The substrate-binding domains of the osmoregulatory ABC importer OpuA transiently interact.

Bacteria utilize various strategies to prevent internal dehydration during hypertonic stress. A common approach to countering the effects of the stress is to import compatible solutes such as glycine betaine, leading to simultaneous passive water fluxes following the osmotic gradient. OpuA from Lactococcus lactis is a type I ABC-importer that uses two substrate-binding domains (SBDs) to capture extracellular glycine betaine and deliver the substrate to the transmembrane domains for subsequent transport. OpuA senses osmotic stress via changes in the internal ionic strength and is furthermore regulated by the 2nd messenger cyclic-di-AMP. We now show, by means of solution-based single-molecule FRET and analysis with multi-parameter photon-by-photon hidden Markov modeling, that the SBDs transiently interact in an ionic strength-dependent manner. The smFRET data are in accordance with the apparent cooperativity in transport and supported by new cryo-EM data of OpuA. We propose that the physical interactions between SBDs and cooperativity in substrate delivery are part of the transport mechanism.

Structural basis for excitatory neuropeptide signaling.

Rapid signaling between neurons is mediated by ligand-gated ion channels, cell-surface proteins with an extracellular ligand-binding domain and a membrane-spanning ion channel domain. The degenerin/epithelial sodium channel (DEG/ENaC) superfamily is diverse in terms of its gating stimuli, with some DEG/ENaCs gated by neuropeptides, and others gated by pH, mechanical force or enzymatic activity. The mechanism by which ligands bind to and activate DEG/ENaCs is poorly understood. Here we dissected the structural basis for neuropeptide-gated activity of a neuropeptide-gated DEG/ENaC, FMRFamide-gated sodium channel 1 (FaNaC1) from the annelid worm Malacoceros fuliginosus, using cryo-electron microscopy. Structures of FaNaC1 in the ligand-free resting state and in several ligand-bound states reveal the ligand-binding site and capture the ligand-induced conformational changes of channel gating, which we verified with complementary mutagenesis experiments. Our results illuminate channel gating in DEG/ENaCs and offer a structural template for experimental dissection of channel pharmacology and ion conduction.

Structures of a sperm-specific solute carrier gated by voltage and cAMP.

The newly characterized sperm-specific Na+/H+ exchanger stands out by its unique tripartite domain composition1,2. It unites a classical solute carrier unit with regulatory domains usually found in ion channels, namely, a voltage-sensing domain and a cyclic-nucleotide binding domain1,3, which makes it a mechanistic chimera and a secondary-active transporter activated strictly by membrane voltage. Our structures of the sea urchin SpSLC9C1 in the absence and presence of ligands reveal the overall domain arrangement and new structural coupling elements. They allow us to propose a gating model, where movements in the voltage sensor indirectly cause the release of the exchanging unit from a locked state through long-distance allosteric effects transmitted by the newly characterized coupling helices. We further propose that modulation by its ligand cyclic AMP occurs by means of disruption of the cytosolic dimer interface, which lowers the energy barrier for S4 movements in the voltage-sensing domain. As SLC9C1 members have been shown to be essential for male fertility, including in mammals2,4,5, our structure represents a potential new platform for the development of new on-demand contraceptives.

Expulsion mechanism of the substrate-translocating subunit in ECF transporters.

Energy-coupling factor (ECF)-type transporters mediate the uptake of micronutrients in many bacteria. They consist of a substrate-translocating subunit (S-component) and an ATP-hydrolysing motor (ECF module) Previous data indicate that the S-component topples within the membrane to alternately expose the binding site to either side of the membrane. In many ECF transporters, the substrate-free S-component can be expelled from the ECF module. Here we study this enigmatic expulsion step by cryogenic electron microscopy and reveal that ATP induces a concave-to-convex shape change of two long helices in the motor, thereby destroying the S-component's docking site and allowing for its dissociation. We show that adaptation of the membrane morphology to the conformational state of the motor may favour expulsion of the substrate-free S-component when ATP is bound and docking of the substrate-loaded S-component after hydrolysis. Our work provides a picture of bilayer-assisted chemo-mechanical coupling in the transport cycle of ECF transporters.

