RESUMEN
ATP-sensitive potassium channels (KATP) are inhibited by ATP but activated by Mg-ADP, coupling the intracellular ATP/ADP ratio to the potassium conductance of the plasma membrane. Although there has been progress in determining the structure of KATP, the functional significance of the domain-domain interface in the gating properties of KATP channels remains incompletely understood. In this study, we define the structure of KATP as two modules: KATPcore and SURABC. Based on this model, we identified two functionally important interfaces between these two modules, namely interface I and interface II. Further structure-guided mutagenesis experiments indicate that destabilizing interface II by deleting ECL3 on the SUR1 subunit impairs KNtp-independent Mg-ADP activation, demonstrating the essential role of intramolecular interactions between KATPcore and SURABC in Mg-ADP activation. Additionally, interface II is functionally conserved between SUR1 and SUR2, and the hydrophobic residue F351 on ECL3 of SUR1 is crucial for maintaining the stability of this interface.
Asunto(s)
Canales KATP , Canales de Potasio de Rectificación Interna , Canales KATP/genética , Canales KATP/metabolismo , Receptores de Sulfonilureas/genética , Receptores de Sulfonilureas/metabolismo , Canales de Potasio de Rectificación Interna/genética , Canales de Potasio de Rectificación Interna/química , Canales de Potasio de Rectificación Interna/metabolismo , Adenosina Trifosfato/metabolismo , Membrana Celular/metabolismoRESUMEN
In this work, eight van der Waals heterojunctions based on CdSe or CdSe-ZnS quantum dots (QDs) and four commonly used two-dimensional transition metal dichalcogenides (2D-TMDs) are theoretically designed. On the basis of the constructed structures, density functional theory (DFT) method is employed to investigate the structural and optoelectronic related properties of these heterojunctions in detail. Specifically, their electronic properties including charge density differences, density of states, and band offsets are calculated, based on which band alignment types as well as their potentials as novel photovoltaic materials are discussed. According to these calculations, we proposed that several van der Waals heterostructures including MoS2/CdSe, MoTe2/CdSe, WSe2/CdSe, MoTe2/CdSe-ZnS, and WSe2/CdSe-ZnS might be used as potential photovoltaic materials due to their type II band alignment characteristics. Moreover, the WSe2/CdSe-ZnS heterostructure is expected to have optimal photovoltaic performance attributed to their large bond offsets and band gaps, which could not only facilitate charge separation processes, but also slow down charge recombination. Our present theoretical work could be helpful for the future experimental design of novel CdSe QDs and 2D-TMD based van der Waals heterostructures with excellent photovoltaic performances.
RESUMEN
ATP binding cassette (ABC) proteins typically function in active transport of solutes across membranes. The ABC core structure is composed of two transmembrane domains (TMD1 and TMD2) and two cytosolic nucleotide binding domains (NBD1 and NBD2). Some members of the C-subfamily of ABC (ABCC) proteins, including human multidrug resistance proteins (MRPs), also possess an N-terminal transmembrane domain (TMD0) that contains five transmembrane α-helices and is connected to the ABC core by the L0 linker. While TMD0 was resolved in SUR1, the atypical ABCC protein that is part of the hetero-octameric ATP-sensitive K+ channel, little is known about the structure of TMD0 in monomeric ABC transporters. Here, we present the structure of yeast cadmium factor 1 protein (Ycf1p), a homolog of human MRP1, determined by electron cryo-microscopy (cryo-EM). A comparison of Ycf1p, SUR1, and a structure of MRP1 that showed TMD0 at low resolution demonstrates that TMD0 can adopt different orientations relative to the ABC core, including a â¼145° rotation between Ycf1p and SUR1. The cryo-EM map also reveals that segments of the regulatory (R) region, which links NBD1 to TMD2 and was poorly resolved in earlier ABCC structures, interacts with the L0 linker, NBD1, and TMD2. These interactions, combined with fluorescence quenching experiments of isolated NBD1 with and without the R region, suggest how posttranslational modifications of the R region modulate ABC protein activity. Mapping known mutations from MRP2 and MRP6 onto the Ycf1p structure explains how mutations involving TMD0 and the R region of these proteins lead to disease.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/química , Procesamiento Proteico-Postraduccional , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Sitios de Unión , Membrana Celular/metabolismo , Clonación Molecular , Microscopía por Crioelectrón , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Modelos Moleculares , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos/química , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos/genética , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Fosforilación , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de Aminoácido , Receptores de Sulfonilureas/química , Receptores de Sulfonilureas/genética , Receptores de Sulfonilureas/metabolismoRESUMEN
