Molecular dynamics simulations of Na⁺ and leucine transport by LeuT

Molecular dynamics simulations are used to gain insight into the binding of Na⁺ and leucine substrate to the bacterial amino acid transporter LeuT, focusing on the crystal structures of LeuT in the outward-open and inward-open states. For both conformations of LeuT, a third Na⁺ binding site involving Glu290 in addition to the two sites identified from the crystal structures is observed. Once the negative charge from Glu290 in the inward-open LeuT is removed, the ion bound to the third site is ejected from LeuT rapidly, suggesting that the protonation state of Glu290 regulates Na⁺ binding and release. In Cl⁻-dependent transporters where Glu290 is replaced by a neutral serine, a Cl⁻ ion would be required to replace the role of Glu290. Thus, the simulations provide insights into understanding Na⁺ and substrate transport as well as Cl⁻-independence of LeuT. - Highlights: • Ion binding site involving Glu290 is identified in the outward- and inward-open LeuT. • Sodium is released from inward-open LeuT once the side chain of Glu290 is protonated. • Protonation state of Glu290 regulates sodium binding and transport in LeuT