Structural basis for the activation of the lipid scramblase TMEM16F.

TMEM16F, a member of the conserved TMEM16 family, plays a central role in the initiation of blood coagulation and the fusion of trophoblasts. The protein mediates passive ion and lipid transport in response to an increase in intracellular Ca2+. However, the mechanism of how the protein facilitates both processes has remained elusive. Here we investigate the basis for TMEM16F activation. In a screen of residues lining the proposed site of conduction, we identify mutants with strongly activating phenotype. Structures of these mutants determined herein by cryo-electron microscopy show major rearrangements leading to the exposure of hydrophilic patches to the membrane, whose distortion facilitates lipid diffusion. The concomitant opening of a pore promotes ion conduction in the same protein conformation. Our work has revealed a mechanism that is distinct for this branch of the family and that will aid the development of a specific pharmacology for a promising drug target.

Inhibited KdpFABC transitions into an E1 off-cycle state.

KdpFABC is a high-affinity prokaryotic K+ uptake system that forms a functional chimera between a channel-like subunit (KdpA) and a P-type ATPase (KdpB). At high K+ levels, KdpFABC needs to be inhibited to prevent excessive K+ accumulation to the point of toxicity. This is achieved by a phosphorylation of the serine residue in the TGES162 motif in the A domain of the pump subunit KdpB (KdpBS162-P). Here, we explore the structural basis of inhibition by KdpBS162 phosphorylation by determining the conformational landscape of KdpFABC under inhibiting and non-inhibiting conditions. Under turnover conditions, we identified a new inhibited KdpFABC state that we termed E1P tight, which is not part of the canonical Post-Albers transport cycle of P-type ATPases. It likely represents the biochemically described stalled E1P state adopted by KdpFABC upon KdpBS162 phosphorylation. The E1P tight state exhibits a compact fold of the three cytoplasmic domains and is likely adopted when the transition from high-energy E1P states to E2P states is unsuccessful. This study represents a structural characterization of a biologically relevant off-cycle state in the P-type ATPase family and supports the emerging discussion of P-type ATPase regulation by such states.

Cryo-EM studies of membrane proteins at 200 keV.

Single-particle cryogenic electron-microscopy (cryo-EM) has emerged as a powerful technique for the structural characterisation of membrane proteins, especially for targets previously thought to be intractable. Taking advantage of the latest hard- and software developments, high-resolution three-dimensional (3D) reconstructions of membrane proteins by cryo-EM has become routine, with 300-kV transmission electron microscopes (TEMs) being the current standard. The use of 200-kV cryo-TEMs is gaining increasingly prominence, showing the capabilities of reaching better than 2 Å resolution for soluble proteins and better than 3 Å resolution for membrane proteins. Here, we highlight the challenges working with membrane proteins and the impact of cryo-EM, and review the technical and practical benefits, achievements and limitations of imaging at lower electron acceleration voltages.

Algae as Food in Europe: An Overview of Species Diversity and Their Application.

Algae have been consumed for millennia in several parts of the world as food, food supplements, and additives, due to their unique organoleptic properties and nutritional and health benefits. Algae are sustainable sources of proteins, minerals, and fiber, with well-balanced essential amino acids, pigments, and fatty acids, among other relevant metabolites for human nutrition. This review covers the historical consumption of algae in Europe, developments in the current European market, challenges when introducing new species to the market, bottlenecks in production technology, consumer acceptance, and legislation. The current algae species that are consumed and commercialized in Europe were investigated, according to their status under the European Union (EU) Novel Food legislation, along with the market perspectives in terms of the current research and development initiatives, while evaluating the interest and potential in the European market. The regular consumption of more than 150 algae species was identified, of which only 20% are approved under the EU Novel Food legislation, which demonstrates that the current legislation is not broad enough and requires an urgent update. Finally, the potential of the European algae market growth was indicated by the analysis of the trends in research, technological advances, and market initiatives to promote algae commercialization and consumption.

