RESUMEN
The inhibitory neurotransmitter γ-aminobutyric acid (GABA) is cleared from the synaptic cleft by the sodium- and chloride-coupled GABA transporter GAT1. Inhibition of GAT1 prolongs the GABAergic signaling at the synapse and is a strategy to treat certain forms of epilepsy. In this study, we present the cryo-electron microscopy structure of Rattus norvegicus GABA transporter 1 (rGAT1) at a resolution of 3.1 Å. The structure elucidation was facilitated by epitope transfer of a fragment-antigen binding (Fab) interaction site from the Drosophila dopamine transporter (dDAT) to rGAT1. The structure reveals rGAT1 in a cytosol-facing conformation, with a linear density in the primary binding site that accommodates a molecule of GABA, a displaced ion density proximal to Na site 1 and a bound chloride ion. A unique insertion in TM10 aids the formation of a compact, closed extracellular gate. Besides yielding mechanistic insights into ion and substrate recognition, our study will enable the rational design of specific antiepileptics.
Asunto(s)
Cloruros , Ácido gamma-Aminobutírico , Ratas , Animales , Proteínas Transportadoras de GABA en la Membrana Plasmática/química , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Microscopía por Crioelectrón , Cloruros/metabolismo , Cloruros/farmacología , Ácido gamma-Aminobutírico/metabolismo , Sitios de UniónRESUMEN
Nucleotide Sugar Transporters (NSTs) belong to the SLC35 family (human solute carrier) of membrane transport proteins and are crucial components of the glycosylation machinery. NSTs are localized in the ER and Golgi apparatus membranes, where they accumulate nucleotide sugars from the cytosol for subsequent polysaccharide biosynthesis. Loss of NST function impacts the glycosylation of cell surface molecules. Mutations in NSTs cause several developmental disorders, immune disorders, and increased susceptibility to infection. Atomic resolution structures of three NSTs have provided a blueprint for a detailed molecular interpretation of their biochemical properties. In this work, we have identified, cloned, and expressed 18 members of the SLC35 family from various eukaryotic organisms in Saccharomyces cerevisiae. Out of 18 clones, we determined Vrg4 from Chaetomium thermophilum (CtVrg4) is a GDP-mannose transporter with an enhanced melting point temperature (Tm) of 56.9°C, which increases with the addition of substrates, GMP and GDP-mannose. In addition, we report-for the first time-that the CtVrg4 shows an affinity to bind to phosphatidylinositol lipids.
Asunto(s)
Proteínas Portadoras , Proteínas de Saccharomyces cerevisiae , Humanos , Proteínas Portadoras/metabolismo , Transporte Biológico , Saccharomyces cerevisiae/genética , Glicosilación , Nucleótidos/metabolismo , Aparato de Golgi/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
γ-aminobutyric acid (GABA) is the major inhibitory neurotransmitter, and its levels in the synaptic space are controlled by the GABA transporter isoforms (GATs). GATs are structurally related to biogenic amine transporters but display interactions with distinct inhibitors used as anti-epileptics. In this study, we engineer the binding pocket of Drosophila melanogaster dopamine transporter to resemble GAT1 and determine high-resolution X-ray structures of the modified transporter in the substrate-free state and in complex with GAT1 inhibitors NO711 and SKF89976a that are analogs of tiagabine, a medication prescribed for the treatment of partial seizures. We observe that the primary binding site undergoes substantial shifts in subsite architecture in the modified transporter to accommodate the two GAT1 inhibitors. We also observe that SKF89976a additionally interacts at an allosteric site in the extracellular vestibule, yielding an occluded conformation. Interchanging SKF89976a interacting residue in the extracellular loop 4 between GAT1 and dDAT suggests a role for this motif in the selective control of neurotransmitter uptake. Our findings, therefore, provide vital insights into the organizational principles dictating GAT1 activity and inhibition.
Asunto(s)
Drosophila melanogaster , Ácido gamma-Aminobutírico , Animales , Transporte Biológico , Drosophila melanogaster/metabolismo , Proteínas Transportadoras de GABA en la Membrana Plasmática/química , Proteínas Transportadoras de GABA en la Membrana Plasmática/genética , Proteínas Transportadoras de GABA en la Membrana Plasmática/metabolismo , Neurotransmisores , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Norepinephrine is a biogenic amine neurotransmitter that has widespread effects on alertness, arousal and pain sensation. Consequently, blockers of norepinephrine uptake have served as vital tools to treat depression and chronic pain. Here, we employ the Drosophila melanogaster dopamine transporter as a surrogate for the norepinephrine transporter and determine X-ray structures of the transporter in its substrate-free and norepinephrine-bound forms. We also report structures of the transporter in complex with inhibitors of chronic pain including duloxetine, milnacipran and a synthetic opioid, tramadol. When compared to dopamine, we observe that norepinephrine binds in a different pose, in the vicinity of subsite C within the primary binding site. Our experiments reveal that this region is the binding site for chronic pain inhibitors and a determinant for norepinephrine-specific reuptake inhibition, thereby providing a paradigm for the design of specific inhibitors for catecholamine neurotransmitter transporters.
