Membrane glycoproteins frequently adopt different conformations when altering between active and inactive states. Here, we discover a molecular switch that exploits dynamic spatial rearrangements of N-glycans during such conformational transitions to control protein function. For the conformationally switchable cell adhesion glycoprotein α5β1 integrin, we find that only the bent-closed state arranges N-glycans to nucleate the formation of up to tetrameric oligomers of the glycan-binding protein galectin-3. We propose a structural model of how these galectin-3 oligomers are built and how they clamp the bent-closed state to select it for endocytic uptake and subsequent retrograde trafficking to the Golgi for polarized distribution in cells. Our findings reveal the dynamic regulation of the glycan landscape at the cell surface to achieve oligomerization of galectin-3. Galectin-3 oligomers are thereby identified as functional decoders of defined spatial patterns of N-glycans on specifically the bent-closed conformational state of α5β1 integrin and possibly other integrin family members.

Galactocerebroside lipid nanotubes are membrane-mimicking systems for studying the function and structure of proteins involved in membrane shape remodeling, such as in intracellular trafficking, cell division, and migration or involved in the formation of membrane contact sites. They exhibit a constant and small diameter of 30 nm and a length of up to 2 μm. They can be functionalized with lipid ligands, providing a large binding surface for protein without membrane shape remodeling. These features make it possible to study protein assemblies on membranes different from those accessible with vesicular systems. This chapter describes the process of galactocerebroside nanotube formation, the incorporation of different lipid ligands, factors influencing protein binding, and the experimental conditions for their use in flotation assay and imaging by transmission electron and cryo-electron microscopy.

Electron microscopy has played a pivotal role in elucidating the ultrastructure of membrane contact sites between cellular organelles. The advent of cryo-electron microscopy has ushered in the ability to determine atomic models of constituent proteins or protein complexes within sites of membrane contact through single particle analysis. Furthermore, it enables the visualization of the three-dimensional architecture of membrane contact sites, encompassing numerous copies of proteins, whether in vitro reconstituted or directly observed in situ using cryo-electron tomography. Nevertheless, there exists a scarcity of cryo-electron microscopy studies focused on the site of membrane contact and their constitutive proteins. This review provides an overview of the contributions made by cryo-electron microscopy to our understanding of membrane contact sites, outlines the associated limitations, and explores prospects in this field.

Myosin VI (Myo6) is the only minus-end directed nanomotor on actin, allowing it to uniquely contribute to numerous cellular functions. As for other nanomotors, the proper functioning of Myo6 relies on precise spatiotemporal control of motor activity via a poorly defined off-state and interactions with partners. Our structural, functional, and cellular studies reveal key features of myosin regulation and indicate that not all partners can activate Myo6. TOM1 and Dab2 cannot bind the off-state, while GIPC1 binds Myo6, releases its auto-inhibition and triggers proximal dimerization. Myo6 partners thus differentially recruit Myo6. We solved a crystal structure of the proximal dimerization domain, and show that its disruption compromises endocytosis in HeLa cells, emphasizing the importance of Myo6 dimerization. Finally, we show that the L926Q deafness mutation disrupts Myo6 auto-inhibition and indirectly impairs proximal dimerization. Our study thus demonstrates the importance of partners in the control of Myo6 auto-inhibition, localization, and activation.

ABSTRACTCells secrete membrane-enclosed extracellular vesicles (EVs) and non-vesicular nanoparticles (ENPs) that may play a role in intercellular communication. Tumor-derived EVs have been proposed either to induce immune priming of antigen presenting cells, or, to be immuno-suppressive agents promoting tumor immune escape. We suspect that such disparate functions are due to variable composition in EV subtypes and ENPs of the analyzed EV preparations. We aimed to exhaustively characterize the array of secreted EVs and ENPs of murine tumor cell lines. Unexpectedly, we identified virus-like particles (VLPs) from endogenous murine leukemia virus in preparations of EVs produced by tumor cells. We established a robust protocol to separate small (s)EVs from VLPs and ENPs. We compared their protein composition and analyzed their functional interaction with target dendritic cells (DCs). ENPs were poorly captured and did not affect DCs. sEVs specifically induced DC death. A mixed EV/VLP preparation was the most efficient to induce DC maturation and antigen presentation. Our results call for systematic re-evaluation of the respective proportions and functions of non-viral EVs and VLPs produced by tumors and their contribution to anti-tumor immune responses and to tumor progression.

