RESUMEN
Polar auxin transport is unique to plants and coordinates their growth and development1,2. The PIN-FORMED (PIN) auxin transporters exhibit highly asymmetrical localizations at the plasma membrane and drive polar auxin transport3,4; however, their structures and transport mechanisms remain largely unknown. Here, we report three inward-facing conformation structures of Arabidopsis thaliana PIN1: the apo state, bound to the natural auxin indole-3-acetic acid (IAA), and in complex with the polar auxin transport inhibitor N-1-naphthylphthalamic acid (NPA). The transmembrane domain of PIN1 shares a conserved NhaA fold5. In the substrate-bound structure, IAA is coordinated by both hydrophobic stacking and hydrogen bonding. NPA competes with IAA for the same site at the intracellular pocket, but with a much higher affinity. These findings inform our understanding of the substrate recognition and transport mechanisms of PINs and set up a framework for future research on directional auxin transport, one of the most crucial processes underlying plant development.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Ácidos Indolacéticos , Proteínas de Transporte de Membrana , Apoproteínas/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Transporte Biológico , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Ácidos Indolacéticos/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Ftalimidas/metabolismo , Conformación Proteica , Especificidad por SustratoRESUMEN
Neutralizing antibodies (nAbs) are important assets to fight COVID-19, but most existing nAbs lose the activities against Omicron subvariants. Here, we report a human monoclonal antibody (Ab08) isolated from a convalescent patient infected with the prototype strain (Wuhan-Hu-1). Ab08 binds to the receptor-binding domain (RBD) with pico-molar affinity (230 pM), effectively neutralizes SARS-CoV-2 and variants of concern (VOCs) including Alpha, Beta, Gamma, Mu, Omicron BA.1 and BA.2, and to a lesser extent for Delta and Omicron BA.4/BA.5 which bear the L452R mutation. Of medical importance, Ab08 shows therapeutic efficacy in SARS-CoV-2-infected hACE2 mice. X-ray crystallography of the Ab08-RBD complex reveals an antibody footprint largely in the ß-strand core and away from the ACE2-binding motif. Negative staining electron-microscopy suggests a neutralizing mechanism through which Ab08 destructs the Spike trimer. Together, our work identifies a nAb with therapeutic potential for COVID-19.
Asunto(s)
Anticuerpos Monoclonales , COVID-19 , SARS-CoV-2 , Animales , Humanos , Ratones , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Neutralizantes/uso terapéutico , Anticuerpos Antivirales/uso terapéutico , Glicoproteína de la Espiga del Coronavirus/genéticaRESUMEN
The COVID pandemic fueled by emerging SARS-CoV-2 new variants of concern remains a major global health concern, and the constantly emerging mutations present challenges to current therapeutics. The spike glycoprotein is not only essential for the initial viral entry, but is also responsible for the transmission of SARS-CoV-2 components via syncytia formation. Spike-mediated cell-cell transmission is strongly resistant to extracellular therapeutic and convalescent antibodies via an unknown mechanism. Here, we describe the antibody-mediated spike activation and syncytia formation on cells displaying the viral spike. We found that soluble antibodies against receptor binding motif (RBM) are capable of inducing the proteolytic processing of spike at both the S1/S2 and S2' cleavage sites, hence triggering ACE2-independent cell-cell fusion. Mechanistically, antibody-induced cell-cell fusion requires the shedding of S1 and exposure of the fusion peptide at the cell surface. By inhibiting S1/S2 proteolysis, we demonstrated that cell-cell fusion mediated by spike can be re-sensitized towards antibody neutralization in vitro. Lastly, we showed that cytopathic effect mediated by authentic SARS-CoV-2 infection remain unaffected by the addition of extracellular neutralization antibodies. Hence, these results unveil a novel mode of antibody evasion and provide insights for antibody selection and drug design strategies targeting the SARS-CoV-2 infected cells.
