RESUMEN
Wnts are evolutionarily conserved ligands that signal at short range to regulate morphogenesis, cell fate, and stem cell renewal. The first and essential steps in Wnt secretion are their O-palmitoleation and subsequent loading onto the dedicated transporter Wntless/evenness interrupted (WLS/Evi). We report the 3.2 Å resolution cryogenic electron microscopy (cryo-EM) structure of palmitoleated human WNT8A in complex with WLS. This is accompanied by biochemical experiments to probe the physiological implications of the observed association. The WLS membrane domain has close structural homology to G protein-coupled receptors (GPCRs). A Wnt hairpin inserts into a conserved hydrophobic cavity in the GPCR-like domain, and the palmitoleate protrudes between two helices into the bilayer. A conformational switch of highly conserved residues on a separate Wnt hairpin might contribute to its transfer to receiving cells. This work provides molecular-level insights into a central mechanism in animal body plan development and stem cell biology.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Wnt/metabolismo , Secuencia de Aminoácidos , Animales , Disulfuros/metabolismo , Glicosilación , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Péptidos y Proteínas de Señalización Intracelular/aislamiento & purificación , Modelos Moleculares , Unión Proteica , Dominios Proteicos , Estructura Secundaria de Proteína , Transporte de Proteínas , Receptores Acoplados a Proteínas G/aislamiento & purificación , Receptores Acoplados a Proteínas G/ultraestructura , Homología Estructural de Proteína , Relación Estructura-Actividad , Proteínas Wnt/química , Proteínas Wnt/aislamiento & purificación , Proteínas Wnt/ultraestructuraRESUMEN
Choline is an essential nutrient that the human body needs in vast quantities for cell membrane synthesis, epigenetic modification and neurotransmission. The brain has a particularly high demand for choline, but how it enters the brain remains unknown1-3. The major facilitator superfamily transporter FLVCR1 (also known as MFSD7B or SLC49A1) was recently determined to be a choline transporter but is not highly expressed at the blood-brain barrier, whereas the related protein FLVCR2 (also known as MFSD7C or SLC49A2) is expressed in endothelial cells at the blood-brain barrier4-7. Previous studies have shown that mutations in human Flvcr2 cause cerebral vascular abnormalities, hydrocephalus and embryonic lethality, but the physiological role of FLVCR2 is unknown4,5. Here we demonstrate both in vivo and in vitro that FLVCR2 is a BBB choline transporter and is responsible for the majority of choline uptake into the brain. We also determine the structures of choline-bound FLVCR2 in both inward-facing and outward-facing states using cryo-electron microscopy. These results reveal how the brain obtains choline and provide molecular-level insights into how FLVCR2 binds choline in an aromatic cage and mediates its uptake. Our work could provide a novel framework for the targeted delivery of therapeutic agents into the brain.
Asunto(s)
Encéfalo , Colina , Proteínas de Transporte de Membrana , Animales , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , Transporte Biológico , Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Colina/metabolismo , Microscopía por Crioelectrón , Técnicas In Vitro , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Membrana/ultraestructura , Modelos MolecularesRESUMEN
The HCN1-4 channel family is responsible for the hyperpolarization-activated cation current If/Ih that controls automaticity in cardiac and neuronal pacemaker cells. We present cryoelectron microscopy (cryo-EM) structures of HCN4 in the presence or absence of bound cAMP, displaying the pore domain in closed and open conformations. Analysis of cAMP-bound and -unbound structures sheds light on how ligand-induced transitions in the channel cytosolic portion mediate the effect of cAMP on channel gating and highlights the regulatory role of a Mg2+ coordination site formed between the C-linker and the S4-S5 linker. Comparison of open/closed pore states shows that the cytosolic gate opens through concerted movements of the S5 and S6 transmembrane helices. Furthermore, in combination with molecular dynamics analyses, the open pore structures provide insights into the mechanisms of K+/Na+ permeation. Our results contribute mechanistic understanding on HCN channel gating, cyclic nucleotide-dependent modulation, and ion permeation.