Biparatopic sybodies neutralize SARS-CoV-2 variants of concern and mitigate drug resistance.

The ongoing COVID-19 pandemic represents an unprecedented global health crisis. Here, we report the identification of a synthetic nanobody (sybody) pair, Sb#15 and Sb#68, that can bind simultaneously to the SARS-CoV-2 spike RBD and efficiently neutralize pseudotyped and live viruses by interfering with ACE2 interaction. Cryo-EM confirms that Sb#15 and Sb#68 engage two spatially discrete epitopes, influencing rational design of bispecific and tri-bispecific fusion constructs that exhibit up to 100- and 1,000-fold increase in neutralization potency, respectively. Cryo-EM of the sybody-spike complex additionally reveals a novel up-out RBD conformation. While resistant viruses emerge rapidly in the presence of single binders, no escape variants are observed in the presence of the bispecific sybody. The multivalent bispecific constructs further increase the neutralization potency against globally circulating SARS-CoV-2 variants of concern. Our study illustrates the power of multivalency and biparatopic nanobody fusions for the potential development of therapeutic strategies that mitigate the emergence of new SARS-CoV-2 escape mutants.

Rational design of ASCT2 inhibitors using an integrated experimental-computational approach.

ASCT2 (SLC1A5) is a sodium-dependent neutral amino acid transporter that controls amino acid homeostasis in peripheral tissues. In cancer, ASCT2 is up-regulated where it modulates intracellular glutamine levels, fueling cell proliferation. Nutrient deprivation via ASCT2 inhibition provides a potential strategy for cancer therapy. Here, we rationally designed stereospecific inhibitors exploiting specific subpockets in the substrate binding site using computational modeling and cryo-electron microscopy (cryo-EM). The final structures combined with molecular dynamics simulations reveal multiple pharmacologically relevant conformations in the ASCT2 binding site as well as a previously unknown mechanism of stereospecific inhibition. Furthermore, this integrated analysis guided the design of a series of unique ASCT2 inhibitors. Our results provide a framework for future development of cancer therapeutics targeting nutrient transport via ASCT2, as well as demonstrate the utility of combining computational modeling and cryo-EM for solute carrier ligand discovery.

Insights into the bilayer-mediated toppling mechanism of a folate-specific ECF transporter by cryo-EM.

Energy-coupling factor (ECF)-type transporters are small, asymmetric membrane protein complexes (∼115 kDa) that consist of a membrane-embedded, substrate-binding protein (S component) and a tripartite ATP-hydrolyzing module (ECF module). They import micronutrients into bacterial cells and have been proposed to use a highly unusual transport mechanism, in which the substrate is dragged across the membrane by a toppling motion of the S component. However, it remains unclear how the lipid bilayer could accommodate such a movement. Here, we used cryogenic electron microscopy at 200 kV to determine structures of a folate-specific ECF transporter in lipid nanodiscs and detergent micelles at 2.7- and 3.4-Šresolution, respectively. The structures reveal an irregularly shaped bilayer environment around the membrane-embedded complex and suggest that toppling of the S component is facilitated by protein-induced membrane deformations. In this way, structural remodeling of the lipid bilayer environment is exploited to guide the transport process.

Deciphering ion transport and ATPase coupling in the intersubunit tunnel of KdpFABC.

KdpFABC, a high-affinity K+ pump, combines the ion channel KdpA and the P-type ATPase KdpB to secure survival at K+ limitation. Here, we apply a combination of cryo-EM, biochemical assays, and MD simulations to illuminate the mechanisms underlying transport and the coupling to ATP hydrolysis. We show that ions are transported via an intersubunit tunnel through KdpA and KdpB. At the subunit interface, the tunnel is constricted by a phenylalanine, which, by polarized cation-π stacking, controls K+ entry into the canonical substrate binding site (CBS) of KdpB. Within the CBS, ATPase coupling is mediated by the charge distribution between an aspartate and a lysine. Interestingly, individual elements of the ion translocation mechanism of KdpFABC identified here are conserved among a wide variety of P-type ATPases from different families. This leads us to the hypothesis that KdpB might represent an early descendant of a common ancestor of cation pumps.

