RESUMEN
The ATP-binding cassette transporter TAPL translocates polypeptides from the cytosol into the lysosomal lumen. TAPL can be divided into two functional units: coreTAPL, active in ATP-dependent peptide translocation, and the N-terminal membrane spanning domain, TMD0, responsible for cellular localization and interaction with the lysosomal associated membrane proteins LAMP-1 and LAMP-2. Although the structure and function of ABC transporters were intensively analyzed in the past, the knowledge about accessory membrane embedded domains is limited. Therefore, we expressed the TMD0 of TAPL via a cell-free expression system and confirmed its correct folding by NMR and interaction studies. In cell as well as cell-free expressed TMD0 forms oligomers, which were assigned as dimers by PELDOR spectroscopy and static light scattering. By NMR spectroscopy of uniformly and selectively isotope labeled TMD0 we performed a complete backbone and partial side chain assignment. Accordingly, TMD0 has a four transmembrane helix topology with a short helical segment in a lysosomal loop. The topology of TMD0 was confirmed by paramagnetic relaxation enhancement with paramagnetic stearic acid as well as by nuclear Overhauser effects with c6-DHPC and cross-peaks with water.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Péptidos/metabolismo , Sistema Libre de Células , Células HEK293 , Humanos , Péptidos/química , Unión Proteica , Dominios Proteicos , Pliegue de Proteína , Multimerización de Proteína , Estructura Secundaria de Proteína , Transporte de ProteínasRESUMEN
Methanogenic archaea share one ion gradient forming reaction in their energy metabolism catalyzed by the membrane-spanning multisubunit complex N(5)-methyl-tetrahydromethanopterin: coenzyme M methyltransferase (MtrABCDEFGH or simply Mtr). In this reaction the methyl group transfer from methyl-tetrahydromethanopterin to coenzyme M mediated by cobalamin is coupled with the vectorial translocation of Na(+) across the cytoplasmic membrane. No detailed structural and mechanistic data are reported about this process. In the present work we describe a procedure to provide a highly pure and homogenous Mtr complex on the basis of a selective removal of the only soluble subunit MtrH with the membrane perturbing agent dimethyl maleic anhydride and a subsequent two-step chromatographic purification. A molecular mass determination of the Mtr complex by laser induced liquid bead ion desorption mass spectrometry (LILBID-MS) and size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) resulted in a (MtrABCDEFG)3 heterotrimeric complex of ca. 430kDa with both techniques. Taking into account that the membrane protein complex contains various firmly bound small molecules, predominantly detergent molecules, the stoichiometry of the subunits is most likely 1:1. A schematic model for the subunit arrangement within the MtrABCDEFG protomer was deduced from the mass of Mtr subcomplexes obtained by harsh IR-laser LILBID-MS.
Asunto(s)
Proteínas Arqueales/química , Coenzimas/química , Proteínas de la Membrana/química , Methanobacteriaceae/química , Metiltransferasas/química , Pterinas/química , Proteínas Arqueales/metabolismo , Coenzimas/metabolismo , Espectrometría de Masas , Proteínas de la Membrana/metabolismo , Methanobacteriaceae/metabolismo , Metiltransferasas/metabolismo , Pterinas/metabolismoRESUMEN
The peptide-loading complex plays a pivotal role in Ag processing and is thus central to the efficient immune recognition of virally and malignantly transformed cells. The underlying mechanism by which MHC class I (MHC I) molecules sample immunodominant peptide epitopes, however, remains poorly understood. In this article, we delineate the interaction between tapasin (Tsn) and MHC I molecules. We followed the process of peptide editing in real time after ultra-fast photoconversion to pseudoempty MHC I molecules. Tsn discriminates between MHC I loaded with optimal and MHC I bound to suboptimal cargo. This differential interaction is key to understanding the kinetics of epitope proofreading. To elucidate the underlying mechanism at the atomic level, we modeled the Tsn/MHC I complex using all-atom molecular dynamics simulations. We present a catalytic working cycle, in which Tsn binds to MHC I with suboptimal cargo and thereby adjusts the energy landscape in favor of MHC I complexes with immunodominant epitopes.
