Phenylalanine Transport through LAT1: Insights from Molecular Dynamics, Steered Molecular Dynamics, and Targeted Molecular Dynamics.

L-type amino acid transporter 1 (LAT1) is a heterodimeric membrane protein that primarily facilitates the transport of phenylalanine, along with other amino acids such as valine, leucine, isoleucine, tryptophan, and tyrosine across the cell membrane. It is predominantly expressed at the blood-brain barrier. While LAT1 has been extensively studied in the context of drug delivery, its specific transport pathway for phenylalanine and the related conformational dynamics remains largely unexplored. Investigation of the transport pathway of phenylalanine via LAT1 is also very important, as excessive amounts of phenylalanine can cause phenylketonuria (PKU). Inhibiting LAT1's transport could reduce phenylalanine's entry into the brain, aiding in the management of its levels and alleviating the adverse effects of PKU. To investigate the phenylalanine transport mechanism and the conformational dynamics of LAT1, microsecond-long molecular dynamics (MD) as well as steered molecular dynamics (SMD) and targeted molecular dynamics (TMD) simulations were performed. The results of this study identify essential structural motifs that enable the isomerization of LAT1 among outward-facing, inward-facing, and occluded states. A significant conclusion drawn from this research is the identification of seven critical transmembrane helices that play central roles along the transport pathway, namely, TM1, TM4, TM6, TM7, TM8, TM9, and TM12. Root-mean-square deviation (RMSD) analyses across the structural models further indicate that TM1 and TM6 are consistently engaged during both extracellular and intracellular phenylalanine transitions, whereas TM9 and TM12 contribute more specifically to the outward and inward opening events, respectively. The observed conformational changes offer fresh insights into the alternating-access mechanism of LAT1 during phenylalanine transport, contrasting with conclusions from structural models based on other substrates transport through LAT1. By elucidating this transport mechanism, the study will contribute to developing LAT1 inhibitors that can selectively regulate brain phenylalanine levels without disrupting the transport of other essential amino acids.