Asunto(s)
Permeabilidad de la Membrana Celular/fisiología , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Activación del Canal Iónico/fisiología , Iones/metabolismo , Proteínas Musculares/metabolismo , Canales de Potasio/metabolismo , Línea Celular , Microscopía por Crioelectrón/métodos , AMP Cíclico/metabolismo , Células HEK293 , HumanosRESUMEN
The outer membrane of Gram-negative bacteria has an external leaflet that is largely composed of lipopolysaccharide, which provides a selective permeation barrier, particularly against antimicrobials1. The final and crucial step in the biosynthesis of lipopolysaccharide is the addition of a species-dependent O-antigen to the lipid A core oligosaccharide, which is catalysed by the O-antigen ligase WaaL2. Here we present structures of WaaL from Cupriavidus metallidurans, both in the apo state and in complex with its lipid carrier undecaprenyl pyrophosphate, determined by single-particle cryo-electron microscopy. The structures reveal that WaaL comprises 12 transmembrane helices and a predominantly α-helical periplasmic region, which we show contains many of the conserved residues that are required for catalysis. We observe a conserved fold within the GT-C family of glycosyltransferases and hypothesize that they have a common mechanism for shuttling the undecaprenyl-based carrier to and from the active site. The structures, combined with genetic, biochemical, bioinformatics and molecular dynamics simulation experiments, offer molecular details on how the ligands come in apposition, and allows us to propose a mechanistic model for catalysis. Together, our work provides a structural basis for lipopolysaccharide maturation in a member of the GT-C superfamily of glycosyltransferases.
Asunto(s)
Ligasas , Lipopolisacáridos , Antígenos O , Proteínas Bacterianas/química , Ligasas de Carbono-Oxígeno/química , Ligasas de Carbono-Oxígeno/genética , Microscopía por Crioelectrón , Glicosiltransferasas , Bacterias Gramnegativas , Lipopolisacáridos/química , Lipopolisacáridos/metabolismoRESUMEN
Mycobacterium tuberculosis causes tuberculosis, a disease that kills over 1 million people each year. Its cell envelope is a common antibiotic target and has a unique structure due, in part, to two lipidated polysaccharides-arabinogalactan and lipoarabinomannan. Arabinofuranosyltransferase D (AftD) is an essential enzyme involved in assembling these glycolipids. We present the 2.9-Å resolution structure of M. abscessus AftD, determined by single-particle cryo-electron microscopy. AftD has a conserved GT-C glycosyltransferase fold and three carbohydrate-binding modules. Glycan array analysis shows that AftD binds complex arabinose glycans. Additionally, AftD is non-covalently complexed with an acyl carrier protein (ACP). 3.4- and 3.5-Å structures of a mutant with impaired ACP binding reveal a conformational change, suggesting that ACP may regulate AftD function. Mutagenesis experiments using a conditional knockout constructed in M. smegmatis confirm the essentiality of the putative active site and the ACP binding for AftD function.
Asunto(s)
Proteína Transportadora de Acilo/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Microscopía por Crioelectrón/métodos , Glicosiltransferasas/metabolismo , Mycobacterium smegmatis/enzimología , Proteína Transportadora de Acilo/genética , Proteínas Bacterianas/genética , Dominio Catalítico , Pared Celular/metabolismo , Galactanos/metabolismo , Glicosiltransferasas/genética , Lipopolisacáridos/metabolismo , Mutación , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/crecimiento & desarrollo , Filogenia , Conformación Proteica , Especificidad por SustratoRESUMEN
Docosahexaenoic acid is an omega-3 fatty acid that is essential for neurological development and function, and it is supplied to the brain and eyes predominantly from dietary sources1-6. This nutrient is transported across the blood-brain and blood-retina barriers in the form of lysophosphatidylcholine by major facilitator superfamily domain containing 2A (MFSD2A) in a Na+-dependent manner7,8. Here we present the structure of MFSD2A determined using single-particle cryo-electron microscopy, which reveals twelve transmembrane helices that are separated into two pseudosymmetric domains. The transporter is in an inward-facing conformation and features a large amphipathic cavity that contains the Na+-binding site and a bound lysolipid substrate, which we confirmed using native mass spectrometry. Together with our functional analyses and molecular dynamics simulations, this structure reveals details of how MFSD2A interacts with substrates and how Na+-dependent conformational changes allow for the release of these substrates into the membrane through a lateral gate. Our work provides insights into the molecular mechanism by which this atypical major facility superfamily transporter mediates the uptake of lysolipids into the brain, and has the potential to aid in the delivery of neurotherapeutic agents.