Optimized cryo-EM data-acquisition workflow by sample-thickness determination.

Sample thickness is a known key parameter in cryo-electron microscopy (cryo-EM) and can affect the amount of high-resolution information retained in the image. Yet, common data-acquisition approaches in single-particle cryo-EM do not take it into account. Here, it is demonstrated how the sample thickness can be determined before data acquisition, allowing the identification of optimal regions and the restriction of automated data collection to images with preserved high-resolution details. This quality-over-quantity approach almost entirely eliminates the time- and storage-consuming collection of suboptimal images, which are discarded after a recorded session or during early image processing due to a lack of high-resolution information. It maximizes the data-collection efficiency and lowers the electron-microscopy time required per data set. This strategy is especially useful if the speed of data collection is restricted by the microscope hardware and software, or if microscope access time, data transfer, data storage and computational power are a bottleneck.

The Groovy TMEM16 Family: Molecular Mechanisms of Lipid Scrambling and Ion Conduction.

The TMEM16 family of membrane proteins displays a remarkable functional dichotomy - while some family members function as Ca2+-activated anion channels, the majority of characterized TMEM16 homologs are Ca2+-activated lipid scramblases, which catalyze the exchange of phospholipids between the two membrane leaflets. Furthermore, some TMEM16 scramblases can also function as channels. Due to their involvement in important physiological processes, the family has been actively studied ever since their molecular identity was unraveled. In this review, we will summarize the recent advances in the field and how they influenced our view of TMEM16 family function and evolution. Structural, functional and computational studies reveal how relatively small rearrangements in the permeation pathway are responsible for the observed functional duality: while TMEM16 scramblases can adopt both ion- and lipid conductive conformations, TMEM16 channels can only populate the former. Recent data further provides the molecular details of a stepwise activation mechanism, which is initiated by Ca2+ binding and modulated by various cellular factors, including lipids. TMEM16 function and the surrounding membrane properties are inextricably intertwined, with the protein inducing bilayer deformations associated with scrambling, while the surrounding lipids modulate TMEM16 conformation and activity.

Gating the pore of the calcium-activated chloride channel TMEM16A.

The binding of cytoplasmic Ca2+ to the anion-selective channel TMEM16A triggers a conformational change around its binding site that is coupled to the release of a gate at the constricted neck of an hourglass-shaped pore. By combining mutagenesis, electrophysiology, and cryo-electron microscopy, we identified three hydrophobic residues at the intracellular entrance of the neck as constituents of this gate. Mutation of each of these residues increases the potency of Ca2+ and results in pronounced basal activity. The structure of an activating mutant shows a conformational change of an α-helix that contributes to Ca2+ binding as a likely cause for the basal activity. Although not in physical contact, the three residues are functionally coupled to collectively contribute to the stabilization of the gate in the closed conformation of the pore, thus explaining the low open probability of the channel in the absence of Ca2+.

Gating by ionic strength and safety check by cyclic-di-AMP in the ABC transporter OpuA.

(Micro)organisms are exposed to fluctuating environmental conditions, and adaptation to stress is essential for survival. Increased osmolality (hypertonicity) causes outflow of water and loss of turgor and is dangerous if the cell is not capable of rapidly restoring its volume. The osmoregulatory adenosine triphosphate-binding cassette transporter OpuA restores the cell volume by accumulating large amounts of compatible solute. OpuA is gated by ionic strength and inhibited by the second messenger cyclic-di-AMP, a molecule recently shown to affect many cellular processes. Despite the master regulatory role of cyclic-di-AMP, structural and functional insights into how the second messenger regulates (transport) proteins on the molecular level are lacking. Here, we present high-resolution cryo-electron microscopy structures of OpuA and in vitro activity assays that show how the osmoregulator OpuA is activated by high ionic strength and how cyclic-di-AMP acts as a backstop to prevent unbridled uptake of compatible solutes.

