X-ray structure of a calcium-activated TMEM16 lipid scramblase.

The TMEM16 family of proteins, also known as anoctamins, features a remarkable functional diversity. This family contains the long sought-after Ca(2+)-activated chloride channels as well as lipid scramblases and cation channels. Here we present the crystal structure of a TMEM16 family member from the fungus Nectria haematococca that operates as a Ca(2+)-activated lipid scramblase. Each subunit of the homodimeric protein contains ten transmembrane helices and a hydrophilic membrane-traversing cavity that is exposed to the lipid bilayer as a potential site of catalysis. This cavity harbours a conserved Ca(2+)-binding site located within the hydrophobic core of the membrane. Mutations of residues involved in Ca(2+) coordination affect both lipid scrambling in N. haematococca TMEM16 and ion conduction in the Cl(-) channel TMEM16A. The structure reveals the general architecture of the family and its mode of Ca(2+) activation. It also provides insight into potential scrambling mechanisms and serves as a framework to unravel the conduction of ions in certain TMEM16 proteins.

Generation and Characterization of Anti-VGLUT Nanobodies Acting as Inhibitors of Transport.

The uptake of glutamate by synaptic vesicles is mediated by vesicular glutamate transporters (VGLUTs). The central role of these transporters in excitatory neurotransmission underpins their importance as pharmacological targets. Although several compounds inhibit VGLUTs, highly specific inhibitors were so far unavailable, thus limiting applications to in vitro experiments. Besides their potential in pharmacology, specific inhibitors would also be beneficial for the elucidation of transport mechanisms. To overcome this shortage, we generated nanobodies (Nbs) by immunization of a llama with purified rat VGLUT1 and subsequent selection of binders from a phage display library. All identified Nbs recognize cytosolic epitopes, and two of the binders greatly reduced the rate of uptake of glutamate by reconstituted liposomes and subcellular fractions enriched with synaptic vesicles. These Nbs can be expressed as functional green fluorescent protein fusion proteins in the cytosol of HEK cells for intracellular applications as immunocytochemical and biochemical agents. The selected binders thus provide valuable tools for cell biology and neuroscience.

Cryo-EM structures of a LptDE transporter in complex with Pro-macrobodies offer insight into lipopolysaccharide translocation.

Lipopolysaccharides are major constituents of the extracellular leaflet in the bacterial outer membrane and form an effective physical barrier for environmental threats and for antibiotics in Gram-negative bacteria. The last step of LPS insertion via the Lpt pathway is mediated by the LptD/E protein complex. Detailed insights into the architecture of LptDE transporter complexes have been derived from X-ray crystallography. However, no structure of a laterally open LptD transporter, a transient state that occurs during LPS release, is available to date. Here, we report a cryo-EM structure of a partially opened LptDE transporter in complex with rigid chaperones derived from nanobodies, at 3.4 Å resolution. In addition, a subset of particles allows to model a structure of a laterally fully opened LptDE complex. Our work offers insights into the mechanism of LPS insertion, provides a structural framework for the development of antibiotics targeting LptD and describes a highly rigid chaperone scaffold to enable structural biology of challenging protein targets.

Production and Application of Nanobodies for Membrane Protein Structural Biology.

Nanobodies, small recombinant binders derived from camelid single chain antibodies, have become widely used tools in a diversity of disciplines related to membrane proteins. They are applied as chaperones in crystallization and blockers or modifiers of protein activity among numerous other applications. Their simple architecture as a single polypeptide chain, in contrast to classical antibodies, enables straightforward cloning, library generation, and recombinant expression. The small diameter and the pointed wedge-like shape of the antigen-binding site underlies binding to hollows and crevices of membrane proteins and renders nanobodies often conformation specific making them a preferred type of chaperone. Here we describe a simple protocol for the recombinant production of nanobodies in E. coli and their purification. We expand the current repertoire of usage further by describing a procedure for enlarging nanobodies on their C-terminal end to generate "macrobodies," without interfering with their original characteristics. These enlarged nanobodies extend the application as a chaperone in crystallography and can serve to increase the mass for small targets in single particle electron cryo-microscopy, a field where nanobodies had so far only limited effect because of their small size.

Structural basis for ion selectivity in TMEM175 K+ channels.

The TMEM175 family constitutes recently discovered K+channels that are important for autophagosome turnover and lysosomal pH regulation and are associated with the early onset of Parkinson Disease. TMEM175 channels lack a P-loop selectivity filter, a hallmark of all known K+ channels, raising the question how selectivity is achieved. Here, we report the X-ray structure of a closed bacterial TMEM175 channel in complex with a nanobody fusion-protein disclosing bound K+ ions. Our analysis revealed that a highly conserved layer of threonine residues in the pore conveys a basal K+ selectivity. An additional layer comprising two serines in human TMEM175 increases selectivity further and renders this channel sensitive to 4-aminopyridine and Zn2+. Our findings suggest that large hydrophobic side chains occlude the pore, forming a physical gate, and that channel opening by iris-like motions simultaneously relocates the gate and exposes the otherwise concealed selectivity filter to the pore lumen.

Preparation of Proteoliposomes with Purified TMEM16 Protein for Accurate Measures of Lipid Scramblase Activity.