Membrane contact sites (MCSs) are heterogeneous in shape, composition, and dynamics. Despite this diversity, VAP proteins act as receptors for multiple FFAT motif-containing proteins and drive the formation of most MCSs that involve the endoplasmic reticulum (ER). Although the VAP-FFAT interaction is well characterized, no model explains how VAP adapts to its partners in various MCSs. We report that VAP-A localization to different MCSs depends on its intrinsically disordered regions (IDRs) in human cells. VAP-A interaction with PTPIP51 and VPS13A at ER-mitochondria MCS conditions mitochondria fusion by promoting lipid transfer and cardiolipin buildup. VAP-A also enables lipid exchange at ER-Golgi MCS by interacting with oxysterol-binding protein (OSBP) and CERT. However, removing IDRs from VAP-A restricts its distribution and function to ER-mitochondria MCS. Our data suggest that IDRs do not modulate VAP-A preference toward specific partners but do adjust their geometry to MCS organization and lifetime constraints. Thus, IDR-mediated VAP-A conformational flexibility ensures membrane tethering plasticity and efficiency.

Fundamental cell physiology originates from conformational changes and diffusion dynamics of membrane proteins. However, studying the interplay between nanoscale protein conformations and membrane mechanics is inherently challenging in cell-based experiments. In this work, we investigate the diffusion dynamics of multidrug resistance ABC transporter BmrA as the function of protein conformations: apo (open) or post-hydrolytic (closed) upon in-vitro membrane reconstitution. We perform single-particle tracking measurements on BmrA reconstituted in giant unilamellar vesicles with controlled and varying membrane tensions.

ABCB1 is the best-characterized member of the ABC superfamily of transmembrane proteins. ABCB1 transports structurally diverse substrates, including anticancer drugs. This, in combination with protein overexpression in cancer cells, hampers interactions with the drugs intracellular targets and jeopardizes chemotherapy treatments. The ATP-driven transport mechanism allows ABCB1 to alternate between an inward-facing (IF) conformation, where the protein adopts an inverted conical shape, and an outward-facing (OF) conformation, with a cylinder-like shape.

Transmembrane proteins (or integral membrane proteins) are synthesized in the endoplasmic reticulum where most of them are core glycosylated prior to folding and in some cases assembly into quaternary structures. Correctly glycosylated, folded, and assembled transmembrane proteins are then shuttled to the Golgi apparatus for additional posttranslational modifications such as complex-type glycosylations, sulfation or proteolytic clipping. At the plasma membrane, the glycosylated extracellular domains are key to communicate with the cellular environment for a variety of functions, such as binding to the extracellular matrix for cell adhesion and migration, to neighboring cells for cell-cell interaction, or to extracellular components for nutrient uptake and cell signaling. Intracellular domains are essential to mediate signaling cascades, or to connect to cytosolic adaptors for internalization and intracellular compartmentalization. Despite its importance for the understanding of molecular mechanisms of transmembrane protein function, the determination of their structures has remained a challenging task. In recent years, their reconstitution in lipid nanodiscs in combination with high resolution cryo-electron microscopy has provided novel avenues to render this process more accessible. Here, we describe detailed protocols for the solubilization of heavily glycosylated α5β1 integrin from rat livers, its purification and reconstitution into nanodiscs. At the plasma membrane of many cells, including tumor metastases, this essential heterodimeric transmembrane protein mediates the communication between extracellular matrix and cytosolic cytoskeleton in processes of cell adhesion and migration. We expect that the protocols that are described here will provide new opportunities for the determination of the full structure of α5β1 integrin, as well as for the understanding of how interacting partners can regulate function and activity of this transmembrane protein.