Asunto(s)
Transporte Biológico , Barrera Hematoencefálica/metabolismo , Microscopía por Crioelectrón , Ácidos Grasos Omega-3/metabolismo , Simportadores/química , Simportadores/metabolismo , Animales , Sitios de Unión , Pollos , Ácidos Grasos Omega-3/química , Espectrometría de Masas , Modelos Moleculares , Simulación de Dinámica Molecular , Dominios Proteicos , Sodio/metabolismo , Simportadores/ultraestructuraRESUMEN
The emergence and spread of drug-resistant Plasmodium falciparum impedes global efforts to control and eliminate malaria. For decades, treatment of malaria has relied on chloroquine (CQ), a safe and affordable 4-aminoquinoline that was highly effective against intra-erythrocytic asexual blood-stage parasites, until resistance arose in Southeast Asia and South America and spread worldwide1. Clinical resistance to the chemically related current first-line combination drug piperaquine (PPQ) has now emerged regionally, reducing its efficacy2. Resistance to CQ and PPQ has been associated with distinct sets of point mutations in the P. falciparum CQ-resistance transporter PfCRT, a 49-kDa member of the drug/metabolite transporter superfamily that traverses the membrane of the acidic digestive vacuole of the parasite3-9. Here we present the structure, at 3.2 Å resolution, of the PfCRT isoform of CQ-resistant, PPQ-sensitive South American 7G8 parasites, using single-particle cryo-electron microscopy and antigen-binding fragment technology. Mutations that contribute to CQ and PPQ resistance localize primarily to moderately conserved sites on distinct helices that line a central negatively charged cavity, indicating that this cavity is the principal site of interaction with the positively charged CQ and PPQ. Binding and transport studies reveal that the 7G8 isoform binds both drugs with comparable affinities, and that these drugs are mutually competitive. The 7G8 isoform transports CQ in a membrane potential- and pH-dependent manner, consistent with an active efflux mechanism that drives CQ resistance5, but does not transport PPQ. Functional studies on the newly emerging PfCRT F145I and C350R mutations, associated with decreased PPQ susceptibility in Asia and South America, respectively6,9, reveal their ability to mediate PPQ transport in 7G8 variant proteins and to confer resistance in gene-edited parasites. Structural, functional and in silico analyses suggest that distinct mechanistic features mediate the resistance to CQ and PPQ in PfCRT variants. These data provide atomic-level insights into the molecular mechanism of this key mediator of antimalarial treatment failures.
