RESUMEN
Elucidating the mechanism of sugar import requires a molecular understanding of how transporters couple sugar binding and gating events. Whereas mammalian glucose transporters (GLUTs) are specialists1, the hexose transporter from the malaria parasite Plasmodium falciparum PfHT12,3 has acquired the ability to transport both glucose and fructose sugars as efficiently as the dedicated glucose (GLUT3) and fructose (GLUT5) transporters. Here, to establish the molecular basis of sugar promiscuity in malaria parasites, we determined the crystal structure of PfHT1 in complex with D-glucose at a resolution of 3.6 Å. We found that the sugar-binding site in PfHT1 is very similar to those of the distantly related GLUT3 and GLUT5 structures4,5. Nevertheless, engineered PfHT1 mutations made to match GLUT sugar-binding sites did not shift sugar preferences. The extracellular substrate-gating helix TM7b in PfHT1 was positioned in a fully occluded conformation, providing a unique glimpse into how sugar binding and gating are coupled. We determined that polar contacts between TM7b and TM1 (located about 15 Å from D-glucose) are just as critical for transport as the residues that directly coordinate D-glucose, which demonstrates a strong allosteric coupling between sugar binding and gating. We conclude that PfHT1 has achieved substrate promiscuity not by modifying its sugar-binding site, but instead by evolving substrate-gating dynamics.
Asunto(s)
Malaria/parasitología , Proteínas de Transporte de Monosacáridos/química , Proteínas de Transporte de Monosacáridos/metabolismo , Plasmodium falciparum/química , Plasmodium falciparum/metabolismo , Azúcares/metabolismo , Regulación Alostérica , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sitios de Unión , Transporte Biológico , Cristalografía por Rayos X , Glucosa/química , Glucosa/metabolismo , Proteínas Facilitadoras del Transporte de la Glucosa/química , Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Humanos , Modelos Moleculares , Conformación Proteica , Especificidad por SustratoRESUMEN
Glucose transporters (GLUTs) are essential for organism-wide glucose homeostasis in mammals, and their dysfunction is associated with numerous diseases, such as diabetes and cancer. Despite structural advances, transport assays using purified GLUTs have proven to be difficult to implement, hampering deeper mechanistic insights. Here, we have optimized a transport assay in liposomes for the fructose-specific isoform GLUT5. By combining lipidomic analysis with native MS and thermal-shift assays, we replicate the GLUT5 transport activities seen in crude lipids using a small number of synthetic lipids. We conclude that GLUT5 is only active under a specific range of membrane fluidity, and that human GLUT1-4 prefers a similar lipid composition to GLUT5. Although GLUT3 is designated as the high-affinity glucose transporter, in vitro D-glucose kinetics demonstrates that GLUT1 and GLUT3 actually have a similar KM, but GLUT3 has a higher turnover. Interestingly, GLUT4 has a high KM for D-glucose and yet a very slow turnover, which may have evolved to ensure uptake regulation by insulin-dependent trafficking. Overall, we outline a much-needed transport assay for measuring GLUT kinetics and our analysis implies that high-levels of free fatty acid in membranes, as found in those suffering from metabolic disorders, could directly impair glucose uptake.
Asunto(s)
Ácidos Grasos no Esterificados , Liposomas , Humanos , Animales , Cinética , Transportador de Glucosa de Tipo 1/genética , Transportador de Glucosa de Tipo 3/genética , Glucosa , MamíferosRESUMEN
Amyloid ß-peptide (Aß) misfolding into ß-sheet structures triggers neurotoxicity inducing Alzheimer's disease (AD). Molecules able to reduce or to impair Aß aggregation are highly relevant as possible AD treatments since they should protect against Aß neurotoxicity. We have studied the effects of the interaction of dynorphins, a family of opioid neuropeptides, with Aß40 the most abundant species of Aß. Biophysical measurements indicate that Aß40 interacts with Big Dynorphin (BigDyn), lowering the amount of hydrophobic aggregates, and slowing down the aggregation kinetics. As expected, we found that BigDyn protects against Aß40 aggregates when studied in human neuroblastoma cells by cell survival assays. The cross-interaction between BigDyn and Aß40 provides insight into the mechanism of amyloid pathophysiology and may open up new therapy possibilities.