Asunto(s)
COVID-19 , SARS-CoV-2 , Humanos , Anticuerpos , Membrana Celular , Glicoproteína de la Espiga del Coronavirus/genéticaRESUMEN
Glycosylphosphatidylinositol (GPI) anchoring of proteins is a ubiquitous posttranslational modification in eukaryotic cells. GPI-anchored proteins (GPI-APs) play critical roles in enzymatic, signaling, regulatory, and adhesion processes. Over 20 enzymes are involved in GPI synthesis, attachment to client proteins, and remodeling after attachment. The GPI transamidase (GPI-T), a large complex located in the endoplasmic reticulum membrane, catalyzes the attachment step by replacing a C-terminal signal peptide of proproteins with GPI. In the last three decades, extensive research has been conducted on the mechanism of the transamidation reaction, the components of the GPI-T complex, the role of each subunit, and the substrate specificity. Two recent studies have reported the three-dimensional architecture of GPI-T, which represent the first structures of the pathway. The structures provide detailed mechanisms for assembly that rationalizes previous biochemical results and subunit-dependent stability data. While the structural data confirm the catalytic role of PIGK, which likely uses a caspase-like mechanism to cleave the proproteins, they suggest that unlike previously proposed, GPAA1 is not a catalytic subunit. The structures also reveal a shared cavity for GPI binding. Somewhat unexpectedly, PIGT, a single-pass membrane protein, plays a crucial role in GPI recognition. Consistent with the assembly mechanisms and the active site architecture, most of the disease mutations occur near the active site or the subunit interfaces. Finally, the catalytic dyad is located ~22 Å away from the membrane interface of the GPI-binding site, and this architecture may confer substrate specificity through topological matching between the substrates and the elongated active site. The research conducted thus far sheds light on the intricate processes involved in GPI anchoring and paves the way for further mechanistic studies of GPI-T.
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Glicosilfosfatidilinositoles , Humanos , Glicosilfosfatidilinositoles/metabolismo , Glicosilfosfatidilinositoles/química , Animales , Especificidad por Sustrato , Aminoaciltransferasas/metabolismo , Aminoaciltransferasas/química , Aminoaciltransferasas/genética , Retículo Endoplásmico/metabolismo , Relación Estructura-Actividad , AciltransferasasRESUMEN
A key step to the SARS-CoV-2 infection is the attachment of its Spike receptor-binding domain (S RBD) to the host receptor ACE2. Considerable research has been devoted to the development of neutralizing antibodies, including llama-derived single-chain nanobodies, to target the receptor-binding motif (RBM) and to block ACE2-RBD binding. Simple and effective strategies to increase potency are desirable for such studies when antibodies are only modestly effective. Here, we identify and characterize a high-affinity synthetic nanobody (sybody, SR31) as a fusion partner to improve the potency of RBM-antibodies. Crystallographic studies reveal that SR31 binds to RBD at a conserved and 'greasy' site distal to RBM. Although SR31 distorts RBD at the interface, it does not perturb the RBM conformation, hence displaying no neutralizing activities itself. However, fusing SR31 to two modestly neutralizing sybodies dramatically increases their affinity for RBD and neutralization activity against SARS-CoV-2 pseudovirus. Our work presents a tool protein and an efficient strategy to improve nanobody potency.
Asunto(s)
Enzima Convertidora de Angiotensina 2/inmunología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , SARS-CoV-2/inmunología , Anticuerpos de Dominio Único/inmunología , Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/genética , Anticuerpos Antivirales/química , Anticuerpos Antivirales/genética , Afinidad de Anticuerpos , Sitios de Unión , Cristalografía por Rayos X , Células HEK293 , Humanos , Modelos Moleculares , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/genéticaRESUMEN
G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a â¼20° rotation between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology.