The human lysosomal polypeptide ABC transporter TAPL (ABC subfamily B member 9, ABCB9) transports 6-59-amino-acid-long polypeptides from the cytosol into lysosomes. The subcellular localization of TAPL depends solely on its N-terminal transmembrane domain, TMD0, which lacks conventional targeting sequences. However, the intracellular route and the molecular mechanisms that control TAPL localization remain unclear. Here, we delineated the route of TAPL to lysosomes and investigated the determinants of single trafficking steps. By synchronizing trafficking events by a retention using selective hooks (RUSH) assay and visualizing individual intermediate steps through immunostaining and confocal microscopy, we demonstrate that TAPL takes the direct route to lysosomes. We further identified conserved charged residues within TMD0 transmembrane helices that are essential for individual steps of lysosomal targeting. Substitutions of these residues retained TAPL in the endoplasmic reticulum (ER) or Golgi. We also observed that for release from the ER, a salt bridge between Asp-17 and Arg-57 is essential. An interactome analysis revealed that Yip1-interacting factor homolog B membrane-trafficking protein (YIF1B) interacts with TAPL. We also found that YIF1B is involved in ER-to-Golgi trafficking and interacts with TMD0 of TAPL via its transmembrane domain and that this interaction strongly depends on the newly identified salt bridge within TMD0. These results expand our knowledge about lysosomal trafficking of TAPL and the general function of extra transmembrane domains of ABC transporters.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/química , Membrana Celular/metabolismo , Células HeLa , Humanos , Chaperonas Moleculares/metabolismo , Unión Proteica , Pliegue de Proteína , Transporte de Proteínas , Fracciones Subcelulares/metabolismoRESUMEN
ATP-Binding cassette (ABC) transporters play an extensive role in the translocation of diverse sets of biologically important molecules across membrane. EchnocandinB (antifungal) and EcdL protein of Aspergillus rugulosus are encoded by the same cluster of genes. Co-expression of EcdL and echinocandinB reflects tightly linked biological functions. EcdL belongs to Multidrug Resistance associated Protein (MRP) subfamily of ABC transporters with an extra transmembrane domain zero (TMD0). Complete structure of MRP subfamily comprising of TMD0 domain, at atomic resolution is not known. We hypothesized that the transportation of echonocandinB is mediated via EcdL protein. Henceforth, it is pertinent to know the topological arrangement of TMD0, with other domains of protein and its possible role in transportation of echinocandinB. Absence of effective template for TMD0 domain lead us to model by I-TASSER, further structure has been refined by multiple template modelling using homologous templates of remaining domains (TMD1, NBD1, TMD2, NBD2). The modelled structure has been validated for packing, folding and stereochemical properties. MD simulation for 0.1 µs has been carried out in the biphasic environment for refinement of modelled protein. Non-redundant structures have been excavated by clustering of MD trajectory. The structural alignment of modelled structure has shown Z-score -37.9; 31.6, 31.5 with RMSD; 2.4, 4.2, 4.8 with ABC transporters; PDB ID 4F4C, 4M1 M, 4M2T, respectively, reflecting the correctness of structure. EchinocandinB has been docked to the modelled as well as to the clustered structures, which reveals interaction of echinocandinB with TMD0 and other TM helices in the translocation path build of TMDs.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Proteínas Fúngicas/química , Modelos Moleculares , Conformación Proteica , Humanos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Relación Estructura-ActividadRESUMEN
ABCC6 is a human ATP binding cassette (ABC) transporter of the plasma membrane associated with Pseudoxanthoma elasticum (PXE), an autosomal recessive disease characterized by ectopic calcification of elastic fibers in dermal, ocular and vascular tissues. Similar to other ABC transporters, ABCC6 encloses the core structure of four domains: two transmembrane domains (TMDs) and two nucleotide binding domains (NBDs) but also an additional N-terminal extension, including a transmembrane domain (TMD0) and a cytosolic loop (L0), which is only found in some members of ABCC subfamily, and for which the function remains to be established. To investigate the functional roles of this N-terminal region, we generated several domain deletion constructs of ABCC6, expressed in HEK293 and polarized LLC-PK1 cells. ABCC6 lacking TMD0 displayed full transport activity as the wild type protein. Unlike the wild type protein, ABCC6 without L0 was not targeted to the basolateral membrane. Moreover, homology modeling of L0 suggests that it forms an ATPase regulatory domain. Furthermore, we show that the expression of ABCC6 is linked to a cellular influx of Ca(2+). The results suggest that TMD0 is not required for transport function and that L0 maintains ABCC6 in a targeting-competent state for the basolateral membrane and might be involved in regulating the NBDs. These findings shed new light on a possible physiological function of ABCC6 and may explain some of the hallmarks of the clinical features associated with PXE that could contribute to the identification of novel pharmacological targets.