assembly and strategically interacts with the transmembrane core, likely serving as a physical substrate for extracellular stimuli to allosterically gate the channel. Finally, our structure establishes the molecular basis for the majority of pathogenic mutations in Pkd2-related ADPKD.

Relaxation of Loaded ESCRT-III Spiral Springs Drives Membrane Deformation.

ESCRT-III is required for lipid membrane remodeling in many cellular processes, from abscission to viral budding and multi-vesicular body biogenesis. However, how ESCRT-III polymerization generates membrane curvature remains debated. Here, we show that Snf7, the main component of ESCRT-III, polymerizes into spirals at the surface of lipid bilayers. When covering the entire membrane surface, these spirals stopped growing when densely packed: they had a polygonal shape, suggesting that lateral compression could deform them. We reasoned that Snf7 spirals could function as spiral springs. By measuring the polymerization energy and the rigidity of Snf7 filaments, we showed that they were deformed while growing in a confined area. Furthermore, we observed that the elastic expansion of compressed Snf7 spirals generated an area difference between the two sides of the membrane and thus curvature. This spring-like activity underlies the driving force by which ESCRT-III could mediate membrane deformation and fission.

Ion and lipid orchestration of secondary active transport.

Transporting small molecules across cell membranes is an essential process in cell physiology. Many structurally diverse, secondary active transporters harness transmembrane electrochemical gradients of ions to power the uptake or efflux of nutrients, signalling molecules, drugs and other ions across cell membranes. Transporters reside in lipid bilayers on the interface between two aqueous compartments, where they are energized and regulated by symported, antiported and allosteric ions on both sides of the membrane and the membrane bilayer itself. Here we outline the mechanisms by which transporters couple ion and solute fluxes and discuss how structural and mechanistic variations enable them to meet specific physiological needs and adapt to environmental conditions. We then consider how general bilayer properties and specific lipid binding modulate transporter activity. Together, ion gradients and lipid properties ensure the effective transport, regulation and distribution of small molecules across cell membranes.

SlyB encapsulates outer membrane proteins in stress-induced lipid nanodomains.

The outer membrane in Gram-negative bacteria consists of an asymmetric phospholipid-lipopolysaccharide bilayer that is densely packed with outer-membrane ß-barrel proteins (OMPs) and lipoproteins1. The architecture and composition of this bilayer is closely monitored and is essential to cell integrity and survival2-4. Here we find that SlyB, a lipoprotein in the PhoPQ stress regulon, forms stable stress-induced complexes with the outer-membrane proteome. SlyB comprises a 10 kDa periplasmic ß-sandwich domain and a glycine zipper domain that forms a transmembrane α-helical hairpin with discrete phospholipid- and lipopolysaccharide-binding sites. After loss in lipid asymmetry, SlyB oligomerizes into ring-shaped transmembrane complexes that encapsulate ß-barrel proteins into lipid nanodomains of variable size. We find that the formation of SlyB nanodomains is essential during lipopolysaccharide destabilization by antimicrobial peptides or acute cation shortage, conditions that result in a loss of OMPs and compromised outer-membrane barrier function in the absence of a functional SlyB. Our data reveal that SlyB is a compartmentalizing transmembrane guard protein that is involved in cell-envelope proteostasis and integrity, and suggest that SlyB represents a larger family of broadly conserved lipoproteins with 2TM glycine zipper domains with the ability to form lipid nanodomains.

Lipids and extracellular materials.

This article introduces the Lipids and Extracellular Materials theme of the Annual Review of Biochemistry, Volume 83.

Cryo-EM structure of SARS-CoV-2 postfusion spike in membrane.

The entry of SARS-CoV-2 into host cells depends on the refolding of the virus-encoded spike protein from a prefusion conformation, which is metastable after cleavage, to a lower-energy stable postfusion conformation1,2. This transition overcomes kinetic barriers for fusion of viral and target cell membranes3,4. Here we report a cryogenic electron microscopy (cryo-EM) structure of the intact postfusion spike in a lipid bilayer that represents the single-membrane product of the fusion reaction. The structure provides structural definition of the functionally critical membrane-interacting segments, including the fusion peptide and transmembrane anchor. The internal fusion peptide forms a hairpin-like wedge that spans almost the entire lipid bilayer and the transmembrane segment wraps around the fusion peptide at the last stage of membrane fusion. These results advance our understanding of the spike protein in a membrane environment and may guide development of intervention strategies.

Structure and desensitization of AMPA receptor complexes with type II TARP γ5 and GSG1L.

