RESUMEN
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 â¼0.5 nm, â¼1.0 nm, and â¼1.6 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.
Asunto(s)
Membrana Celular , Proteínas de Escherichia coli , Escherichia coli , Microscopía de Fuerza Atómica , Simportadores , Membrana Celular/genética , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/ultraestructura , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/ultraestructura , Simportadores/genética , Simportadores/metabolismo , Simportadores/ultraestructuraRESUMEN
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.
Asunto(s)
Tratamiento Farmacológico de COVID-19 , Anticuerpos de Dominio Único , Anticuerpos Neutralizantes , Anticuerpos Antivirales/metabolismo , Resistencia a Medicamentos , Humanos , Pandemias , Unión Proteica , SARS-CoV-2/genética , Anticuerpos de Dominio Único/genética , Anticuerpos de Dominio Único/metabolismo , Anticuerpos de Dominio Único/farmacología , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
Energy-coupling factor (ECF)-type transporters are small, asymmetric membrane protein complexes (â¼115 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-Å resolution, 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.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Microscopía por Crioelectrón/métodos , Ácido Fólico/metabolismo , Membrana Dobles de Lípidos/metabolismo , Microdominios de Membrana/metabolismo , Transportadoras de Casetes de Unión a ATP/química , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/química , Transporte Biológico , Cristalografía por Rayos X , Lactobacillus delbrueckii/metabolismo , Modelos Moleculares , Unión Proteica , Conformación ProteicaRESUMEN
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.
Asunto(s)
Sistema de Transporte de Aminoácidos ASC/antagonistas & inhibidores , Unión Competitiva , Química Computacional , Microscopía por Crioelectrón/métodos , Glutamina/metabolismo , Preparaciones Farmacéuticas/administración & dosificación , Sistema de Transporte de Aminoácidos ASC/metabolismo , Sitios de Unión , Diseño de Fármacos , Humanos , Antígenos de Histocompatibilidad Menor/metabolismo , Simulación del Acoplamiento Molecular , Preparaciones Farmacéuticas/química , Unión Proteica , Dominios Proteicos , Estructura Terciaria de Proteína , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Sondas Moleculares , Proteínas , Proteínas/química , Sondas Moleculares/químicaRESUMEN
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.
Asunto(s)
Metionina , Sodio , Transporte Biológico , Iones/metabolismo , Metionina/metabolismo , Proteínas de Transporte de Neurotransmisores/metabolismoRESUMEN
Photopharmacology addresses the challenge of drug selectivity and side effects through creation of photoresponsive molecules activated with light with high spatiotemporal precision. This is achieved through incorporation of molecular photoswitches and photocages into the pharmacophore. However, the structural basis for the light-induced modulation of inhibitory potency in general is still missing, which poses a major design challenge for this emerging field of research. Here we solved crystal structures of the glutamate transporter homologue GltTk in complex with photoresponsive transport inhibitors-azobenzene derivative of TBOA (both in trans and cis configuration) and with the photocaged compound ONB-hydroxyaspartate. The essential role of glutamate transporters in the functioning of the central nervous system renders them potential therapeutic targets in the treatment of neurodegenerative diseases. The obtained structures provide a clear structural insight into the origins of photocontrol in photopharmacology and lay the foundation for application of photocontrolled ligands to study the transporter dynamics by using time-resolved X-ray crystallography.
Asunto(s)
Sistema de Transporte de Aminoácidos X-AG/antagonistas & inhibidores , Sistema de Transporte de Aminoácidos X-AG/metabolismo , Ácido Aspártico/análogos & derivados , Ácido Aspártico/metabolismo , Compuestos Azo/metabolismo , Sistema de Transporte de Aminoácidos X-AG/química , Ácido Aspártico/efectos de la radiación , Compuestos Azo/química , Compuestos Azo/efectos de la radiación , Cristalografía por Rayos X , Unión Proteica , Estereoisomerismo , Thermococcus/química , Rayos UltravioletaRESUMEN
Chemical probes that covalently modify proteins of interest are powerful tools for the research of biological processes. Important in the design of a probe is the choice of reactive group that forms the covalent bond, as it decides the success of a probe. However, choosing the right reactive group is not a simple feat and methodologies for expedient screening of different groups are needed. We herein report a modular approach that allows easy coupling of a reactive group to a ligand. α-Nucleophile ligands are combined with 2-formylphenylboronic acid derived reactive groups to form iminoboronate probes that selectively label their target proteins. A transimination reaction on the labeled proteins with an α-amino hydrazide provides further modification, for example to introduce a fluorophore.
RESUMEN
Membrane transporters are integral membrane proteins that mediate the passage of solutes across lipid bilayers. These proteins undergo conformational transitions between outward- and inward-facing states, which lead to alternating access of the substrate-binding site to the aqueous environment on either side of the membrane. Dozens of different transporter families have evolved, providing a wide variety of structural solutions to achieve alternating access. A sub-set of structurally diverse transporters operate by mechanisms that are collectively named 'elevator-type'. These transporters have one common characteristic: they contain a distinct protein domain that slides across the membrane as a rigid body, and in doing so it 'drags" the transported substrate along. Analysis of the global conformational changes that take place in membrane transporters using elevator-type mechanisms reveals that elevator-type movements can be achieved in more than one way. Molecular dynamics simulations and experimental data help to understand how lipid bilayer properties may affect elevator movements and vice versa.