Asunto(s)
Arrestina/química , Arrestina/metabolismo , Rodopsina/química , Rodopsina/metabolismo , Animales , Sitios de Unión , Cristalografía por Rayos X , Disulfuros/química , Disulfuros/metabolismo , Humanos , Rayos Láser , Ratones , Modelos Moleculares , Complejos Multiproteicos/biosíntesis , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Unión Proteica , Reproducibilidad de los Resultados , Transducción de Señal , Rayos XRESUMEN
Recombinant expression of human membrane proteins in large quantities remains a major challenge. Expression host is an important variable to screen for high-level production of membrane proteins. Using the green fluorescent protein (GFP) as a reporter, we screened the expression of a human multi-pass membrane protein called sterol Δ8-Δ7 isomerase in three different hosts: Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris. The expression of the His-tagged isomerase was exceptionally high in P. pastoris, reaching ~200â¯mgâ¯L-1 in standard flasks, and ~1,000â¯mgâ¯L-1 in condensed culture that mimics fermentation. The heterogeneously expressed isomerase could be extracted fully with dodecyl maltoside, and the solubilized protein in the form of GFP fusion showed a sharp and symmetric peak on fluorescence-detection size exclusion chromatography. Our work provides a useful source for the purification of the recombinant isomerase.
Asunto(s)
Pichia/genética , Esteroide Isomerasas/química , Esteroide Isomerasas/genética , Cromatografía en Gel , Expresión Génica , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , SolubilidadRESUMEN
Diacylglycerol kinase catalyses the ATP-dependent phosphorylation of diacylglycerol to phosphatidic acid for use in shuttling water-soluble components to membrane-derived oligosaccharide and lipopolysaccharide in the cell envelope of Gram-negative bacteria. For half a century, this 121-residue kinase has served as a model for investigating membrane protein enzymology, folding, assembly and stability. Here we present crystal structures for three functional forms of this unique and paradigmatic kinase, one of which is wild type. These reveal a homo-trimeric enzyme with three transmembrane helices and an amino-terminal amphiphilic helix per monomer. Bound lipid substrate and docked ATP identify the putative active site that is of the composite, shared site type. The crystal structures rationalize extensive biochemical and biophysical data on the enzyme. They are, however, at variance with a published solution NMR model in that domain swapping, a key feature of the solution form, is not observed in the crystal structures.
Asunto(s)
Proteínas Bacterianas/química , Membrana Celular/metabolismo , Diacilglicerol Quinasa/química , Diacilglicerol Quinasa/metabolismo , Proteínas de la Membrana/química , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Diacilglicerol Quinasa/genética , Activación Enzimática/efectos de los fármacos , Estabilidad de Enzimas , Lípidos , Magnesio/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Zinc/farmacologíaRESUMEN
We describe a data collection method that uses a single crystal to solve X-ray structures by native SAD (single-wavelength anomalous diffraction). We solved the structures of 11 real-life examples, including a human membrane protein, a protein-DNA complex and a 266-kDa multiprotein-ligand complex, using this method. The data collection strategy is suitable for routine structure determination and can be implemented at most macromolecular crystallography synchrotron beamlines.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de la Membrana/química , Complejos Multiproteicos/química , Difracción de Rayos X/métodos , Animales , Humanos , Modelos Moleculares , Conformación Proteica , Programas Informáticos , SincrotronesAsunto(s)
COVID-19 , SARS-CoV-2 , Humanos , Infección Irruptiva , Inmunoglobulinas , Vacunas contra la COVID-19 , Vacunación , Anticuerpos AntiviralesRESUMEN
We developed a method that allows release of intact membrane protein complexes from amphipols, bicelles and nanodiscs in the gas phase for observation by mass spectrometry (MS). Current methods involve release of membrane protein complexes from detergent micelles, which reveals subunit composition and lipid binding. We demonstrated that oligomeric complexes or proteins requiring defined lipid environments are stabilized to a greater extent in the absence of detergent.