Asunto(s)
Epítopos/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Péptidos/metabolismo , Secuencia de Aminoácidos , Epítopos/química , Epítopos/genética , Polarización de Fluorescencia , Antígeno HLA-B44/química , Antígeno HLA-B44/genética , Antígeno HLA-B44/metabolismo , Antígenos de Histocompatibilidad Clase I/química , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Epítopos Inmunodominantes/química , Epítopos Inmunodominantes/genética , Epítopos Inmunodominantes/metabolismo , Cinética , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/genética , Simulación de Dinámica Molecular , Mutación , Péptidos/química , Péptidos/genética , Unión Proteica , Proteína Disulfuro Isomerasas/química , Proteína Disulfuro Isomerasas/genética , Proteína Disulfuro Isomerasas/metabolismo , Estructura Terciaria de Proteína , TermodinámicaRESUMEN
Solution NMR studies of α-helical membrane proteins are often complicated by severe spectral crowding. In addition, hydrophobic environments like detergent micelles, isotropic bicelles or nanodiscs lead to considerably reduced molecular tumbling rates which translates into line-broadening and low sensitivity. Both difficulties can be addressed by selective isotope labeling methods. In this publication, we propose a combinatorial protocol that utilizes four different classes of labeled amino acids, in which the three backbone heteronuclei (amide nitrogen, α-carbon and carbonyl carbon) are enriched in (15)N or (13)C isotopes individually as well as simultaneously. This results in eight different combinations of dipeptides giving rise to cross peaks in (1)H-(15)N correlated spectra. Their differentiation is achieved by recording a series of HN-detected 2D triple-resonance spectra. The utility of this new scheme is demonstrated with a homodimeric 142-residue membrane protein in DHPC micelles. Restricting the number of selectively labeled samples to three allowed the identification of the amino-acid type for 77 % and provided sequential information for 47 % of its residues. This enabled us to complete the backbone resonance assignment of the uniformly labeled protein merely with the help of a 3D HNCA spectrum, which can be collected with reasonable sensitivity even for relatively large, non-deuterated proteins.
Asunto(s)
Isótopos de Carbono/química , Proteínas de la Membrana/química , Isótopos de Nitrógeno/química , Resonancia Magnética Nuclear Biomolecular/métodos , Isótopos de Carbono/metabolismo , Proteínas de la Membrana/metabolismo , Isótopos de Nitrógeno/metabolismo , Estructura Secundaria de ProteínaRESUMEN
ATP-binding cassette (ABC) transporters use ATP to drive solute transport across biological membranes. Members of this superfamily have crucial roles in cell physiology, and some of the transporters are linked to severe diseases. However, understanding of the transport mechanism, especially of human ABC exporters, is scarce. We reconstituted the human lysosomal polypeptide ABC transporter TAPL, expressed in Pichia pastoris, into lipid vesicles (liposomes) and performed explicit transport measurements. We analyzed solute transport at the single liposome level by monitoring the coincident fluorescence of solutes and proteoliposomes in the focal volume of a confocal microscope. We determined a turnover number of eight peptides per minute, which is two orders of magnitude higher than previously estimated from macroscopic measurements. Moreover, we show that TAPL translocates peptides against a large concentration gradient. Maximal filling is not limited by an electrochemical gradient but by trans-inhibition. Countertransport and reversibility studies demonstrate that peptide translocation is a strictly unidirectional process. Altogether, these data are included in a refined model of solute transport by ABC exporters.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Liposomas , Péptidos/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Humanos , Pichia/genética , Transporte de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Combinatorial triple-selective labeling facilitates the NMR assignment process for proteins that are subject to signal overlap and insufficient signal-to-noise in standard triple-resonance experiments. Aiming at maximum amino-acid type and sequence-specific information, the method represents a trade-off between the number of selectively labeled samples that have to be prepared and the number of spectra to be recorded per sample. In order to address the demand of long measurement times, we here propose pulse sequences in which individual phase-shifted transients are stored separately and recombined later to produce several 2D HN(CX) type spectra that are usually acquired sequentially. Sign encoding by the phases of (13)C 90° pulses allows to either select or discriminate against (13)C' or (13)C(α) spins coupled to (15)N. As a result, (1)H-(15)N correlation maps of the various isotopomeric species present in triple-selectively labeled proteins are deconvoluted which in turn reduces problems due to spectral overlap. The new methods are demonstrated with four different membrane proteins with rotational correlation times ranging from 18 to 52 ns.
Asunto(s)
Algoritmos , Espectroscopía de Resonancia Magnética/métodos , Proteínas de la Membrana/química , Proteínas de la Membrana/ultraestructura , Mapeo Peptídico/métodos , Procesamiento de Señales Asistido por Computador , Secuencia de Aminoácidos , Datos de Secuencia Molecular , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Relación Señal-Ruido , Marcadores de SpinRESUMEN
5-Lipoxygenase (5-LOX) is the key player of pro-inflammatory leukotriene biosynthesis. Its regulatory or so-called PLAT (polycystin-1, lipoxygenase, α-toxin) domain binds allosteric modulators like calcium, membranes, coactosin-like protein and Dicer, thereby influencing 5-LOX activity at the nuclear membrane by mediating translocation. The PLAT domain may also regulate cytosolic 5-LOX activity and possibly influence microRNA metabolism. Hence, it has also evolved as a promising target for anti-inflammatory therapy. Research focusing on this substructure of 5-LOX requires an assay system based on the isolated domain. However, we found that the isolated PLAT domain was highly prone to aggregation and therefore unsuitable for interaction studies. Substitution of the single, membrane-binding tryptophan 75 with glycine reduced aggregation and substantially increased its thermal stability. Calcium interaction of the single mutant was confirmed by differential scanning fluorimetry. Moreover, crosslinking experiments demonstrated the ability of the isolated PLAT domain to bind Dicer C-terminus whereas the interaction with coactosin-like protein required the interplay of the catalytic and the PLAT domain.
