Stabilization of a prokaryotic LAT transporter by random mutagenesis

Rodríguez-Banqueri, Arturo; Errasti-Murugarren, Ekaitz; Bartoccioni, Paola; Kowalczyk, Lukasz; Perálvarez-Marín, Àlex; Palacı́n, Manuel; Vázquez-Ibar, José Luís

doi:10.1085/jgp.201511510

Cited by 10 publications

(13 citation statements)

References 50 publications

(95 reference statements)

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“…In contrast, a fraction of PcATP2 and PcCdc50B from P3 membranes was solubilized in N-dodecyl-β-D-maltopyranoside (DDM) and, with less efficiency, in Lauryl maltose neopentyl-glycol (LMNG) or Octaethylene glycol monodecyl ether (C12E8) (Figure supplement 3). The fact that only the PcATP2 and PcCdc50B fractions present in P3 could be solubilized in DDM strongly suggests a folding defect of the P2-containing proteins because DDM is know to be poorly efficient fo solubilize unfolded or partially folded integral membrane proteins[ 41 ]. Importantly, the solubilization efficiency of these detergents was greatly improved by the presence of the cholesterol derivative, cholesteryl hemisuccinate (CHS) (Figure supplement 3).…”

Section: Resultsmentioning

confidence: 99%

“…We fused the superfolder green fluorescent protein (GFP) at the C-terminal end of PcATP2 followed by the BAD (PcATP2-GFP-BAD). We chose to tag PcATP2 at the C-terminal end rather than the N-terminal like the previous experiments because the GFP is a more sensitive folding reporter when fused at the C-terminal end of the target protein [ 41 ]. The presence of both co-expressed proteins in P3 membranes was confirmed by western blot (Figure supplement 5, lanes 1 and 2).…”

Section: Resultsmentioning

confidence: 99%

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ATP2, The essential P4-ATPase of malaria parasites, catalyzes lipid-stimulated ATP hydrolysis in complex with a Cdc50 β-subunit

Lamy

Macarini-Bruzaferro

Dieudonné

et al. 2021

Emerging Microbes & Infections

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Gene targeting approaches have demonstrated the essential role for the malaria parasite of membrane transport proteins involved in lipid transport and in the maintenance of membrane lipid asymmetry, representing emerging oportunites for therapeutical intervention. This is the case of ATP2, a Plasmodium -encoded 4 P-type ATPase (P4-ATPase or lipid flippase), whose activity is completely irreplaceable during the asexual stages of the parasite. Moreover, a recent chemogenomic study has situated ATP2 as the possible target of two antimalarial drug candidates. In eukaryotes, P4-ATPases assure the asymmetric phospholipid distribution in membranes by translocating phospholipids from the outer to the inner leaflet. In this work, we have used a recombinantly-produced P. chabaudi ATP2 (PcATP2), to gain insights into the function and structural organization of this essential transporter. Our work demonstrates that PcATP2 associates with two of the three Plasmodium- encoded Cdc50 proteins: PcCdc50B and PcCdc50A. Purified PcATP2/PcCdc50B complex displays ATPase activity in the presence of either phosphatidylserine or phosphatidylethanolamine. In addition, this activity is upregulated by phosphatidylinositol 4-phosphate. Overall, our work describes the first biochemical characterization of a Plasmodium lipid flippase, a first step towards the understanding of the essential physiological role of this transporter and towards its validation as a potential antimalarial drug target.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

ATP2, The essential P4-ATPase of malaria parasites, catalyzes lipid-stimulated ATP hydrolysis in complex with a Cdc50 β-subunit

Lamy

Macarini-Bruzaferro

Dieudonné

et al. 2021

Emerging Microbes & Infections

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“…For this reason, SteT emerged as a good bacterial paradigm of LATs. Nevertheless, although a great deal of effort in recent years has been channeled into improving SteT stability (Rodríguez-Banqueri et al, 2016), crystallization assays have failed to render results, thus fueling the search for a new prokaryotic paradigm suitable for atomic structure determination.…”

