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Laboratory | Molecular mechanisms


Structure, function and regulation of disease-related membrane transport proteins

​The physiological and therapeutical relevance of Membrane Transport Proteins (MTPs) is reflected by the fact that MTPs comprise ~15 % of the FDA-approved human targets, and that ~18 % of total FDA-approved drugs target MTPs. We study the structure-function relationships of disease-related amino acid transporters and transporters encoded by malaria parasites. Our goal is to understand their biological role and pathophysiology, as well as to find new mechanisms for pharmaceutical intervention. To this end we combine structural biology, spectroscopy and biochemical tools, including protein engineering.

Published on 17 November 2020

Group leader 
José-Luis Vazquez-Ibar

Amino acid transporters 

Amino acid transporters are essentials players in human physiology as they participate on protein synthesis, energy metabolism, gene expression, redox balance or signal transduction pathways. One of the largest families of amino acid transporters is the L-amino acid transporter (LAT) family, where congenital mutatios of two members of this family cause two pathological disorders: cystinuria and lysinuric protein intolerance. In collaboration with Prof Manuel Palacín (IRB, Barcelona, Spain) we solved the X-ray structure of a LAT prokaryotic homolog: Adic (Kowalczyk, L et al, 2011, Figure 1), revealing for the first time an induced-fit mechanism during the first step of substrate-protein interaction. While two structures of mammalian LATs have recently appeared, prokaryotic LATs are still very valuable tools to continue deciphering the transport mechanism and regulation of their mammalian homologs. Using a directed evolution strategy (Rodriguez-Banqueri et al 2016), we have generated engineered versions of SteT (a prokaryotic paradigm of LATs) trapped in different stages along the catalytic cycle. Structure-function analyses of these mutants are expected to improve our understanding of the transport mechanism and lipid regulation of LATs.

Figure 1: Structure of a mutated version of Adic bound to the substrate, Arg+. (A) The transporter forms a homodimer (in purple, protomer 1 and in orange, protomer 2). The bound substrate is depicted with a ball-and-stick model. (B) Detail of the binding site. The structure shows the delocalization of the substrate in the binding site as a consequence of removing one hydrogen bond interaction between the substrate and the transporter, providing structural evidences of a substrate’s induce fit mechanism of these transporters.

In collaboration with Dr Bruno Gasnier (Paris-Descartes) and Dr Liang Feng (Stanford University, USA), we are also studying the lysosomal cationic amino acid transporter PQLC2. PQLC2 activity is likely to be linked with mTORC1 signaling by tightly controlling intralysosomal arginine levels. In addition, PQLC2 mediates the recruitment of the C9orf72 complex to the lysosomal surface. Mutations in the gene encoding C9orf72 causes amyotrophic lateral sclerosis and frontotemporal dementia. Our aims include unraveling the transport mechanism and regulation of PQLC2 as well as the study of the structural and functional consequences of PQLC2 interaction with the C9orf72 complex. 

Membrane transporters from malaria parasites 

Due to the intracellular mode of living of Plasmodium parasites, MTPs encoded by these parasites are a source of potential candidates for drug targeting, although most of them are still considered as putative. Notably, a high proportion of transporters with predicted role in lipid transport and in the maintenance of membrane lipid asymmetry are essential for the parasite; like ATP2, the Plasmodium P4-ATPase (or lipid flippase), recently identified as the possible target of two antimalarial drug candidates. Recombinant ATP2 associates with two of the three Plasmodium-encoded Cdc50 proteins (Cdc50A and Cdc50B), and functional analysis of purified ATP2/Cdc50B complex have identified possible physiological phospholipid substrates of ATP2 (Lamy et al, 2020).  Our next goals include a better understanding of the catalytic activity of ATP2 and the physiological meaning of ATP2 association with each of the Cdc50 subunits, and the finding of ATP2 inhibitors. We also aim at determining the 3D atomic structure of ATP2 in complex with these Cdc50 subunits.

Development of new methodologies to  study MTPs

MTPs are notoriously challenging proteins to produce in heterologous host. Also, their metastable nature is often an obstacle for their structural and functional characterization. We have developed new approaches and methodologies to circumvent some of these problems:

  1. We developed a strategy to engineer stability in a LAT transporter combining random mutagenesis, and a split-GFP complementation assay as reporter of protein expression and membrane insertion (Rodriguez-Banqueri A et al, 2012), (Rodriguez-Banqueri A et al, 2016), (Errasti-Murugarren E et al, 2017).

  2. We have used nanobodies (the heavy chain domains of camelide antibodies) against the GFP to purify the GFP-tagged ATP2 in complex with a Cdc50 beta-subunit, and to immobilize them in agarose beads for functional analysis (Lamy et al, 2020). In collaboration with Leandro Tabares and Sun Un, we are now aiming to extend the use of nanobodies for in vivo and in vitro structural characterization of MTPs (and complexes) using spectroscopic tools. 


  • Doñate-Macián P, Álvarez-Marimon E, Sepulcre F, Vázquez-Ibar JL, Perálvarez-Marín A (2019). Int J Mol Sci 20:682
  • Errasti-Murugarren E, Rodríguez-Banqueri A, Vázquez-Ibar JL (2017). Split GFP Complementation as Reporter of Membrane Protein Expression and Stability in E. coli: A Tool to Engineer Stability in a LAT Transporter. Methods Mol Biol 1586:181
  • Rodríguez-Banqueri A, Errasti-Murugarren E, Bartoccioni P, Kowalczyk L, Perálvarez-Marín A, Palacín M, Vázquez-Ibar JL (2016). Stabilization of a prokaryotic LAT transporter by random mutagenesis. J Gen Physiol 147:353
  • Doñate-Macian P, Bañó-Polo M, Vázquez-Ibar JL, Mingarro I, Perálvarez-Marín A (2015). Molecular and topological membrane folding determinants of transient receptor potential vanilloid 2 channel. Biochem Biophys Res Commun 462:221