Functional Consequences of Incomplete Hydrophobic Matching at TM1 of the LeuT Transporter

Abstract

The Leucine Transporter (LeuT) is the prototype for structure-function studies of mammalian Neurotransmitter: Sodium Symporters such as DAT, SERT and NET, the transporters for dopamine, serotonin, and norepinephrine, respectively. Its functional sensitivity to the environment, i.e., membranes or detergents in various compositions, has engaged much recent research. As the role of the environment in the function and organization of transmembrane proteins has been shown to involve hydrophobic mismatch, we investigated the membrane deformation and extent of hydrophobic matching for LeuT with the recently described hybrid Continuum-Molecular Dynamics (CTMD) method that combines elastic continuum formulations with an atomistic description of the lipid-protein interface from molecular dynamics simulations. The analysis was performed for functionally relevant conformations of LeuT embedded in two different model membranes: a POPC lipid bilayer and a model bacterial bilayer composed of a 3:1 mixture of POPE and POPG lipids. In both bilayers we found significant membrane thinning and water penetration near the membrane-facing Lys288 of TM7, a positively charged residue embedded deep inside the bilayer. This generates a polar environment near Lys288, but leads to unfavorable hydrophobic-polar interactions at neighboring membrane-facing hydrophobic residues in the cytoplasmic-end segments of TM1 (TM1a) and TM7. Analysis of the K288A mutant MD simulations showed that both membrane deformation and water penetration were eliminated, together with the unfavorable hydrophobic-polar interaction at TM1a and TM7. These results connect the hydrophobic mismatch due to the non-conserved Lys288 residue to a key structural element mediating transport, the TM1a segment that has been shown to move outward during substrate transport. In so doing, the results also provide mechanistic insights into how the K288A mutation, a background mutation used in some recent experimental studies, leads to significantly improved transport properties in LeuT.