Chemiosmotic nutrient transport in synthetic cells powered by electrogenic antiport coupled to decarboxylation.

Cellular homeostasis depends on the supply of metabolic energy in the form of ATP and electrochemical ion gradients. The construction of synthetic cells requires a constant supply of energy to drive membrane transport and metabolism. Here, we provide synthetic cells with long-lasting metabolic energy in the form of an electrochemical proton gradient. Leveraging the L-malate decarboxylation pathway we generate a stable proton gradient and electrical potential in lipid vesicles by electrogenic L-malate/L-lactate exchange coupled to L-malate decarboxylation. By co-reconstitution with the transporters GltP and LacY, the synthetic cells maintain accumulation of L-glutamate and lactose over periods of hours, mimicking nutrient feeding in living cells. We couple the accumulation of lactose to a metabolic network for the generation of intermediates of the glycolytic and pentose phosphate pathways. This study underscores the potential of harnessing a proton motive force via a simple metabolic network, paving the way for the development of more complex synthetic systems.

Structural basis of the obligatory exchange mode of human neutral amino acid transporter ASCT2.

ASCT2 is an obligate exchanger of neutral amino acids, contributing to cellular amino acid homeostasis. ASCT2 belongs to the same family (SLC1) as Excitatory Amino Acid Transporters (EAATs) that concentrate glutamate in the cytosol. The mechanism that makes ASCT2 an exchanger rather than a concentrator remains enigmatic. Here, we employ cryo-electron microscopy and molecular dynamics simulations to elucidate the structural basis of the exchange mechanism of ASCT2. We establish that ASCT2 binds three Na+ ions per transported substrate and visits a state that likely acts as checkpoint in preventing Na+ ion leakage, both features shared with EAATs. However, in contrast to EAATs, ASCT2 retains one Na+ ion even under Na+-depleted conditions. We demonstrate that ASCT2 cannot undergo the structural transition in TM7 that is essential for the concentrative transport cycle of EAATs. This structural rigidity and the high-affinity Na+ binding site effectively confine ASCT2 to an exchange mode.

Fitness landscape of substrate-adaptive mutations in evolved amino acid-polyamine-organocation transporters.

The emergence of new protein functions is crucial for the evolution of organisms. This process has been extensively researched for soluble enzymes, but it is largely unexplored for membrane transporters, even though the ability to acquire new nutrients from a changing environment requires evolvability of transport functions. Here, we demonstrate the importance of environmental pressure in obtaining a new activity or altering a promiscuous activity in members of the amino acid-polyamine-organocation (APC)-type yeast amino acid transporters family. We identify APC members that have broader substrate spectra than previously described. Using in vivo experimental evolution, we evolve two of these transporter genes, AGP1 and PUT4, toward new substrate specificities. Single mutations on these transporters are found to be sufficient for expanding the substrate range of the proteins, while retaining the capacity to transport all original substrates. Nonetheless, each adaptive mutation comes with a distinct effect on the fitness for each of the original substrates, illustrating a trade-off between the ancestral and evolved functions. Collectively, our findings reveal how substrate-adaptive mutations in membrane transporters contribute to fitness and provide insights into how organisms can use transporter evolution to explore new ecological niches.

Cobalamin decyanation by the membrane transporter BtuM.

BtuM is a bacterial cobalamin transporter that binds the transported substrate in the base-off state, with a cysteine residue providing the α-axial coordination of the central cobalt ion via a sulfur-cobalt bond. Binding leads to decyanation of cobalamin variants with a cyano group as the ß-axial ligand. Here, we report the crystal structures of untagged BtuM bound to two variants of cobalamin, hydroxycobalamin and cyanocobalamin, and unveil the native residue responsible for the ß-axial coordination, His28. This coordination had previously been obscured by non-native histidines of His-tagged BtuM. A model in which BtuM initially binds cobinamide reversibly with low affinity (KD = 4.0 µM), followed by the formation of a covalent bond (rate constant of 0.163 s-1), fits the kinetics data of substrate binding and decyanation of the cobalamin precursor cobinamide by BtuM. The covalent binding mode suggests a mechanism not used by any other transport protein.

Bidirectional ATP-driven transport of cobalamin by the mycobacterial ABC transporter BacA.

BacA is a mycobacterial ATP-binding cassette (ABC) transporter involved in the translocation of water-soluble compounds across the lipid bilayer. Whole-cell-based assays have shown that BacA imports cobalamin as well as unrelated hydrophilic compounds such as the antibiotic bleomycin and the antimicrobial peptide Bac7 into the cytoplasm. Surprisingly, there are indications that BacA also mediates the export of different antibacterial compounds, which is difficult to reconcile with the notion that ABC transporters generally operate in a strictly unidirectional manner. Here we resolve this conundrum by developing a fluorescence-based transport assay to monitor the transport of cobalamin across liposomal membranes. We find that BacA transports cobalamin in both the import and export direction. This highly unusual bidirectionality suggests that BacA is mechanistically distinct from other ABC transporters and facilitates ATP-driven diffusion, a function that may be important for the evolvability of specific transporters, and may bring competitive advantages to microbial communities.