Asunto(s)
Membrana Dobles de Lípidos/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Simulación de Dinámica Molecular , Adenosina Difosfato/química , Adenosina Trifosfato/química , Sitio Alostérico , Ácidos y Sales Biliares/química , Transporte Biológico , Humanos , Cinética , Lípidos/química , Dominios Proteicos , Pliegue de Proteína , Receptores de Somatostatina/metabolismoRESUMEN
Energy coupling factor (ECF) transporters take up micronutrients in Bacteria and Archaea. They consist of a membrane-embedded S-component that provides substrate specificity and a three-subunit ECF module that couples ATP hydrolysis to transport. The S-components ThiT (for thiamin) and NiaX (for niacin) from Lactococcus lactis form complexes with the same ECF module. Here, we assayed the uptake of thiamin and niacin in Escherichia coli cells expressing the transporter genes. We demonstrate that the two different S-components compete for the ECF module, and that competition is more efficient in the presence of the transported substrate. The data suggest that binding and release of the S-components is a step in the transport cycle.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Lactococcus lactis/metabolismo , Niacina/metabolismo , Tiamina/metabolismo , Adenosina Trifosfato/genética , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/genética , Transporte Biológico Activo/fisiología , Proteínas Portadoras/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Hidrólisis , Lactococcus lactis/genética , Niacina/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tiamina/genéticaRESUMEN
Energy-coupling factor (ECF) transporters are membrane-protein complexes that mediate vitamin uptake in prokaryotes. They bind the substrate through the action of a specific integral membrane subunit (S-component) and power transport by hydrolysis of ATP in the three-subunit ECF module. Here, we have studied the binding of thiamine derivatives to ThiT, a thiamine-specific S-component. We designed and synthesized derivatives of thiamine that bind to ThiT with high affinity; this allowed us to evaluate the contribution of the functional groups to the binding affinity. We determined six crystal structures of ThiT in complex with our derivatives. The structure of the substrate-binding site in ThiT remains almost unchanged despite substantial differences in affinity. This work indicates that the structural organization of the binding site is robust and suggests that substrate release, which is required for transport, requires additional changes in conformation in ThiT that might be imposed by the ECF module.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Bacterianas/metabolismo , Diseño de Fármacos , Bibliotecas de Moléculas Pequeñas/metabolismo , Tiamina/metabolismo , Transportadoras de Casetes de Unión a ATP/química , Proteínas Bacterianas/química , Transporte Biológico , Lactococcus lactis/química , Modelos Moleculares , Estructura Molecular , Bibliotecas de Moléculas Pequeñas/química , Tiamina/síntesis química , Tiamina/químicaRESUMEN
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.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Vitamina B 12 , Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Membrana Dobles de Lípidos , Adenosina Trifosfato , Transporte BiológicoRESUMEN
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.
Asunto(s)
Sistemas de Transporte de Aminoácidos , Mutación , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sistemas de Transporte de Aminoácidos/genética , Sistemas de Transporte de Aminoácidos/metabolismo , Especificidad por Sustrato , Evolución Molecular , Poliaminas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Aptitud Genética , Aminoácidos/metabolismo , Aminoácidos/genéticaRESUMEN
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.
Asunto(s)
Proteínas Bacterianas , Streptococcus pneumoniae , Modelos Moleculares , Proteínas Bacterianas/químicaRESUMEN
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.
Asunto(s)
Sistema de Transporte de Aminoácidos ASC , Microscopía por Crioelectrón , Antígenos de Histocompatibilidad Menor , Simulación de Dinámica Molecular , Sodio , Humanos , Sistema de Transporte de Aminoácidos ASC/metabolismo , Sistema de Transporte de Aminoácidos ASC/química , Sistema de Transporte de Aminoácidos ASC/genética , Antígenos de Histocompatibilidad Menor/metabolismo , Antígenos de Histocompatibilidad Menor/química , Sodio/metabolismo , Sitios de Unión , Células HEK293 , Unión ProteicaRESUMEN
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.
Asunto(s)
Proteínas Bacterianas , Modelos Moleculares , Unión Proteica , Vitamina B 12 , Vitamina B 12/metabolismo , Vitamina B 12/química , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Sitios de Unión , Cobalto/química , Cobalto/metabolismo , Cobamidas/metabolismo , Cobamidas/química , Cinética , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/metabolismo , Histidina/metabolismo , Histidina/químicaRESUMEN
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.
Asunto(s)
Células Artificiales , Células Artificiales/metabolismo , Membrana Dobles de Lípidos/metabolismo , Proteínas de la Membrana/genética , Liposomas Unilamelares/metabolismoRESUMEN
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.
Asunto(s)
Bacterias , Proteínas Bacterianas , Proteínas Bacterianas/metabolismo , Conformación Proteica , Bacterias/metabolismo , Transporte Biológico , Adenosina Trifosfato/metabolismoRESUMEN
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.