Asunto(s)
Microscopía por Crioelectrón , Resistencia a Medicamentos/efectos de los fármacos , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/ultraestructura , Plasmodium falciparum/química , Proteínas Protozoarias/química , Proteínas Protozoarias/ultraestructura , Cloroquina/metabolismo , Cloroquina/farmacología , Resistencia a Medicamentos/genética , Concentración de Iones de Hidrógeno , Malaria Falciparum/tratamiento farmacológico , Malaria Falciparum/parasitología , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Modelos Moleculares , Mutación , Plasmodium falciparum/genética , Plasmodium falciparum/ultraestructura , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Quinolinas/metabolismo , Quinolinas/farmacologíaRESUMEN
BACKGROUND: Structural birth defects occur in approximately 3% of live births; most such defects lack defined genetic or environmental causes. Despite advances in surgical approaches, pharmacologic prevention remains largely out of reach. METHODS: We queried worldwide databases of 20,248 families that included children with neurodevelopmental disorders and that were enriched for parental consanguinity. Approximately one third of affected children in these families presented with structural birth defects or microcephaly. We performed exome or genome sequencing of samples obtained from the children, their parents, or both to identify genes with biallelic pathogenic or likely pathogenic mutations present in more than one family. After identifying disease-causing variants, we generated two mouse models, each with a pathogenic variant "knocked in," to study mechanisms and test candidate treatments. We administered a small-molecule Wnt agonist to pregnant animals and assessed their offspring. RESULTS: We identified homozygous mutations in WLS, which encodes the Wnt ligand secretion mediator (also known as Wntless or WLS) in 10 affected persons from 5 unrelated families. (The Wnt ligand secretion mediator is essential for the secretion of all Wnt proteins.) Patients had multiorgan defects, including microcephaly and facial dysmorphism as well as foot syndactyly, renal agenesis, alopecia, iris coloboma, and heart defects. The mutations affected WLS protein stability and Wnt signaling. Knock-in mice showed tissue and cell vulnerability consistent with Wnt-signaling intensity and individual and collective functions of Wnts in embryogenesis. Administration of a pharmacologic Wnt agonist partially restored embryonic development. CONCLUSIONS: Genetic variations affecting a central Wnt regulator caused syndromic structural birth defects. Results from mouse models suggest that what we have named Zaki syndrome is a potentially preventable disorder. (Funded by the National Institutes of Health and others.).
Asunto(s)
Anomalías Múltiples/genética , Anomalías Congénitas/genética , Pleiotropía Genética , Péptidos y Proteínas de Señalización Intracelular/genética , Mutación , Receptores Acoplados a Proteínas G/genética , Proteínas Wnt/metabolismo , Animales , Modelos Animales de Enfermedad , Fibroblastos/metabolismo , Técnicas de Sustitución del Gen , Genes Recesivos , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ratones , Ratones Transgénicos , Linaje , Fenotipo , Receptores Acoplados a Proteínas G/metabolismo , Síndrome , Vía de Señalización WntRESUMEN
Multidrug-resistant Plasmodium falciparum parasites have emerged in Cambodia and neighboring countries in Southeast Asia, compromising the efficacy of first-line antimalarial combinations. Dihydroartemisinin + piperaquine (PPQ) treatment failure rates have risen to as high as 50% in some areas in this region. For PPQ, resistance is driven primarily by a series of mutant alleles of the P. falciparum chloroquine resistance transporter (PfCRT). PPQ resistance was reported in China three decades earlier, but the molecular driver remained unknown. Herein, we identify a PPQ-resistant pfcrt allele (China C) from Yunnan Province, China, whose genotypic lineage is distinct from the PPQ-resistant pfcrt alleles currently observed in Cambodia. Combining gene editing and competitive growth assays, we report that PfCRT China C confers moderate PPQ resistance while re-sensitizing parasites to chloroquine (CQ) and incurring a fitness cost that manifests as a reduced rate of parasite growth. PPQ transport assays using purified PfCRT isoforms, combined with molecular dynamics simulations, highlight differences in drug transport kinetics and in this transporter's central cavity conformation between China C and the current Southeast Asian PPQ-resistant isoforms. We also report a novel computational model that incorporates empirically determined fitness landscapes at varying drug concentrations, combined with antimalarial susceptibility profiles, mutation rates, and drug pharmacokinetics. Our simulations with PPQ-resistant or -sensitive parasite lines predict that a three-day regimen of PPQ combined with CQ can effectively clear infections and prevent the evolution of PfCRT variants. This work suggests that including CQ in combination therapies could be effective in suppressing the evolution of PfCRT-mediated multidrug resistance in regions where PPQ has lost efficacy.