Hit optimization by dynamic combinatorial chemistry on Streptococcus pneumoniae energy-coupling factor transporter ECF-PanT.

Herein, we present the first application of target-directed dynamic combinatorial chemistry (tdDCC) to the whole complex of the highly dynamic transmembrane, energy-coupling factor (ECF) transporter ECF-PanT in Streptococcus pneumoniae. In addition, we successfully employed the tdDCC technique as a hit-identification and -optimization strategy that led to the identification of optimized ECF inhibitors with improved activity. We characterized the best compounds regarding cytotoxicity and performed computational modeling studies on the crystal structure of ECF-PanT to rationalize their binding mode. Notably, docking studies showed that the acylhydrazone linker is able to maintain the crucial interactions.

Identification of inhibitors targeting the energy-coupling factor (ECF) transporters.

The energy-coupling factor (ECF) transporters are a family of transmembrane proteins involved in the uptake of vitamins in a wide range of bacteria. Inhibition of the activity of these proteins could reduce the viability of pathogens that depend on vitamin uptake. The central role of vitamin transport in the metabolism of bacteria and absence from humans make the ECF transporters an attractive target for inhibition with selective chemical probes. Here, we report on the identification of a promising class of inhibitors of the ECF transporters. We used coarse-grained molecular dynamics simulations on Lactobacillus delbrueckii ECF-FolT2 and ECF-PanT to profile the binding mode and mechanism of inhibition of this novel chemotype. The results corroborate the postulated mechanism of transport and pave the way for further drug-discovery efforts.

The linkage-type and the exchange molecule affect the protein-labeling efficiency of iminoboronate probes.

Reversible bioorthogonal conjugation reactions have been exploited in the chemoproteomic field to prepare protein labeling reagents and to visualize labeled proteins. We recently demonstrated that reversible iminoboronates can be used to prepare probes from fragment libraries and that the linkage subsequently can be used to detect the labeled proteins. In this study, we determined the effect of the stability of the iminoboronate linkage on the efficiency of the labeling protocol. Our study reveals that the linkage should be stable enough to allow for efficient targeting, but should be labile enough to detect the labeled protein. Acyl hydrazides were identified as the most suitable handles for the probe synthesis step. Anthranilic hydrazides and N-hydroxy semicarbazides were found to be the most efficient read-out molecules. With these novel exchange molecules, native probe-labeled proteins could be visualized under physiological conditions.

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.

Minimal Out-of-Equilibrium Metabolism for Synthetic Cells: A Membrane Perspective.

Life-like systems need to maintain a basal metabolism, which includes importing a variety of building blocks required for macromolecule synthesis, exporting dead-end products, and recycling cofactors and metabolic intermediates, while maintaining steady internal physical and chemical conditions (physicochemical homeostasis). A compartment, such as a unilamellar vesicle, functionalized with membrane-embedded transport proteins and metabolic enzymes encapsulated in the lumen meets these requirements. Here, we identify four modules designed for a minimal metabolism in a synthetic cell with a lipid bilayer boundary: energy provision and conversion, physicochemical homeostasis, metabolite transport, and membrane expansion. We review design strategies that can be used to fulfill these functions with a focus on the lipid and membrane protein composition of a cell. We compare our bottom-up design with the equivalent essential modules of JCVI-syn3a, a top-down genome-minimized living cell with a size comparable to that of large unilamellar vesicles. Finally, we discuss the bottlenecks related to the insertion of a complex mixture of membrane proteins into lipid bilayers and provide a semiquantitative estimate of the relative surface area and lipid-to-protein mass ratios (i.e., the minimal number of membrane proteins) that are required for the construction of a synthetic cell.

Mutation in glutamate transporter homologue GltTk provides insights into pathologic mechanism of episodic ataxia 6.

Episodic ataxias (EAs) are rare neurological conditions affecting the nervous system and typically leading to motor impairment. EA6 is linked to the mutation of a highly conserved proline into an arginine in the glutamate transporter EAAT1. In vitro studies showed that this mutation leads to a reduction in the substrates transport and an increase in the anion conductance. It was hypothesised that the structural basis of these opposed functional effects might be the straightening of transmembrane helix 5, which is kinked in the wild-type protein. In this study, we present the functional and structural implications of the mutation P208R in the archaeal homologue of glutamate transporters GltTk. We show that also in GltTk the P208R mutation leads to reduced aspartate transport activity and increased anion conductance, however a cryo-EM structure reveals that the kink is preserved. The arginine side chain of the mutant points towards the lipidic environment, where it may engage in interactions with the phospholipids, thereby potentially interfering with the transport cycle and contributing to stabilisation of an anion conducting state.