Intracellular trafficking is mediated by transport carriers that originate by membrane remodeling from donor organelles. Tubular carriers contribute to the flux of membrane lipids and proteins to acceptor organelles, but how lipids and proteins impose a tubular geometry on the carriers is incompletely understood. Using imaging approaches on cells and in vitro membrane systems, we show that phosphatidylinositol-4-phosphate (PI4P) and biogenesis of lysosome-related organelles complex 1 (BLOC-1) govern the formation, stability, and functions of recycling endosomal tubules. In vitro, BLOC-1 binds and tubulates negatively charged membranes, including those containing PI4P. In cells, endosomal PI4P production by type II PI4-kinases is needed to form and stabilize BLOC-1-dependent recycling endosomal tubules. Decreased PI4KIIs expression impairs the recycling of endosomal cargoes and the life cycles of intracellular pathogens such as Chlamydia bacteria and influenza virus that exploit the membrane dynamics of recycling endosomes. This study demonstrates how a phospholipid and a protein complex coordinate the remodeling of cellular membranes into functional tubules.

Galectin-3 (Gal3) is a β-galactoside binding lectin that mediates many physiological functions, including the binding of cells to the extracellular matrix for which the glycoprotein α5β1 integrin is of critical importance. The mechanisms by which Gal3 interacts with membranes have not been widely explored to date due to the complexity of cell membranes and the difficulty of integrin reconstitution within model membranes. Herein, to study their interaction, Gal3 and α5β1 were purified, and the latter reconstituted into pore-suspended lipid bilayers comprised eggPC:eggPA. Using electrochemical impedance and fluorescence lifetime correlation spectroscopy, we found that on incubation with low nanomolar concentrations of wild-type Gal3, the membrane's admittance and fluidity, as well as integrin's lateral diffusivity, were enhanced. These effects were diminished in the following conditions: (i) absence of integrin, (ii) presence of lactose as a competitive inhibitor of glycan-Gal3 interaction, and (iii) use of a Gal3 mutant that lacked the N-terminal oligomerization domain (Gal3ΔNter). These findings indicated that WTGal3 oligomerized on α5β1 integrin in a glycan-dependent manner and that the N-terminal domain interacted directly with membranes in a way that is yet to be fully understood. At concentrations above 10 nM of WTGal3, membrane capacitance started to decrease and very slowly diffusing molecular species appeared, which indicated the formation of protein clusters made from WTGal3-α5β1 integrin assemblies. Overall, our study demonstrates the capacity of WTGal3 to oligomerize in a cargo protein-dependent manner at low nanomolar concentrations. Of note, these WTGal3 oligomers appeared to have membrane active properties that could only be revealed using our sensitive methods. At slightly higher WTGal3 concentrations, the capacity to generate lateral assemblies between cargo proteins was observed. In cells, this could lead to the construction of tubular endocytic pits according to the glycolipid-lectin (GL-Lect) hypothesis or to the formation of galectin lattices, depending on cargo glycoprotein stability at the membrane, the local Gal3 concentration, or plasma membrane intrinsic parameters. The study also demonstrates the utility of microcavity array-suspended lipid bilayers to address the biophysics of transmembrane proteins.

Cryo-electron microscopy (cryo-EM) is a technique for imaging biological samples that plays a central role in structural biology, with high impact on research fields such as cell and developmental biology, bioinformatics, cell physics and applied mathematics. It allows the determination of structures of purified proteins within cells. This review describes the main recent advances in cryo-EM, illustrated by examples of proteins of biomedical interest, and the avenues for future development.

