Computational Account of the First Stages in the Elevator Motion of the Human EAAT3 Glutamate Transporter

Abstract

The plasma membrane excitatory amino acid transporters (EAATs) belong to mammalian glutamate transporter family that regulates levels of L-Glu in the synaptic cleft. The transport cycle mechanism of the EAATs is believed to be similar to that in its prokaryotic homolog GltPh and involves elevator motion of the transport domain that travels ∼ 2 nm across the membrane from the outward to inward facing state (OFS and IFS). Although EAAT3 has the highest turnover rate among the members of the family (∼ 100 s−1), a thorough molecular dynamical (MD) study of the full transport cycle requires significant computational effort. From extensive MD simulations (∼ 1 millisecond) of WT in fully loaded and apo- states we have extracted structural time-correlations, and along with results obtained with the N-body Information Theory (NbIT) analysis concluded on the main structural elements of the elevator motion. These were verified with in silico mutagenesis of the residues within these structural elements in a set of additional MD simulations. The dynamics of two hairpin motifs in the transport domain (HP1 and HP2) and conformations that favor the motion emerged from these studies, and the role of elongated extracellular loop of EAAT3 that is absent in the GltPh in initiating the elevator motion was outlined. Notably, application of our new equilibrium constant pH method (ECpH) to reveal the pH dependence of EAAT3 function yielded structural insight into the motion of the HP2 and HP1 gates, the role of the Glu374 protonation state, and the interactions influencing the gate opening in HP2 that hinders the substrate transportation.