Asunto(s)
Artemisininas/uso terapéutico , Cloroquina/uso terapéutico , Resistencia a Múltiples Medicamentos , Proteínas de Transporte de Membrana/genética , Piperazinas/uso terapéutico , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/genética , Proteínas Protozoarias/genética , Quinolinas/uso terapéutico , Alelos , Animales , Antimaláricos/uso terapéutico , Simulación por Computador , Humanos , Malaria Falciparum/parasitologíaRESUMEN
The mycobacteria genus is responsible for numerous infectious diseases that have afflicted the human race since antiquity-tuberculosis and leprosy in particular. An important contributor to their evolutionary success is their unique cell envelope, which constitutes a quasi-impermeable barrier, protecting the microorganism from external threats, antibiotics included. The arabinofuranosyltransferases are a family of enzymes, unique to the Actinobacteria family that mycobacteria genus belongs to, that are critical to building of this cell envelope. In this chapter, we will analyze available structures of members of the mycobacterial arabinofuranosyltransferase, clarify their function, as well as explore the common themes present amongst this family of enzymes, as revealed by recent research.
Asunto(s)
Mycobacterium tuberculosis , Mycobacterium , Antibacterianos , Membrana Celular , Pared Celular , HumanosRESUMEN
Vitamin A has diverse biological functions and is essential for human survival at every point from embryogenesis to adulthood. Vitamin A and its derivatives have been used to treat human diseases including vision diseases, skin diseases, and cancer. Both insufficient and excessive vitamin A uptake are detrimental, but how its transport is regulated is poorly understood. STRA6 is a multitransmembrane domain cell-surface receptor and mediates vitamin A uptake from plasma retinol binding protein (RBP). STRA6 can mediate both cellular vitamin A influx and efflux, but what regulates these opposing activities is unknown. To answer this question, we purified and identified STRA6-associated proteins in a native mammalian cell type that takes up vitamin A through STRA6 using mass spectrometry. We found that the major protein repeatedly identified as STRA6-associated protein is calmodulin, consistent with the cryogenic electron microscopy (cryo-EM) study of zebrafish STRA6 associated with calmodulin. Using radioactivity-based, high-performance liquid chromatography (HPLC)-based and real-time fluorescence techniques, we found that calmodulin profoundly affects STRA6's vitamin A transport activity. Increased calcium/calmodulin promotes cellular vitamin A efflux and suppresses vitamin A influx through STRA6. Further mechanistic studies revealed that calmodulin enhances the binding of apo-RBP to STRA6, and this enhancement is much more pronounced for apo-RBP than holo-RBP. This study revealed that calmodulin regulates STRA6's vitamin A influx or efflux activity by modulating its preferential interaction with apo-RBP or holo-RBP. This molecular mechanism of regulating vitamin A transport may point to new directions to treat human diseases associated with insufficient or excessive vitamin A uptake.
Asunto(s)
Transporte Biológico/genética , Calmodulina/genética , Proteínas de la Membrana/genética , Proteínas Plasmáticas de Unión al Retinol/genética , Vitamina A/metabolismo , Animales , Apoproteínas/genética , Apoproteínas/metabolismo , Calcio/metabolismo , Bovinos , Línea Celular , Cromatografía Líquida de Alta Presión , Microscopía por Crioelectrón , Humanos , Proteínas de la Membrana/metabolismo , Unión Proteica/genética , Receptores de Superficie Celular/genética , Proteínas Plasmáticas de Unión al Retinol/metabolismo , Vitamina A/genética , Pez Cebra/genéticaRESUMEN
SARS-CoV-2 epidemics quickly propagated worldwide, sorting virus genomic variants in newly established propagules of infections. Stochasticity in transmission within and between countries or an actual selective advantage could explain the global high frequency reached by some genomic variants. Using statistical analyses, demographic reconstructions, and molecular dynamics simulations, we show that the globally invasive G614 spike variant 1) underwent a significant demographic expansion in most countries explained neither by stochastic effects nor by overrepresentation in clinical samples, 2) increases the spike S1/S2 furin-like site conformational plasticity (short-range effect), and 3) modifies the internal motion of the receptor-binding domain affecting its cross-connection with other functional domains (long-range effect). Our results support the hypothesis of a selective advantage at the basis of the spread of the G614 variant, which we suggest may be due to structural modification of the spike protein at the S1/S2 proteolytic site, and provide structural information to guide the design of variant-specific drugs.