Asunto(s)
Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Proteínas de Transporte de Neurotransmisores/química , Proteínas de Transporte de Neurotransmisores/metabolismo , Norepinefrina/química , Norepinefrina/metabolismo , Analgésicos Opioides/química , Animales , Sitios de Unión , Cristalografía por Rayos X , Dopamina , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática , Proteínas de Drosophila/genética , Drosophila melanogaster , Células HEK293 , Humanos , Ratones Noqueados , Modelos Moleculares , Proteínas de Transporte de Neurotransmisores/genética , Células Sf9 , TranscriptomaRESUMEN
Neurotransmitters released at the neural synapse through vesicle exocytosis are spatiotemporally controlled by the action of neurotransmitter transporters. Integral membrane proteins of the solute carrier 6 (SLC6) family are involved in the sodium and chloride coupled uptake of biogenic amine neurotransmitters including dopamine, serotonin, noradrenaline and inhibitory neurotransmitters including glycine and γ-amino butyric acid. This ion-coupled symport works through a well-orchestrated gating of substrate through alternating-access, which is mediated through movements of helices that resemble a rocking-bundle. A large array of commercially prescribed drugs and psychostimulants selectively target neurotransmitter transporters thereby modulating their levels in the synaptic space. Drug-induced changes in the synaptic neurotransmitter levels can be used to treat depression or neuropathic pain whereas in some instances prolonged usage can lead to habituation. Earlier structural studies of bacterial neurotransmitter transporter homolog LeuT and recent structure elucidation of the Drosophila dopamine transporter (dDAT) and human serotonin transporter (hSERT) have yielded a wealth of information in understanding the transport and inhibition mechanism of neurotransmitter transporters. Computational studies based on the structures of dDAT and hSERT have shed light on the dynamics of varied components of these molecular gates in affecting the uphill transport of neurotransmitters. This review seeks to address structural dynamics of neurotransmitter transporters at the extracellular and intracellular gates and the effect of inhibitors on the ligand-binding pocket. We also delve into the effect of additional factors including lipids and cytosolic domains that influence the translocation of neurotransmitters across the membrane.
RESUMEN
Several pathogenic bacteria utilize sialic acid, including host-derived N-acetylneuraminic acid (Neu5Ac), in at least two ways: they use it as a nutrient source and as a host-evasion strategy by coating themselves with Neu5Ac. Given the significant role of sialic acid in pathogenesis and host-gut colonization by various pathogenic bacteria, including Neisseria meningitidis, Haemophilus influenzae, Pasteurella multocida and Vibrio cholerae, several enzymes of the sialic acid catabolic, biosynthetic and incorporation pathways are considered to be potential drug targets. In this work, findings on the structural and functional characterization of CMP-N-acetylneuraminate synthetase (CMAS), a key enzyme in the incorporation pathway, from Vibrio cholerae are reported. CMAS catalyzes the synthesis of CMP-sialic acid by utilizing CTP and sialic acid. Crystal structures of the apo and the CDP-bound forms of the enzyme were determined, which allowed the identification of the metal cofactor Mg2+ in the active site interacting with CDP and the invariant Asp215 residue. While open and closed structural forms of the enzyme from eukaryotic and other bacterial species have already been characterized, a partially closed structure of V. cholerae CMAS (VcCMAS) observed upon CDP binding, representing an intermediate state, is reported here. The kinetic data suggest that VcCMAS is capable of activating the two most common sialic acid derivatives, Neu5Ac and Neu5Gc. Amino-acid sequence and structural comparison of the active site of VcCMAS with those of eukaryotic and other bacterial counterparts reveal a diverse hydrophobic pocket that interacts with the C5 substituents of sialic acid. Analyses of the thermodynamic signatures obtained from the binding of the nucleotide (CTP) and the product (CMP-sialic acid) to VcCMAS provide fundamental information on the energetics of the binding process.
Asunto(s)
Proteínas Bacterianas/química , N-Acilneuraminato Citidililtransferasa/química , Vibrio cholerae/enzimología , Secuencia de Aminoácidos , Proteínas Bacterianas/farmacología , Proteínas Bacterianas/fisiología , Sitios de Unión , Dominio Catalítico , Cristalización , Cristalografía por Rayos X/métodos , Citidina Difosfato/química , Citidina Difosfato/metabolismo , Ácido N-Acetilneuramínico Citidina Monofosfato/química , Ácido N-Acetilneuramínico Citidina Monofosfato/metabolismo , Citidina Trifosfato/química , Citidina Trifosfato/metabolismo , N-Acilneuraminato Citidililtransferasa/farmacología , N-Acilneuraminato Citidililtransferasa/fisiología , Dominios y Motivos de Interacción de Proteínas , Estructura Cuaternaria de Proteína , Ácidos Siálicos/metabolismoRESUMEN
Valproic acid (VA) is a pharmacologically important histone deacetylase inhibitor that recently garnered attention as an anticancer agent. Since the molecular mechanisms behind the multiple effects of VA are unclear, this study was aimed to unravel the comprehensive cellular processes affected by VA and its molecular targets in vivo using budding yeast as a model organism. Interestingly, genome-wide transcriptome analysis of cells treated with VA showed differential regulation of 30% of the genome. Functional enrichment analysis of VA transcriptome evidenced alteration of various cellular processes including cell cycle, cell wall biogenesis, DNA repair, ion homeostasis, metabolism, stress response, transport and ribosomal biogenesis, etc. Moreover, our genetic screening analysis revealed VA molecular targets belonging to oxidative and osmotic stress, DNA repair, cell wall integrity, and iron homeostasis. Further, our results demonstrated the activation of mitogen-activated protein kinases (MAPKs) Hog1 (p38) and Slt2 (p44/42) upon VA treatment. Our results also exhibited that VA acts through alteration of mitochondrial, ER architecture and functions. Especially, VA effects were neutralized in cells lacking lipid particles. Altogether, our results deciphered the novel molecular insights and mechanistic links to strengthen our knowledge on diverse cellular effects of VA along with its probable therapeutic targets and detoxification approaches.