Membrane transport of cobalamin.

A wide variety of organisms encode cobalamin-dependent enzymes catalyzing essential metabolic reactions, but the cofactor cobalamin (vitamin B12) is only synthesized by a subset of bacteria and archaea. The biosynthesis of cobalamin is complex and energetically costly, making cobalamin variants and precursors metabolically valuable. Auxotrophs for these molecules have evolved uptake mechanisms to compensate for the lack of a synthesis pathway. Bacterial transport of cobalamin involves the passage over one or two lipidic membranes in Gram-positive and -negative bacteria, respectively. In higher eukaryotes, a complex system of carriers, receptors and transporters facilitates the delivery of the essential molecule to the tissues. Biochemical and genetic approaches have identified different transporter families involved in cobalamin transport. The majority of the characterized cobalamin transporters are active transport systems that belong to the ATP-binding cassette (ABC) superfamily of transporters. In this chapter, we describe the different cobalamin transport systems characterized to date that are present in bacteria and humans, as well as yet-to-be-identified transporters.

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.

High-speed atomic force microscopy reveals a three-state elevator mechanism in the citrate transporter CitS.

The secondary active transporter CitS shuttles citrate across the cytoplasmic membrane of gram-negative bacteria by coupling substrate translocation to the transport of two Na+ ions. Static crystal structures suggest an elevator type of transport mechanism with two states: up and down. However, no dynamic measurements have been performed to substantiate this assumption. Here, we use high-speed atomic force microscopy for real-time visualization of the transport cycle at the level of single transporters. Unexpectedly, instead of a bimodal height distribution for the up and down states, the experiments reveal movements between three distinguishable states, with protrusions of ∼0.5 nm, ∼1.0 nm, and ∼1.6 nm above the membrane, respectively. Furthermore, the real-time measurements show that the individual protomers of the CitS dimer move up and down independently. A three-state elevator model of independently operating protomers resembles the mechanism proposed for the aspartate transporter GltPh Since CitS and GltPh are structurally unrelated, we conclude that the three-state elevators have evolved independently.

On the Role of a Conserved Methionine in the Na+-Coupling Mechanism of a Neurotransmitter Transporter Homolog.

Excitatory amino acid transporters (EAAT) play a key role in glutamatergic synaptic communication. Driven by transmembrane cation gradients, these transporters catalyze the reuptake of glutamate from the synaptic cleft once this neurotransmitter has been utilized for signaling. Two decades ago, pioneering studies in the Kanner lab identified a conserved methionine within the transmembrane domain as key for substrate turnover rate and specificity; later structural work, particularly for the prokaryotic homologs GltPh and GltTk, revealed that this methionine is involved in the coordination of one of the three Na+ ions that are co-transported with the substrate. Albeit extremely atypical, the existence of this interaction is consistent with biophysical analyses of GltPh showing that mutations of this methionine diminish the binding cooperativity between substrates and Na+. It has been unclear, however, whether this intriguing methionine influences the thermodynamics of the transport reaction, i.e., its substrate:ion stoichiometry, or whether it simply fosters a specific kinetics in the binding reaction, which, while influential for the turnover rate, do not fundamentally explain the ion-coupling mechanism of this class of transporters. Here, studies of GltTk using experimental and computational methods independently arrive at the conclusion that the latter hypothesis is the most plausible, and lay the groundwork for future efforts to uncover the underlying mechanism.

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.

Kinetic mechanism of Na+-coupled aspartate transport catalyzed by GltTk.

It is well-established that the secondary active transporters GltTk and GltPh catalyze coupled uptake of aspartate and three sodium ions, but insight in the kinetic mechanism of transport is fragmentary. Here, we systematically measured aspartate uptake rates in proteoliposomes containing purified GltTk, and derived the rate equation for a mechanism in which two sodium ions bind before and another after aspartate. Re-analysis of existing data on GltPh using this equation allowed for determination of the turnover number (0.14 s-1), without the need for error-prone protein quantification. To overcome the complication that purified transporters may adopt right-side-out or inside-out membrane orientations upon reconstitution, thereby confounding the kinetic analysis, we employed a rapid method using synthetic nanobodies to inactivate one population. Oppositely oriented GltTk proteins showed the same transport kinetics, consistent with the use of an identical gating element on both sides of the membrane. Our work underlines the value of bona fide transport experiments to reveal mechanistic features of Na+-aspartate symport that cannot be observed in detergent solution. Combined with previous pre-equilibrium binding studies, a full kinetic mechanism of structurally characterized aspartate transporters of the SLC1A family is now emerging.