Membrane contact sites (MCS) are subcellular regions where two organelles appose their membranes to exchange small molecules, including lipids. Structural information on how proteins form MCS is scarce. We designed an in vitro MCS with two membranes and a pair of tethering proteins suitable for cryo-tomography analysis. It includes VAP-A, an ER transmembrane protein interacting with a myriad of cytosolic proteins, and oxysterol-binding protein (OSBP), a lipid transfer protein that transports cholesterol from the ER to the trans Golgi network. We show that VAP-A is a highly flexible protein, allowing formation of MCS of variable intermembrane distance. The tethering part of OSBP contains a central, dimeric, and helical T-shape region. We propose that the molecular flexibility of VAP-A enables the recruitment of partners of different sizes within MCS of adjustable thickness, whereas the T geometry of the OSBP dimer facilitates the movement of the two lipid-transfer domains between membranes. Membrane contact sites (MCS) are subcellular regions where two organelles appose their membranes to exchange small molecules, including lipids. Here authors designed an in vitro MCS suitable for cryotomography and sub-tomogram analysis which sheds light on the recruitment of proteins of different sizes within MCS of adjustable thickness.

A species-specific region, denoted SpG8-1b allowing hydroxycinnamic acids (HCAs) degradation is important for the transition between the two lifestyles (rhizospheric versus pathogenic) of the plant pathogen Agrobacterium fabrum. Indeed, HCAs can be either used as trophic resources and/or as induced-virulence molecules. The SpG8-1b region is regulated by two transcriptional regulators, namely, HcaR (Atu1422) and Atu1419. In contrast to HcaR, Atu1419 remains so far uncharacterized. The high-resolution crystal structures of two fortuitous citrate complexes, two DNA complexes and the apoform revealed that the tetrameric Atu1419 transcriptional regulator belongs to the VanR group of Pfam PF07729 subfamily of the large GntR superfamily. Until now, GntR regulators were described as dimers. Here, we showed that Atu1419 represses three genes of the HCAs catabolic pathway. We characterized both the effector and DNA binding sites and identified key nucleotides in the target palindrome. From promoter activity measurement using defective gene mutants, structural analysis and gel-shift assays, we propose N5,N10-methylenetetrahydrofolate as the effector molecule, which is not a direct product/substrate of the HCA degradation pathway. The Zn2+ ion present in the effector domain has both a structural and regulatory role. Overall, our work shed light on the allosteric mechanism of transcription employed by this GntR repressor.

Septins are ubiquitous cytoskeletal filaments that interact with the inner plasma membrane and are essential for cell division in eukaryotes. In cellular contexts, septins are often localized at micrometric Gaussian curvatures, where they assemble onto ring-like structures. The behavior of budding yeast septins depends on their specific interaction with inositol phospholipids, enriched at the inner leaflet of the plasma membrane. Septin filaments are built from the non-polar self-assembly of short rods into filaments. However, the molecular mechanisms regulating the interplay with the inner plasma membrane and the resulting interaction with specific curvatures are not fully understood. In this report, we have imaged dynamical molecular assemblies of budding yeast septins on PIP2-containing supported lipid bilayers using a combination of high-speed AFM and correlative AFM-fluorescence microscopy. Our results clearly demonstrate that septins are able to bind to flat supported lipid bilayers and thereafter induce the remodeling of membranes. Short septin rods (octamers subunits) can indeed destabilize supported lipid bilayers and reshape the membrane to form 3D structures such as rings and tubes, demonstrating that long filaments are not necessary for septin-induced membrane buckling.

Estelle Dransart, Aurélie Di Cicco, Ahmed El Marjou, Daniel Lévy, Staffan Johansson, Ludger Johannes, Massiullah Shafaq-Zadah (2021 Jul 14) Physiological alpha5beta1 integrin transmembrane protein extraction, purification and reconstitution into proteo-lipidic nanodiscs bilayer Methods in Molecular Biology on Heterologous Expression of Membrane ProteinsMethods in Molecular Biology on Heterologous Expression of Membrane Proteins