Ecological traits, genetic diversity and regional distribution of the macroalga Treptacantha elegans along the Catalan coast (NW Mediterranean Sea).

The widespread decline of canopy-forming macroalgal assemblages has been documented in many regions during the last decades. This pattern is often followed by the replacement of structurally complex algal canopies by more simplified habitats (e.g., turfs or sea urchin barren grounds). Against all odds, the fucoid Treptacantha elegans, a large Mediterranean brown macroalga, broadened its depth range to deeper and exposed environments and displayed an unexpected range expansion along the northern coast of Catalonia over the last two decades. Here, we reconstruct the spread of T. elegans in time and space and unravel ecological and demographic traits such as population dynamics and genetic patterns to provide a comprehensive and integrated view of the current status and geographical expansion for this species. Fast-growing dynamics, early fertile maturity, and high turnover rate are the main competitive advantages that allow the exposed populations of T. elegans to colonize available substrata and maintain dense and patchy populations. We also provided evidence that the deeper and exposed populations of T. elegans constitute a single group across the Catalan coast, with little genetic differentiation among populations. This seems to support the hypothesis of a unique source of spread in the last decades from the Medes Islands No-Take Zone towards both southern and northern waters.

Single-Particle Cryo-EM of Membrane Proteins in Lipid Nanodiscs.

Single-particle cryo-electron microscopy has become an indispensable technique in structural biology. In particular when studying membrane proteins, it allows the use of membrane-mimicking tools, which can be crucial for a comprehensive understanding of the structure-function relationship of the protein in its native environment. In this chapter we focus on the application of nanodiscs and use our recent studies on the TMEM16 family as an example.

A one-gate elevator mechanism for the human neutral amino acid transporter ASCT2.

The human Alanine Serine Cysteine Transporter 2 (ASCT2) is a neutral amino acid exchanger that belongs to the solute carrier family 1 (SLC1A). SLC1A structures have revealed an elevator-type mechanism, in which the substrate is translocated across the cell membrane by a large displacement of the transport domain, whereas a small movement of hairpin 2 (HP2) gates the extracellular access to the substrate-binding site. However, it has remained unclear how substrate binding and release is gated on the cytoplasmic side. Here, we present an inward-open structure of the human ASCT2, revealing a hitherto elusive SLC1A conformation. Strikingly, the same structural element (HP2) serves as a gate in the inward-facing as in the outward-facing state. The structures reveal that SLC1A transporters work as one-gate elevators. Unassigned densities near the gate and surrounding the scaffold domain, may represent potential allosteric binding sites, which could guide the design of lipidic-inhibitors for anticancer therapy.

Stepwise activation mechanism of the scramblase nhTMEM16 revealed by cryo-EM.

Scramblases catalyze the movement of lipids between both leaflets of a bilayer. Whereas the X-ray structure of the protein nhTMEM16 has previously revealed the architecture of a Ca2+-dependent lipid scramblase, its regulation mechanism has remained elusive. Here, we have used cryo-electron microscopy and functional assays to address this question. Ca2+-bound and Ca2+-free conformations of nhTMEM16 in detergent and lipid nanodiscs illustrate the interactions with its environment and they reveal the conformational changes underlying its activation. In this process, Ca2+ binding induces a stepwise transition of the catalytic subunit cavity, converting a closed cavity that is shielded from the membrane in the absence of ligand, into a polar furrow that becomes accessible to lipid headgroups in the Ca2+-bound state. Additionally, our structures demonstrate how nhTMEM16 distorts the membrane at both entrances of the subunit cavity, thereby decreasing the energy barrier for lipid movement.