Asunto(s)
COVID-19/genética , Mutación Missense , SARS-CoV-2/genética , Selección Genética , Glicoproteína de la Espiga del Coronavirus/genética , COVID-19/epidemiología , HumanosRESUMEN
Wnts are short-range signaling proteins, expressed in all metazoans from sponges to humans, critical for cell development and fate. There are 19 different Wnts in the human genome with varying expression levels and patterns, and post-translational modifications. Common to essentially all Wnts is the palmitoleation of a conserved serine by the O-acyltransferase PORCN in the endoplasmic reticulum (ER). All lipidated Wnts then bind a dedicated carrier Wntless (WLS), endowed with the task of transporting them from the ER to the plasma membrane, and ultimately facilitating their release to receptors on the Wnt-receiving cell to initiate signaling. Here, we will focus on the WLS-mediated transport step. There are currently two published structures, both obtained by single-particle cryo-electron microscopy of the Wnt/WLS complex: human Wnt8A-bound and human Wnt3A-bound WLS. We analyze the two Wnt/WLS structures - remarkably similar despite the sequence similarity between Wnt8A and Wnt3A being only â¼39% - to begin to understand the conserved nature of this binding mechanism, and ultimately how one carrier can accommodate a family of 19 different Wnts. By comparing how Wnt associates with WLS with how it binds to PORCN and FZD receptors, we can begin to speculate on mechanisms of Wnt transfer from PORCN to WLS, and from WLS to FZD, thus providing molecular-level insight into these essential steps of the Wnt signaling pathway.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular , Vía de Señalización Wnt , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Microscopía por Crioelectrón , Aciltransferasas/metabolismo , Membrana Celular/metabolismo , Proteínas de la Membrana/metabolismoRESUMEN
Ryanodine receptors (RyRs) mediate the rapid release of calcium (Ca(2+)) from intracellular stores into the cytosol, which is essential for numerous cellular functions including excitation-contraction coupling in muscle. Lack of sufficient structural detail has impeded understanding of RyR gating and regulation. Here we report the closed-state structure of the 2.3-megadalton complex of the rabbit skeletal muscle type 1 RyR (RyR1), solved by single-particle electron cryomicroscopy at an overall resolution of 4.8 Å. We fitted a polyalanine-level model to all 3,757 ordered residues in each protomer, defining the transmembrane pore in unprecedented detail and placing all cytosolic domains as tertiary folds. The cytosolic assembly is built on an extended α-solenoid scaffold connecting key regulatory domains to the pore. The RyR1 pore architecture places it in the six-transmembrane ion channel superfamily. A unique domain inserted between the second and third transmembrane helices interacts intimately with paired EF-hands originating from the α-solenoid scaffold, suggesting a mechanism for channel gating by Ca(2+).
Asunto(s)
Canal Liberador de Calcio Receptor de Rianodina/química , Canal Liberador de Calcio Receptor de Rianodina/ultraestructura , Animales , Calcio/deficiencia , Calcio/metabolismo , Calcio/farmacología , Membrana Celular/metabolismo , Microscopía por Crioelectrón , Citosol/metabolismo , Activación del Canal Iónico/efectos de los fármacos , Músculo Esquelético/química , Estructura Terciaria de Proteína , Conejos , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Proteínas de Unión a Tacrolimus/química , Proteínas de Unión a Tacrolimus/metabolismoRESUMEN
The membrane-water interface forms a uniquely heterogeneous and geometrically constrained environment for enzymatic catalysis. Integral membrane enzymes sample three environments - the uniformly hydrophobic interior of the membrane, the aqueous extramembrane region, and the fuzzy, amphipathic interfacial region formed by the tightly packed headgroups of the components of the lipid bilayer. Depending on the nature of the substrates and the location of the site of chemical modification, catalysis may occur in each of these environments. The availability of structural information for alpha-helical enzyme families from each of these classes, as well as several beta-barrel enzymes from the bacterial outer membrane, has allowed us to review here the different ways in which each enzyme fold has adapted to the nature of the substrates, products, and the unique environment of the membrane. Our focus here is on enzymes that process lipidic substrates. This article is part of a Special Issue entitled: Bacterial Lipids edited by Russell E. Bishop.