RESUMEN
Immunosurveillance of tumor cells depends on NKp30, a major activating receptor of human natural killer (NK) cells. The human BCL2-associated athanogene 6 (BAG-6, also known as BAT3; 1126 amino acids) is a cellular ligand of NKp30. To date, little is known about the molecular details of this receptor ligand system. Within the current study, we have located the binding site of NKp30 to a sequence stretch of 250 amino acids in the C-terminal region of BAG-6 (BAG-6(686-936)). BAG-6(686-936) forms a noncovalent dimer of 57-59 kDa, which is sufficient for high affinity interaction with NKp30 (KD < 100 nM). As our most important finding, BAG-6(686-936) inhibits NKp30-dependent signaling, interferon-γ release, and degranulation of NK cells in the presence of malignantly transformed target cells. Based on these data, we show for the first time that BAG-6(686-936) comprises a subdomain of BAG-6, which is sufficient for receptor docking and inhibition of NKp30-dependent NK cell cytotoxicity as part of a tumor immune escape mechanism. These molecular insights provide an access point to restore tumor immunosurveillance by NK cells and to increase the efficacy of cellular therapies.
Asunto(s)
Citotoxicidad Inmunológica , Células Asesinas Naturales/metabolismo , Chaperonas Moleculares/metabolismo , Receptor 3 Gatillante de la Citotoxidad Natural/metabolismo , Neoplasias/genética , Animales , Sitios de Unión , Degranulación de la Célula/inmunología , Células HEK293 , Humanos , Interferón gamma/metabolismo , Células Asesinas Naturales/inmunología , Ligandos , Ratones , Chaperonas Moleculares/genética , Chaperonas Moleculares/inmunología , Receptor 3 Gatillante de la Citotoxidad Natural/genética , Receptor 3 Gatillante de la Citotoxidad Natural/inmunología , Neoplasias/inmunología , Neoplasias/patología , Unión ProteicaRESUMEN
The ATP binding cassette transporter TAPL translocates cytosolic peptides into the lumen of lysosomes driven by the hydrolysis of ATP. Functionally, this transporter can be divided into coreTAPL, comprising the transport function, and an additional N-terminal transmembrane domain called TMD0, which is essential for lysosomal targeting and mediates the interaction with the lysosomal associated membrane proteins LAMP-1 and LAMP-2. To elucidate the structure of this unique domain, we developed protocols for the production of high quantities of cell-free expressed TMD0 by screening different N-terminal expression tags. Independently of the amino acid sequence, high expression was detected for AU-rich sequences in the first seven codons, decreasing the free energy of RNA secondary structure formation at translation initiation. Furthermore, avoiding NGG codons in the region of translation initiation demonstrated a positive effect on expression. For NMR studies, conditions were optimized for high solubilization efficiency, long-term stability, and high quality spectra. A most critical step was the careful exchange of the detergent used for solubilization by the detergent dihexanoylphosphatidylcholine. Several constructs of different size were tested in order to stabilize the fold of TMD0 as well as to reduce the conformation exchange. NMR spectra with sufficient resolution and homogeneity were finally obtained with a TMD0 derivative only modified by a C-terminal His10-tag and containing a codon optimized AT-rich sequence.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Proteínas de la Membrana/química , Resonancia Magnética Nuclear Biomolecular , Sistema Libre de Células , Codón/genética , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Estabilidad Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , ARN Mensajero/química , Solubilidad , SolucionesRESUMEN
The transporter associated with antigen processing-like (TAPL) is a polypeptide transporter translocating cytosolic peptides into the lumen of lysosomes driven by ATP hydrolysis. TAPL belongs to the family of ABC transporters and forms a homodimer. This ABC transporter not only shows a broad tissue but also a wide phylogenetic distribution, because orthologs are still found in nematodes and insects. Here, we present the topology, substrate specificity, and distribution of this intracellular polypeptide transporter. Additionally, we will discuss its proposed physiological functions such as housekeeping together with a specialized factor for metabolite storage as well as for the adaptive immunity.