AMPA receptors (AMPARs) mediate the majority of excitatory neurotransmission. Their surface expression, trafficking, gating, and pharmacology are regulated by auxiliary subunits. Of the two types of TARP auxiliary subunits, type I TARPs assume activating roles, while type II TARPs serve suppressive functions. We present cryo-EM structures of GluA2 AMPAR in complex with type II TARP γ5, which reduces steady-state currents, increases single-channel conductance, and slows recovery from desensitization. Regulation of AMPAR function depends on its ligand-binding domain (LBD) interaction with the γ5 head domain. GluA2-γ5 complex shows maximum stoichiometry of two TARPs per AMPAR tetramer, being different from type I TARPs but reminiscent of the auxiliary subunit GSG1L. Desensitization of both GluA2-GSG1L and GluA2-γ5 complexes is accompanied by rupture of LBD dimer interface, while GluA2-γ5 but not GluA2-GSG1L LBD dimers remain two-fold symmetric. Different structural architectures and desensitization mechanisms of complexes with auxiliary subunits endow AMPARs with broad functional capabilities.

Cryo-EM structure of the periplasmic tunnel of T7 DNA-ejectosome at 2.7 Å resolution.

Hershey and Chase used bacteriophage T2 genome delivery inside Escherichia coli to demonstrate that DNA, not protein, is the genetic material. Seventy years later, our understanding of viral genome delivery in prokaryotes remains limited, especially for short-tailed phages of the Podoviridae family. These viruses expel mysterious ejection proteins found inside the capsid to form a DNA-ejectosome for genome delivery into bacteria. Here, we reconstitute the phage T7 DNA-ejectosome components gp14, gp15, and gp16 and solve the periplasmic tunnel structure at 2.7 Å resolution. We find that gp14 forms an outer membrane pore, gp15 assembles into a 210 Å hexameric DNA tube spanning the host periplasm, and gp16 extends into the host cytoplasm forming a ∼4,200 residue hub. Gp16 promotes gp15 oligomerization, coordinating peptidoglycan hydrolysis, DNA binding, and lipid insertion. The reconstituted gp15:gp16 complex lacks channel-forming activity, suggesting that the pore for DNA passage forms only transiently during genome ejection.

Exploring membrane asymmetry and its effects on membrane proteins.

Plasma membranes utilize free energy to maintain highly asymmetric, non-equilibrium distributions of lipids and proteins between their two leaflets. In this review we discuss recent progress in quantitative research enabled by using compositionally controlled asymmetric model membranes. Both experimental and computational studies have shed light on the nuanced mechanisms that govern the structural and dynamic coupling between compositionally distinct bilayer leaflets. This coupling can increase the membrane bending rigidity and induce order - or lipid domains - across the membrane. Furthermore, emerging evidence indicates that integral membrane proteins not only respond to asymmetric lipid distributions but also exhibit intriguing asymmetric properties themselves. We propose strategies to advance experimental research, aiming for a deeper, quantitative understanding of membrane asymmetry, which carries profound implications for cellular physiology.

Limitations in membrane protein structure determination by lipid nanodiscs.

Lipid nanodiscs are popular mimetics of biological membranes for determining membrane protein structures. However, a recent study revealed that the choice of nanodisc scaffold directly influenced the structure of an ion channel. This finding prompts us to be cautious and calls for improved membrane mimetics for structure determination.

MemPrep, a new technology for isolating organellar membranes provides fingerprints of lipid bilayer stress.

Biological membranes have a stunning ability to adapt their composition in response to physiological stress and metabolic challenges. Little is known how such perturbations affect individual organelles in eukaryotic cells. Pioneering work has provided insights into the subcellular distribution of lipids in the yeast Saccharomyces cerevisiae, but the composition of the endoplasmic reticulum (ER) membrane, which also crucially regulates lipid metabolism and the unfolded protein response, remains insufficiently characterized. Here, we describe a method for purifying organelle membranes from yeast, MemPrep. We demonstrate the purity of our ER membrane preparations by proteomics, and document the general utility of MemPrep by isolating vacuolar membranes. Quantitative lipidomics establishes the lipid composition of the ER and the vacuolar membrane. Our findings provide a baseline for studying membrane protein biogenesis and have important implications for understanding the role of lipids in regulating the unfolded protein response (UPR). The combined preparative and analytical MemPrep approach uncovers dynamic remodeling of ER membranes in stressed cells and establishes distinct molecular fingerprints of lipid bilayer stress.

Stacked endoplasmic reticulum sheets are connected by helicoidal membrane motifs.

The endoplasmic reticulum (ER) often forms stacked membrane sheets, an arrangement that is likely required to accommodate a maximum of membrane-bound polysomes for secretory protein synthesis. How sheets are stacked is unknown. Here, we used improved staining and automated ultrathin sectioning electron microscopy methods to analyze stacked ER sheets in neuronal cells and secretory salivary gland cells of mice. Our results show that stacked ER sheets form a continuous membrane system in which the sheets are connected by twisted membrane surfaces with helical edges of left- or right-handedness. The three-dimensional structure of tightly stacked ER sheets resembles a parking garage, in which the different levels are connected by helicoidal ramps. A theoretical model explains the experimental observations and indicates that the structure corresponds to a minimum of elastic energy of sheet edges and surfaces. The structure allows the dense packing of ER sheets in the restricted space of a cell.