Asunto(s)
Detergentes/química , Lípidos/química , Espectrometría de Masas/métodos , Proteínas de la Membrana/química , Micelas , Diacilglicerol Quinasa/química , Difusión , Escherichia coli/química , Escherichia coli/enzimología , Proteínas de Escherichia coli/química , Proteínas Fluorescentes Verdes/química , Halobacteriaceae/química , Espectroscopía de Resonancia Magnética/métodos , Microscopía Electrónica de Transmisión/métodos , Proteínas de Transporte de Monosacáridos/química , Nanopartículas/química , Plásmidos/metabolismo , Rodopsinas Sensoriales/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Simportadores/químicaRESUMEN
An enigma in the field of peptide transport is the structural basis for ligand promiscuity, as exemplified by PepT1, the mammalian plasma membrane peptide transporter. Here, we present crystal structures of di- and tripeptide-bound complexes of a bacterial homologue of PepT1, which reveal at least two mechanisms for peptide recognition that operate within a single, centrally located binding site. The dipeptide was orientated laterally in the binding site, whereas the tripeptide revealed an alternative vertical binding mode. The co-crystal structures combined with functional studies reveal that biochemically distinct peptide-binding sites likely operate within the POT/PTR family of proton-coupled symporters and suggest that transport promiscuity has arisen in part through the ability of the binding site to accommodate peptides in multiple orientations for transport.
Asunto(s)
Proteínas Bacterianas/química , Streptococcus thermophilus , Simportadores/química , Sitios de Unión , Cristalografía por Rayos X , Dipéptidos/química , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Oligopéptidos/química , Estructura Secundaria de Proteína , Especificidad por SustratoRESUMEN
Despite the marked increase in the number of membrane-protein structures solved using crystals grown by the lipid cubic phase or in meso method, only ten have been determined by SAD/MAD. This is likely to be a consequence of the technical difficulties associated with handling proteins and crystals in the sticky and viscous hosting mesophase that is usually incubated in glass sandwich plates for the purposes of crystallization. Here, a four-year campaign aimed at phasing the in meso structure of the integral membrane diacylglycerol kinase (DgkA) from Escherichia coli is reported. Heavy-atom labelling of this small hydrophobic enzyme was attempted by pre-labelling, co-crystallization, soaking, site-specific mercury binding to genetically engineered single-cysteine mutants and selenomethionine incorporation. Strategies and techniques for special handling are reported, as well as the typical results and the lessons learned for each of these approaches. In addition, an assay to assess the accessibility of cysteine residues in membrane proteins for mercury labelling is introduced. The various techniques and strategies described will provide a valuable reference for future experimental phasing of membrane proteins where crystals are grown by the lipid cubic phase method.
Asunto(s)
Proteínas de la Membrana/química , Cristalización , Diacilglicerol Quinasa/química , Diacilglicerol Quinasa/genética , Escherichia coli/enzimología , Conformación ProteicaRESUMEN
Membrane proteins are key elements in cell physiology and drug targeting, but getting a high-resolution structure by crystallographic means is still enormously challenging. Novel strategies are in big demand to facilitate the structure determination process that will ultimately hasten the day when sequence information alone can provide a three-dimensional model. Cell-free or in vitro expression enables rapid access to large quantities of high-quality membrane proteins suitable for an array of applications. Despite its impressive efficiency, to date only two membrane proteins produced by the in vitro approach have yielded crystal structures. Here, we have analysed synergies of cell-free expression and crystallisation in lipid mesophases for generating an X-ray structure of the integral membrane enzyme diacylglycerol kinase to 2.28-Å resolution. The quality of cellular and cell-free-expressed kinase samples has been evaluated systematically by comparing (1) spectroscopic properties, (2) purity and oligomer formation, (3) lipid content and (4) functionality. DgkA is the first membrane enzyme crystallised based on cell-free expression. The study provides a basic standard for the crystallisation of cell-free-expressed membrane proteins and the methods detailed here should prove generally useful and contribute to accelerating the pace at which membrane protein structures are solved.