Section: Discussionmentioning

confidence: 99%

Functional characterization of the alanine-serine-cysteine exchanger of Carnobacterium sp AT7

Bartoccioni

Fort

Zorzano

et al. 2019

Journal of General Physiology

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Many key cell processes require prior cell uptake of amino acids from the environment, which is facilitated by cell membrane amino acid transporters such as those of the L-type amino acid transporter (LAT) subfamily. Alterations in LAT subfamily amino acid transport are associated with several human diseases, including cancer, aminoacidurias, and neurodegenerative conditions. Therefore, from the perspective of human health, there is considerable interest in obtaining structural information about these transporter proteins. We recently solved the crystal structure of the first LAT transporter, the bacterial alanine-serine-cysteine exchanger of Carnobacterium sp AT7 (BasC). Here, we provide a complete functional characterization of detergent-purified, liposome-reconstituted BasC transporter to allow the extension of the structural insights into mechanistic understanding. BasC is a sodium- and proton-independent small neutral amino acid exchanger whose substrate and inhibitor selectivity are almost identical to those previously described for the human LAT subfamily member Asc-1. Additionally, we show that, like its human counterparts, this transporter has apparent affinity asymmetry for the intra- and extracellular substrate binding sites—a key feature in the physiological role played by these proteins. BasC is an excellent paradigm of human LAT transporters and will contribute to our understanding of the molecular mechanisms underlying substrate recognition and translocation at both sides of the plasma membrane.

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“…Protein engineering is one of the most widely used and successful strategies for conferring desirable physical chemistry properties to a membrane protein for structural studies [ 29 , 30 ]. In particular, the generation of membrane protein mutant libraries and identification of the best-expressed and most stable versions of these have been widely used [ 31 , 32 , 33 ]. Sources of mutation include rational design, random mutagenesis, scanning mutagenesis, and consensus mutation.…”

Section: Membrane Protein Mutagenesismentioning

confidence: 99%

“…Protein engineering by point mutations, especially in transmembrane domains, can overcome membrane protein instability [ 32 , 34 , 35 ]. Given that interactions between transmembrane regions are the major determinants of integral membrane protein folding and insertion [ 36 ], side-chain substitutions within these domains are likely to have a greater impact on protein stability [ 37 , 38 ].…”

Section: Membrane Protein Mutagenesismentioning

confidence: 99%

Membrane Protein Stabilization Strategies for Structural and Functional Studies

2021

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Accounting for nearly two-thirds of known druggable targets, membrane proteins are highly relevant for cell physiology and pharmacology. In this regard, the structural determination of pharmacologically relevant targets would facilitate the intelligent design of new drugs. The structural biology of membrane proteins is a field experiencing significant growth as a result of the development of new strategies for structure determination. However, membrane protein preparation for structural studies continues to be a limiting step in many cases due to the inherent instability of these molecules in non-native membrane environments. This review describes the approaches that have been developed to improve membrane protein stability. Membrane protein mutagenesis, detergent selection, lipid membrane mimics, antibodies, and ligands are described in this review as approaches to facilitate the production of purified and stable membrane proteins of interest for structural and functional studies.

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Stabilization of a prokaryotic LAT transporter by random mutagenesis

Abstract: A fluorescence-based screen was used to analyze 70 LAT transporter mutants and identify variants with improved stability and monodispersity.

Cited by 10 publications

References 50 publications

ATP2, The essential P4-ATPase of malaria parasites, catalyzes lipid-stimulated ATP hydrolysis in complex with a Cdc50 β-subunit

ATP2, The essential P4-ATPase of malaria parasites, catalyzes lipid-stimulated ATP hydrolysis in complex with a Cdc50 β-subunit

Functional characterization of the alanine-serine-cysteine exchanger of Carnobacterium sp AT7

Membrane Protein Stabilization Strategies for Structural and Functional Studies

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