RESUMEN
Electrospray ionization mass spectrometry is increasingly applied to study the structures and interactions of membrane protein complexes. However, the charging mechanism is complicated by the presence of detergent micelles during ionization. Here, we show that the final charge of membrane proteins can be predicted by their molecular weight when released from the non-charge reducing saccharide detergents. Our data indicate that PEG detergents lower the charge depending on the number of detergent molecules in the surrounding micelle, whereas fos-choline detergents may additionally participate in ion-ion reactions after desolvation. The supercharging reagent sulfolane, on the other hand, has no discernible effect on the charge of detergent-free membrane proteins. Taking our observations into the context of protein-detergent interactions in the gas phase, we propose a charge equilibration model for the generation of native-like membrane protein ions. During ionization of the protein-detergent complex, the ESI charges are distributed between detergent and protein according to proton affinity of the detergent, number of detergent molecules, and surface area of the protein. Charge equilibration influenced by detergents determines the final charge state of membrane proteins. This process likely contributes to maintaining a native-like fold after detergent release and can be harnessed to stabilize particularly labile membrane protein complexes in the gas phase.
RESUMEN
Heterologous expression of human membrane proteins is a challenge in structural biology towards drug discovery. Here we report a complete expression and purification process of a functional human sodium/D-glucose co-transporter 1 (hSGLT1) in Pichia pastoris as representative example of a useful strategy for any human membrane protein. hSGLT1 gene was cloned in two different plasmids to develop parallel strategies: one which includes green fluorescent protein fusion for screening optimal conditions, and another for large scale protein production for structural biology and biophysics studies. Our strategy yields at least 1 mg of monodisperse purified recombinant hSGLT1 per liter of culture, which can be characterized by circular dichroism and infrared spectroscopy as an alpha-helical fold protein. This purified hSGLT1 transports co-substrates (Na+ and glucose) and it is inhibited by phlorizin in electrophysiological experiments performed in planar lipid membranes.
Asunto(s)
Ingeniería de Proteínas/métodos , Transportador 1 de Sodio-Glucosa/genética , Transportador 1 de Sodio-Glucosa/aislamiento & purificación , Glucosa/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Monosacáridos/genética , Pichia/genética , Plásmidos/genética , Proteínas Recombinantes/genética , Sodio/metabolismo , Proteínas de Transporte de Sodio-Glucosa/aislamiento & purificación , Proteínas de Transporte de Sodio-Glucosa/metabolismo , Transportador 1 de Sodio-Glucosa/metabolismo , Flujo de TrabajoRESUMEN
Alzheimer's disease (AD) is a neurodegenerative process characterized by the accumulation of extracellular deposits of amyloid ß-peptide (Aß), which induces neuronal death. Monomeric Aß is not toxic but tends to aggregate into ß-sheets that are neurotoxic. Therefore to prevent or delay AD onset and progression one of the main therapeutic approaches would be to impair Aß assembly into oligomers and fibrils and to promote disaggregation of the preformed aggregate. Albumin is the most abundant protein in the cerebrospinal fluid and it was reported to bind Aß impeding its aggregation. In a previous work we identified a 35-residue sequence of clusterin, a well-known protein that binds Aß, that is highly similar to the C-terminus (CTerm) of albumin. In this work, the docking experiments show that the average binding free energy of the CTerm-Aß1-42 simulations was significantly lower than that of the clusterin-Aß1-42 binding, highlighting the possibility that the CTerm retains albumin's binding properties. To validate this observation, we performed in vitro structural analysis of soluble and aggregated 1⯵M Aß1-42 incubated with 5⯵M CTerm, equimolar to the albumin concentration in the CSF. Reversed-phase chromatography and electron microscopy analysis demonstrated a reduction of Aß1-42 aggregates when the CTerm was present. Furthermore, we treated a human neuroblastoma cell line with soluble and aggregated Aß1-42 incubated with CTerm obtaining a significant protection against Aß-induced neurotoxicity. These in silico and in vitro data suggest that the albumin CTerm is able to impair Aß aggregation and to promote disassemble of Aß aggregates protecting neurons.