Abstract
The molecular mechanism of transport mediated by LAT1, a sodium-independent antiporter of large neutral amino acids, was investigated through in silico procedures, specifically making reference to two transported substrates, tyrosine (Tyr) and leucine methyl ester (LME), and to 3,5-diiodo-L-tyrosine (DIT), a well-known LAT1 inhibitor. Two models of the transporter were built by comparative modeling, with LAT1 either in an outward-facing (OF) or in an inward-facing (IF) conformation, based, respectively, on the crystal structure of AdiC and of GadC. As frequently classic Molecular Dynamics (MD) fails to monitor large-scale conformational transitions within a reasonable simulated time, the OF structure was equilibrated for 150 ns then processed through targeted MD (tMD). During this procedure, an elastic force pulled the OF structure to the IF structure and induced, at the same time, substrates/inhibitor to move through the transport channel. This elastic force was modulated by a spring constant (k) value; by decreasing its value from 100 to 70, it was possible to comparatively account for the propensity for transport of the three tested molecules. In line with our expectations, during the tMD simulations, Tyr and LME behaved as substrates, moving down the transport channel, or most of it, for all k values. On the contrary, DIT behaved as an inhibitor, being (almost) transported across the channel only at the highest k value (100). During their transit through the channel, Tyr and LME interacted with specific amino acids (first with Phe252 then with Thr345, Arg348, Tyr259, and Phe262); this suggests that a primary as well as a putative secondary gate may contribute to the transport of substrates. Quite on the opposite, DIT appeared to establish only transient interactions with side chains lining the external part of the transport channel. Our tMD simulations could thus efficiently discriminate between two transported substrates and one inhibitor, and therefore can be proposed as a benchmark for developing novel LAT1 inhibitors of pharmacological interest.
Highlights
The L-type amino acid transporter 1 (LAT1, SLC7A5) is a ubiquitous Na+- and H+-independent antiporter, involved in cellular uptake of essential amino acids
Like other members of the amino-acid-polyamineorganocation (APC) family (Napolitano et al, 2017a), LAT1 is characterized by 12 transmembrane α-helices, which can be grouped in three different domains (Boudker and Verdon, 2010; Diallinas, 2014): α-helices 1–5 and 6–10 are involved in the transport mechanism and have an antiparallel symmetry in their secondary structures, while 11 and 12 are necessary for the homo-dimerization of the transporter
With our research, based on single continuous targeted MD (tMD) simulations, we propose, for the first time and at an atomistic level, the characterization of residues involved in LAT1 transport mechanism
Summary
The L-type amino acid transporter 1 (LAT1, SLC7A5) is a ubiquitous Na+- and H+-independent antiporter, involved in cellular uptake of essential amino acids. Natural mutations of LAT1 impair brain cells amino acid uptake and have been linked to Autism Spectrum Disorder (ASD) (Tarlungeanu et al, 2016) For these reasons, much attention is currently devoted to LAT1 transporter activity (Napolitano et al, 2017a,b; Scalise et al, 2018), and to its inhibition, e.g., with 3,5-diiodo-L-tyrosine (DIT), fenclonine, and acivicine (Geier et al, 2013; Schlessinger et al, 2013). The repetitive symmetry of the first two domains gives to the α-helices a discrete mobility within the cell membrane and defines the binding site for transported substrates, in the middle of the transport channel This structural feature is defined “LeuT fold,” from the name of the first crystallized transporter belonging to the APC family (Krammer et al, 2016; Kazmier et al, 2017).
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