Asunto(s)
Membrana Celular/enzimología , Diacilglicerol Quinasa/química , Proteínas de la Membrana/química , Conformación Proteica , Sistema Libre de Células , Dicroismo Circular , Cristalización , Cristalografía por Rayos X , Diacilglicerol Quinasa/genética , Diacilglicerol Quinasa/metabolismo , Electroforesis en Gel de Poliacrilamida , Pruebas de Enzimas , Regulación Enzimológica de la Expresión Génica , Lípidos/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Multimerización de ProteínaRESUMEN
The exopolysaccharide alginate is an important component of biofilms produced by Pseudomonas aeruginosa, a major pathogen that contributes to the demise of cystic fibrosis patients. Alginate exits the cell via the outer membrane porin AlgE. X-ray structures of several AlgE crystal forms are reported here. Whilst all share a common ß-barrel constitution, they differ in the degree to which loops L2 and T8 are ordered. L2 and T8 have been identified as an extracellular gate (E-gate) and a periplasmic gate (P-gate), respectively, that reside on either side of an alginate-selectivity pore located midway through AlgE. Passage of alginate across the membrane is proposed to be regulated by the sequential opening and closing of the two gates. In one crystal form, the selectivity pore contains a bound citrate. Because citrate mimics the uronate monomers of alginate, its location is taken to highlight a route through AlgE taken by alginate as it crosses the pore. Docking and molecular-dynamics simulations support and extend the proposed transport mechanism. Specifically, the P-gate and E-gate are flexible and move between open and closed states. Citrate can leave the selectivity pore bidirectionally. Alginate docks stably in a linear conformation through the open pore. To translate across the pore, a force is required that presumably is provided by the alginate-synthesis machinery. Accessing the open pore is facilitated by complex formation between AlgE and the periplasmic protein AlgK. Alginate can thread through a continuous pore in the complex, suggesting that AlgK pre-orients newly synthesized exopolysaccharide for delivery to AlgE.
Asunto(s)
Pseudomonas aeruginosa/metabolismo , Alginatos/química , Secuencia de Bases , Membrana Celular/metabolismo , Cristalografía por Rayos X , Cartilla de ADN , Ácido Glucurónico/química , Ácido Glucurónico/metabolismo , Ácidos Hexurónicos/química , Simulación de Dinámica Molecular , Conformación ProteicaRESUMEN
The lipidic cubic mesophase has been used to crystallize important membrane proteins for high-resolution structure determination. To date, however, no integral membrane enzymes have yielded to this method, the in meso. For a crystal structure to be meaningful the target protein must be functional. Using the in meso method with a membrane enzyme requires that the protein is active in the mesophase that grows crystals. Because the cubic phase is sticky and viscous and is bicontinuous topologically, quantitatively assessing enzyme activity in meso is a challenge. Here, we describe a procedure for characterizing the catalytic properties of the integral membrane enzyme, diacylglycerol kinase, reconstituted into the bilayer of the lipidic cubic phase. The kinase activity of this elusive crystallographic target was monitored spectrophotometrically using a coupled assay in a high-throughput, 96-well plate format. In meso, the enzyme exhibits classic Michaelis-Menten kinetics and works with a range of lipid substrates. The fact that the enzyme and its lipid substrate and product remain confined to the porous mesophase while its water-soluble substrate and product are free to partition into the aqueous bathing solution suggests a general and convenient approach for characterizing membrane enzymes that function with lipids in a membrane-like environment. The distinctive rheology of the cubic phase means that a procedural step to physically separate substrate from product is not needed. Because of its open, bicontinuous nature, the cubic phase offers the added benefit that the protein is accessible for assay from both sides of the membrane.
Asunto(s)
Enzimas/química , Lípidos/química , Proteínas de la Membrana/química , Modelos Biológicos , Catálisis , Cristalización , Cristalografía por Rayos X , Diacilglicerol Quinasa/química , Membrana Dobles de Lípidos , Membranas ArtificialesRESUMEN
Neutralizing antibodies (nAbs) play an important role against SARS-CoV-2 infections. Previously, we have reported one potent receptor binding domain (RBD)-binding nAb Ab08 against the SARS-CoV-2 prototype and a panel of variants, but Ab08 showed much less efficacy against the variants harboring the L452R mutation. To overcome the antibody escape caused by the L452R mutation, we generated several structure-based Ab08 derivatives. One derivative, Ab08-K99E, displayed the mostly enhanced neutralizing potency against the Delta pseudovirus bearing the L452R mutation compared to the Ab08 and other derivatives. Ab08-K99E also showed improved neutralizing effects against the prototype, Omicron BA.1, and Omicron BA.4/5 pseudoviruses. In addition, compared to the original Ab08, Ab08-K99E exhibited high binding properties and affinities to the RBDs of the prototype, Delta, and Omicron BA.4/5 variants. Altogether, our findings report an optimized nAb, Ab08-K99E, against SARS-CoV-2 variants and demonstrate structure-based optimization as an effective way for antibody development against pathogens.