The distribution of different lipid species between the two leaflets is tightly regulated and underlies the concerted action of distinct catalytic entities. While flippases and floppases establish membrane asymmetry, scramblases randomize the lipid distribution and play pivotal roles during blood clotting, apoptosis, and in processes such as N-linked glycosylation of proteins. The recent discovery of TMEM16 family members acting as scramblases has led to an increasing demand for developing protocols tailored for TMEM16 proteins to enable functional investigations of their scrambling activity. Here we describe a protocol for the expression, purification, and functional reconstitution of TMEM16 proteins into preformed liposomes and measurement of their scrambling activity using fluorescence-labeled lipid derivatives. The reconstitution involves extrusion of liposomes through a membrane, destabilization of liposomes using Triton X-100, and stepwise detergent removal by adsorption on styryl-beads. The scrambling assay is based on the selective bleaching of nitrobenzoxadiazol fluorescent lipids on the outer leaflet of liposomes by the membrane-impermeant reducing agent sodium dithionite. The assay allows conclusions on the substrate specificity and on the kinetics of the transported lipids as shown with the example of a Ca2+-activated TMEM16 scramblase from the fungus Nectria haematococca (nhTMEM16).

Dual and Direction-Selective Mechanisms of Phosphate Transport by the Vesicular Glutamate Transporter.

Vesicular glutamate transporters (VGLUTs) fill synaptic vesicles with glutamate and are thus essential for glutamatergic neurotransmission. However, VGLUTs were originally discovered as members of a transporter subfamily specific for inorganic phosphate (Pi). It is still unclear how VGLUTs accommodate glutamate transport coupled to an electrochemical proton gradient ΔµH+ with inversely directed Pi transport coupled to the Na+ gradient and the membrane potential. Using both functional reconstitution and heterologous expression, we show that VGLUT transports glutamate and Pi using a single substrate binding site but different coupling to cation gradients. When facing the cytoplasm, both ions are transported into synaptic vesicles in a ΔµH+-dependent fashion, with glutamate preferred over Pi. When facing the extracellular space, Pi is transported in a Na+-coupled manner, with glutamate competing for binding but at lower affinity. We conclude that VGLUTs have dual functions in both vesicle transmitter loading and Pi homeostasis within glutamatergic neurons.

Structural basis for anion conduction in the calcium-activated chloride channel TMEM16A.

The calcium-activated chloride channel TMEM16A is a member of a conserved protein family that comprises ion channels and lipid scramblases. Although the structure of the scramblase nhTMEM16 has defined the architecture of the family, it was unknown how a channel has adapted to cope with its distinct functional properties. Here we have addressed this question by the structure determination of mouse TMEM16A by cryo-electron microscopy and a complementary functional characterization. The protein shows a similar organization to nhTMEM16, except for changes at the site of catalysis. There, the conformation of transmembrane helices constituting a membrane-spanning furrow that provides a path for lipids in scramblases has changed to form an enclosed aqueous pore that is largely shielded from the membrane. Our study thus reveals the structural basis of anion conduction in a TMEM16 channel and it defines the foundation for the diverse functional behavior in the TMEM16 family.

Structural basis for phospholipid scrambling in the TMEM16 family.

Upon activation, lipid scramblases dissipate the lipid asymmetry of membranes, in an ATP-independent manner, by catalyzing flip-flop of lipids between the leaflets. The molecular identities of these proteins long remained obscure, but in recent years the TMEM16 family of proteins has been found to constitute Ca2+-activated scramblases. Recently, the X-ray structure of a fungal TMEM16 homologue has provided insight into the architecture of this protein family and into potential scrambling mechanisms. The protein forms homodimers with each subunit containing a membrane-spanning hydrophilic cleft. This region is of sufficient size to harbor polar headgroups on their way across the membrane and thus may lower the energetic barrier for the diffusion of lipids between the two leaflets of the bilayer. A regulatory Ca2+ binding site located within the membrane adjacent to this hydrophobic cleft is responsible for activation by yet unknown mechanisms.

A chloride conductance in VGLUT1 underlies maximal glutamate loading into synaptic vesicles.

Uptake of glutamate into synaptic vesicles is mediated by vesicular glutamate transporters (VGLUTs). Although glutamate uptake has been shown to depend critically on Cl(-), the precise contribution of this ion to the transport process is unclear. We found that VGLUT1, and not ClC-3 as proposed previously, represents the major Cl(-) permeation pathway in synaptic vesicles. Using reconstituted VGLUT1, we found that the biphasic dependence of glutamate transport on extravesicular Cl(-) is a result of the permeation of this anion through VGLUT1 itself. Moreover, we observed that high luminal Cl(-) concentrations markedly enhanced loading of glutamate by facilitation of membrane potential-driven uptake and discovered a hitherto unrecognized transport mode of VGLUT1. Because a steep Cl(-) gradient across the synaptic vesicle membrane exists in endocytosed synaptic vesicles, our results imply that the transport velocity and the final glutamate content are highly influenced, if not determined, by the extracellular Cl(-) concentration.

Molecular anatomy of a trafficking organelle.

Membrane traffic in eukaryotic cells involves transport of vesicles that bud from a donor compartment and fuse with an acceptor compartment. Common principles of budding and fusion have emerged, and many of the proteins involved in these events are now known. However, a detailed picture of an entire trafficking organelle is not yet available. Using synaptic vesicles as a model, we have now determined the protein and lipid composition; measured vesicle size, density, and mass; calculated the average protein and lipid mass per vesicle; and determined the copy number of more than a dozen major constituents. A model has been constructed that integrates all quantitative data and includes structural models of abundant proteins. Synaptic vesicles are dominated by proteins, possess a surprising diversity of trafficking proteins, and, with the exception of the V-ATPase that is present in only one to two copies, contain numerous copies of proteins essential for membrane traffic and neurotransmitter uptake.