Please use this identifier to cite or link to this item: http://hdl.handle.net/2445/59883
Title: Structural studies of Heteromeric Amino acid Transporters (HATs): Validation of the first 3D structural model of a HAT (human 4F2hc/LAT2) and identification of new HAT targets for 3D‐crystallization
Author: Álvarez Marimón, Mª Elena
Director: Palacín Prieto, Manuel
Rosell Febres, Albert
Keywords: Aminoàcids
Proteïnes de membrana
Visualització tridimensional
Purificació de proteïnes
Amino acids
Membrane proteins
Three-dimensional display systems
Protein purification
Issue Date: 27-Oct-2014
Publisher: Universitat de Barcelona
Abstract: [spa] Los transportadores heteroméricos de aminoácidos (HAT) median el transporte de aminoácidos a través de la membrana plasmática. Representan el único ejemplo de transportadores de solutos formado por dos subunidades distintas unidas por un puente disulfuro. Debido a su gran relevancia en fisiología (asociados a aminoacidurias, infección por virus, cáncer,…) el estudio de su estructura-función resulta clave. Debido a su naturaleza, son proteínas difíciles de cristalizar, de las que sólo se conoce la estructura atómica del ectodominio de 4F2hc humano. En este escenario la tesis se centró en la validación del primer modelo 3D a baja resolución de un HAT humano (4F2hc/LAT2), mediante experimentos de crosslinking entre subunidades, e identificación de nuevos candidatos para cristalización 3D. Para ello se seleccionaron 24 subunidades ligeras de distintas especies eucariotas y se testaron en un proceso de selección para determinar el/los mejores candidatos. El primer objetivo de la tesis concluyó con la determinación de residuos concretos en 4F2hc y LAT2 cercanos a una distancia de 3-14 Å mediante la utilización de crosslinkings de cisteínas. Finalmente, tres líneas distintas: el modelo 3D obtenido por microscopía electrónica de transmisión y tinción negativa de partículas individuales (en colaboración con Dr. Fotiadis), los experimentos de crosslinking, y el docking generado en colaboración con Dr. Fdz-Recio, demostraron que 4F2hc-ED cubre, casi completamente, la superficie extracelular de LAT2. Además, se demostró que el ectodominio de 4F2hc es suficiente para estabilizar LAT2. Como resultados del segundo objetivo, tres subunidades ligeras fueron seleccionadas, tras adaptar el protocolo desarrollado por Drew et al.,2008, como mejores candidatas para estudios de cristalización 3D. Posteriormente, distintas estrategias se siguieron para mejorar la estabilidad de la mejor candidata: eliminación de la cisteína reactiva, adición de lípidos a la muestra, cambio de sistema de expresión para aumentar su expresión a células de insecto Sf9. Además, se generaron mutantes delecionados en N y C terminal para reducir su flexibilidad y aumentar la probabilidad de cristalización. Se concluyó en encontrar un buen candidato para estudios de cristalización.
[eng] Heteromeric amino acid transporters (HATs) mediate the transport of amino acids through the plasma membrane. They are composed of two subunits (a heavy and a light one) linked by a conserved disulfide bridge. Genetic defects in the genes coding these HATs may affect its functionality or expression, leading to inherited aminoacidurias. Thus, solving the structure of the Eukaryotic HATs has become of great importance. However structural information about interactions between the heavy and light subunits of HATs is scarce. In this work, human 4F2hc/L-type amino acid transporter 2 (LAT2) first low resolution 3D model obtained by single particle negative-staining transmission electron microscopy (TEM) was validated. In order to assess the interaction between both subunits of the heterodimer, crosslinking experiments between cysteine residues in both moieties was tried. Namely, two chemical spacers of different length (10.5 and 14.3 Å) were tested and crosslinking was observed for those mutants with pairing positions between 8 and 17.5 Å. Indeed, specific residues that crosslinked 4F2hc and LAT2 nearly completely (>80%). As a result of the positive results (as compared to the appropriate controls) the idea that 4F2hc-ED almost completely covers the extracellular surface of the transporter subunit LAT2 is reasonable. Moreover, further varied evidences (TEM, SPA and docking experiments) were in line with the obtained results, revealing that the extracellular domain of 4F2hc interacts with LAT2, almost completely covering the extracellular face of the transporter. The interaction of 4F2hc with LAT2 gives insights into the structural bases for light subunit recognition and the stabilizing role of the ancillary protein in HATs. In addition, it has been demonstrated that the ectodomain of 4F2hc suffices the stabilization of the light subunit. The second goal of the thesis was to find a suitable HAT candidate to perform crystallization trials and posterior structure elucidation, since human 4F2h/LAT2 was not stable enough for this aim. Until now, only the human 4F2hc ectodomain atomic structure has been solved (Fort et al., 2007), and some low sequence amino acid identity prokaryotic homologues of LATs. In order to identify putative good eukaryotic light subunits for 3D crystallization the adopted GFP‐based Saccharomyces cerevisiae protocol for our transporters resulted successful since it allowed to find three putative good candidate eukaryotic light subunits for 3D crystallization studies. GFP technology allowed quick expression screening, membrane protein-detergent solubilization screening and finally another screening step including assessment of the stability by ultracentrifugation dispersity sedimentation. Once the candidates selected in the best conditions, further purification was required (size exclusion chromatography) before attempting crystallization. In this sense, further efforts were delivered in order to try to enhance the stability (and minimize aggregation). Thus, addition of lipids in the solubilization step and during protein purification was used to mimic the protein membrane environment and reduce the aggregation. Really interesting is the stabilizing effect that cholesterol has on almost all light subunits tested. This is in concordance with the fact that 4F2hc has been found located in lipid rafts where membrane are rich in cholesterol. Removal of reactive cysteine and generation of truncated versions of the protein in the C and N terminal were introduced to increase the crystallization probability. This work ended with the finding of 3 good candidates for crystallization screenings, and the best candidate was optimized in terms of stability and protein flexibility for crystallization studies. Just preliminary crystallization trials were done.
URI: http://hdl.handle.net/2445/59883
Appears in Collections:Tesis Doctorals - Departament - Bioquímica i Biologia Molecular (Biologia)

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