ATP-Binding Cassette (ABC) transporters constitute one of the largest superfamily of transmembrane proteins, present in both prokaryotes and eukaryotes. ATP hydrolysis at the nucleotide binding domains (NBD) drives conformational changes in the transmembrane domains (TMD) allowing substrate translocation. During this process, the structure of the ABC transporter alternates between the inward-facing (IF) conformation and the outward-facing (OF) conformation. The most prevalent and best characterized ABC-member is the mammalian P-glycoprotein (Pgp). Pgp extrudes cytotoxic drugs out of the cell, hampering interactions with their cellular targets, and, in combination with overexpression of the transporter in cancer cells, Pgp leads to failure of cancer chemotherapy. In the IF conformation, for substrate binding, Pgp displays an inverted conical shape, while in the OF conformation, for substrate release, the protein acquires a cylinder-like shape. It has been proposed that proteins with different shapes may not only diffuse differently along the lipid bilayer but also induce different local membrane curvature. In this study, we performed coarse-grained (CG) molecular dynamics (MD) simulations of Pgp in the IF and OF conformations using the Martini force field to assess the influence of protein shape in lateral diffusion and induced membrane curvature under regimes of low and high protein density (LPD, HPD). Our 50μs long simulations reveal that in the IF form, protein diffusion is significantly diminished when in a crowded environment. In contrast, the OF form, remains unaltered. Our findings also indicate that in the IF form, protein-protein interactions are driven by a cooperative effect coupled to the membrane curvature induced by conical shape profile. Our results are aligned with previous computational and experimental reports, reinforcing the hypothesis that shape has a substantial effect in protein diffusion and deformation of the local lipid environment.

Membrane proteins are involved in all major cellular processes such as cell homeostasis, bioenergetics, cell division and communication. When trying to understand their role in cellular functions or when designing drugs it is often beneficial to isolate these proteins and study them in a well-controlled bio-mimetic membrane environment, such as in cell-sized giant unilamellar vesicles (GUVs). In this chapter, we will cover the two most commonly reported classes of formation methods for proteo-GUVs, GUVs incorporating proteins, using either dehydration/rehydration or direct incorporation or fusion.

Lipid transfer proteins (LTPs) acting at membrane contact sites (MCS) between the ER and other organelles contain domains involved in heterotypic (e.g., ER to Golgi) membrane tethering as well as domains involved in lipid transfer. Here, we show that a long ≈90 aa intrinsically unfolded sequence at the N terminus of oxysterol-binding protein (OSBP) controls OSBP orientation and dynamics at MCS. This Gly-Pro-Ala-rich sequence, whose hydrodynamic radius is twice as that of folded domains, prevents the two PH domains of the OSBP dimer from homotypically tethering two Golgi-like membranes and considerably facilitates OSBP in-plane diffusion and recycling at MCS. Although quite distant in sequence, the N terminus of OSBP-related protein-4 (ORP4) has similar effects. We propose that N-terminal sequences of low complexity in ORPs form an entropic barrier that restrains protein orientation, limits protein density, and facilitates protein mobility in the narrow and crowded MCS environment.

ABC (ATP Binding Cassette) transporters represent one of the largest families of transmembrane protein. They use ATP hydrolysis to transport a wide range of substrates such as lipids, antibiotics and drugs. Some ABCs, such as glycoprotein P (PgP), a human ABC transporter, export anti-cancer agents out of the cell, and contribute to a cellular phenotype of multi-drug resistance. ABC transporters consist of two transmembrane domains (TMDs) that contain drug binding sites and two nucleotide binding domains (NBDs) that hydrolyze ATP. Substrate transport is associated with both rearrangements of NBDs and TMDs. We study BmrA, a bacterial homologue of PgP from B. Subtillis, for which a large conformational change is a priori expected. However, the BmrA structure not being solved yet, this change has to be scrutinized. We aim at investigating the interplay between the conformational dynamics of the protein and the mechanical properties of its surrounding membrane, e.g. curvature and tension. We have shown that the purified protein is highly active when incorporated into different membrane model systems. In WT both NBDs contain a single cysteine, labelled with fluorescent dyes. We have used single molecule FRET method (Forster Resonance Energy Transfer) to probe the transporter conformation in immobilized liposomes. Our results show little difference in the FRET efficiency histograms between the apo and the post-hydrolysis conformations, suggesting that the distance between the two NBDs does not change significantly, in contrast with initial expectations. Based on Electron Microscopy observations and other biophysical results from our group, we suggest that a significant conformational change occurs in protein TMDs, in line with recent models proposing a transport mechanism with constant contact between NBDs.