Asunto(s)
Anticuerpos Neutralizantes , Anticuerpos Antivirales , COVID-19 , Mutación , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Anticuerpos Neutralizantes/inmunología , SARS-CoV-2/inmunología , SARS-CoV-2/genética , Humanos , Anticuerpos Antivirales/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/química , COVID-19/inmunología , COVID-19/virología , Pruebas de Neutralización , Unión Proteica , Células HEK293RESUMEN
The secretion and quality control of glycosylphosphatidylinositol-anchored proteins (GPI-APs) necessitates post-attachment remodeling initiated by the evolutionarily conserved PGAP1, which deacylates the inositol in nascent GPI-APs. Impairment of PGAP1 activity leads to developmental diseases in humans and fatality and infertility in animals. Here, we present three PGAP1 structures (2.66-2.84 Å), revealing its 10-transmembrane architecture and product-enzyme interaction details. PGAP1 holds GPI-AP acyl chains in an optimally organized, guitar-shaped cavity with apparent energetic penalties from hydrophobic-hydrophilic mismatches. However, abundant glycan-mediated interactions in the lumen counterbalance these repulsions, likely conferring substrate fidelity and preventing off-target hydrolysis of bulk membrane lipids. Structural and biochemical analyses uncover a serine hydrolase-type catalysis with atypical features and imply mechanisms for substrate entrance and product release involving a drawing compass movement of GPI-APs. Our findings advance the mechanistic understanding of GPI-AP remodeling.
Asunto(s)
Inositol , Proteínas de la Membrana , Animales , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Inositol/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Hidrolasas , Control de Calidad , Glicosilfosfatidilinositoles/químicaRESUMEN
Monoamine neurotransmitters such as serotonin and dopamine are loaded by vesicular monoamine transporter 2 (VMAT2) into synaptic vesicles for storage and subsequent release in neurons. Impaired VMAT2 function underlies various neuropsychiatric diseases. VMAT2 inhibitors reserpine and tetrabenazine are used to treat hypertension, movement disorders associated with Huntington's Disease and Tardive Dyskinesia. Despite its physiological and pharmacological significance, the structural basis underlying VMAT2 substrate recognition and its inhibition by various inhibitors remains unknown. Here we present cryo-EM structures of human apo VMAT2 in addition to states bound to serotonin, tetrabenazine, and reserpine. These structures collectively capture three states, namely the lumen-facing, occluded, and cytosol-facing conformations. Notably, tetrabenazine induces a substantial rearrangement of TM2 and TM7, extending beyond the typical rocker-switch movement. These functionally dynamic snapshots, complemented by biochemical analysis, unveil the essential components responsible for ligand recognition, elucidate the proton-driven exchange cycle, and provide a framework to design improved pharmaceutics targeting VMAT2.
Asunto(s)
Tetrabenazina , Proteínas de Transporte Vesicular de Monoaminas , Humanos , Reserpina , Serotonina/metabolismo , Vesículas Sinápticas/metabolismo , Tetrabenazina/farmacología , Tetrabenazina/metabolismo , Proteínas de Transporte Vesicular de Monoaminas/metabolismoRESUMEN
Green fluorescent proteins (GFPs) have lightened up almost every aspect of biological research including protein sciences. In the field of membrane protein structural biology, GFPs have been used widely to monitor membrane protein localization, expression level, the purification process and yield, and the stability inside the cells and in the test tube. Of particular interest is the fluorescence-detector size-exclusion chromatography-based thermostability assay (FSEC-TS). By simple heating and FSEC, the generally applicable method allows rapid assessment of the thermostability of GFP-fused membrane proteins without purification. Here we describe the experimental details and some typical results for the FSEC-TS method.