Asunto(s)
Bacterias/enzimología , Proteínas Bacterianas/metabolismo , Membrana Celular/enzimología , Lípidos de la Membrana/metabolismo , Agua/metabolismo , Proteínas Bacterianas/química , Catálisis , Dominio Catalítico , Membrana Celular/química , Interacciones Hidrofóbicas e Hidrofílicas , Lípidos de la Membrana/química , Modelos Moleculares , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Pliegue de Proteína , Relación Estructura-Actividad , Especificidad por Sustrato , Agua/químicaRESUMEN
G protein-coupled receptors (GPCRs) are a prominent class of plasma membrane proteins that regulate physiologic responses to a wide variety of stimuli and therapeutic agents. Although GPCR oligomerization has been studied extensively in recombinant cells, it remains uncertain whether native receptors expressed in their natural cellular environment are monomers, dimers, or oligomers. The goal of this study was to determine the monomer/oligomer status of a native GPCR endogenously expressed in its natural cellular environment. Native 5-HT2C receptors in choroid plexus epithelial cells were evaluated using fluorescence correlation spectroscopy (FCS) with photon counting histogram (PCH). An anti-5-HT2C fragment antigen binding protein was used to label native 5-HT2C receptors. A known monomeric receptor (CD-86) served as a control for decoding the oligomer status of native 5-HT2C receptors by molecular brightness analysis. FCS with PCH revealed molecular brightness values for native 5-HT2C receptors equivalent to the molecular brightness of a homodimer. 5-HT2C receptors displayed a diffusion coefficient of 5 × 10(-9) cm(2)/s and were expressed at 32 receptors/µm(2) on the apical surface of choroid plexus epithelial cells. The functional significance and signaling capabilities of the homodimer were investigated in human embryonic kidney 293 cells using agonists that bind in a wash-resistant manner to one or both protomers of the homodimer. Whereas agonist binding to one protomer resulted in G protein activation, maximal stimulation required occupancy of both protomers. This study is the first to demonstrate the homodimeric structure of 5-HT2C receptors endogenously expressed in their native cellular environment, and identifies the homodimer as a functional signaling unit.
Asunto(s)
Plexo Coroideo/metabolismo , Células Epiteliales/metabolismo , Receptor de Serotonina 5-HT2C/metabolismo , Marcadores de Afinidad , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/metabolismo , Células Cultivadas , Proteínas Fluorescentes Verdes/genética , Células HEK293 , Humanos , Fragmentos Fab de Inmunoglobulinas/genética , Fragmentos Fab de Inmunoglobulinas/metabolismo , Regiones Promotoras Genéticas , Multimerización de Proteína , Ensayo de Unión Radioligante , Ratas Sprague-Dawley , Receptor de Serotonina 5-HT2C/genética , Receptor de Serotonina 5-HT2C/inmunología , Transducción de SeñalRESUMEN
Anomalous diffraction signals from typical native macromolecules are very weak, frustrating their use in de novo structure determination. Here, native SAD procedures are described to enhance signal to noise in anomalous diffraction by using multiple crystals in combination with synchrotron X-rays at 6â keV. Increased anomalous signals were obtained at 6â keV compared with 7â keV X-ray energy, which was used for previous native SAD analyses. A feasibility test of multi-crystal-based native SAD phasing was performed at 3.2â Å resolution for a known tyrosine protein kinase domain, and real-life applications were made to two novel membrane proteins at about 3.0â Å resolution. The three applications collectively serve to validate the robust feasibility of native SAD phasing at lower energy.
