Molecular Dynamics Simulation Study of a Mutant Construct of the Archaeal Glutamate Transporter GltPh with Transport Rates as Fast as its Human Counterpart

Abstract

The glutamate transporter GltPh is a homolog of mammalian excitatory amino acid transporters (EAATs) that mediate glutamate re-uptake after discharge at the neuronal synaptic cleft, thereby enabling repeated signaling cycles and preventing excitotoxicity. While the structure of EAATs is not yet known, several stages of the transport cycle have been captured in crystallographic studies of the homotrimeric GltPh, suggesting a mechanism of separate elevator-like motions of three transport domains relative to a scaffold composed of the trimerization domains from each monomer. Dynamic aspects of the transport cycle monitored in single-molecule FRET experiments, revealed quiescent phases in which GltPh appears to be “locked” in the inward- or outward-facing states. Significantly higher transport rates, typical for human EAATs, were recently reported for a GltPh construct in which key residues were mutated to mimic the sequence of mammalian EAAT1. The mutant adopted a novel “unlocked” conformation, in which the transport domains were separated from the trimerization domains. Molecular dynamics simulations of this GltPh mutant in lipid bilayers found the unlocked conformation to be unstable if the transport/trimerization domain interface is solvated by water only, but stabilized by insertion of one or several hydrophobic moieties such as lipid tails. Analysis of the effect of the mutations on the local dynamics in several known stages of the transport cycle showed that a charged side chain introduced at the protein interface with the membrane produces membrane deformation and a destabilizing energy cost due to residual hydrophobic mismatch. Free energy perturbation calculations were used to estimate the impact of the mutation in the explored stages of the transport cycle in order to gain insights on the key structural determinants of EAAT transport efficiency.