Asunto(s)
Proteínas Bacterianas/química , Cristalografía por Rayos X/métodos , Receptores ErbB/química , Conformación Proteica , Bacillus subtilis/química , Humanos , Listeria monocytogenes/química , Proteínas de Transporte de Membrana/química , Modelos Moleculares , Estructura Terciaria de Proteína , Reproducibilidad de los Resultados , SincrotronesRESUMEN
IMPORTANCE: Our study leverages gene editing techniques in Plasmodium falciparum asexual blood stage parasites to profile novel mutations in mutant PfCRT, an important mediator of piperaquine resistance, which developed in Southeast Asian field isolates or in parasites cultured for long periods of time. We provide evidence that increased parasite fitness of these lines is the primary driver for the emergence of these PfCRT variants. These mutations differentially impact parasite susceptibility to piperaquine and chloroquine, highlighting the multifaceted effects of single point mutations in this transporter. Molecular features of drug resistance and parasite physiology were examined in depth using proteoliposome-based drug uptake studies and peptidomics, respectively. Energy minimization calculations, showing how these novel mutations might impact the PfCRT structure, suggested a small but significant effect on drug interactions. This study reveals the subtle interplay between antimalarial resistance, parasite fitness, PfCRT structure, and intracellular peptide availability in PfCRT-mediated parasite responses to changing drug selective pressures.
Asunto(s)
Antimaláricos , Malaria Falciparum , Parásitos , Piperazinas , Quinolinas , Animales , Plasmodium falciparum , Quinolinas/farmacología , Quinolinas/química , Cloroquina/farmacología , Antimaláricos/farmacología , Resistencia a Medicamentos/genética , Mutación , Proteínas Protozoarias/genética , Proteínas Protozoarias/química , Malaria Falciparum/parasitologíaRESUMEN
Major Facilitator Superfamily Domain containing 2 A (MFSD2A) is a transporter that is highly enriched at the blood-brain and blood-retinal barriers, where it mediates Na+-dependent uptake of ω-3 fatty acids in the form of lysolipids into the brain and eyes, respectively. Despite recent structural insights, it remains unclear how this process is initiated, and driven by Na+. Here, we perform Molecular Dynamics simulations which demonstrate that substrates enter outward facing MFSD2A from the outer leaflet of the membrane via lateral openings between transmembrane helices 5/8 and 2/11. The substrate headgroup enters first and engages in Na+ -bridged interactions with a conserved glutamic acid, while the tail is surrounded by hydrophobic residues. This binding mode is consistent with a "trap-and-flip" mechanism and triggers transition to an occluded conformation. Furthermore, using machine learning analysis, we identify key elements that enable these transitions. These results advance our molecular understanding of the MFSD2A transport cycle.
Asunto(s)
Ácidos Grasos Omega-3 , Simportadores , Ácidos Grasos Omega-3/metabolismo , Simportadores/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Encéfalo/metabolismo , Transporte Biológico , Simulación de Dinámica MolecularRESUMEN
There are two main families of G protein-coupled receptors that detect odours in humans, the odorant receptors (ORs) and the trace amine-associated receptors (TAARs). Their amino acid sequences are distinct, with the TAARs being most similar to the aminergic receptors such as those activated by adrenaline, serotonin and histamine. To elucidate the structural determinants of ligand recognition by TAARs, we have determined the cryo-EM structure of a murine receptor, mTAAR7f, coupled to the heterotrimeric G protein Gs and bound to the odorant N,N-dimethylcyclohexylamine (DMCH) to an overall resolution of 2.9 Å. DMCH is bound in a hydrophobic orthosteric binding site primarily through van der Waals interactions and a strong charge-charge interaction between the tertiary amine of the ligand and an aspartic acid residue. This site is distinct and non-overlapping with the binding site for the odorant propionate in the odorant receptor OR51E2. The structure, in combination with mutagenesis data and molecular dynamics simulations suggests that the activation of the receptor follows a similar pathway to that of the ß-adrenoceptors, with the significant difference that DMCH interacts directly with one of the main activation microswitch residues.