The mechanism of substrate release by the aspartate transporter Glt_Ph: insights from simulations

Abstract

Glutamate transporters regulate excitatory amino acid neurotransmission across neuronal and glial cell membranes by coupling the translocation of their substrate (aspartate or glutamate) into the intracellular (IC) medium to the energetically favorable transport of sodium ions or other cations. The first crystallographically resolved structure of this family, the archaeal aspartate transporter, GltPh, has served as a structural paradigm for elucidating the mechanism of substrate translocation by these transporters. Two helical hairpins, HP2 and HP1, at the core domains of the three subunits that form this membrane protein have been proposed to act as the respective extracellular and IC gates for substrate intake and release. Molecular dynamics simulations using the outward-facing structure have confirmed that the HP2 loop acts as an EC gate. The mechanism of substrate release at atomic scale, however, remained unknown due to the lack of structural data until the recent determination of the inward-facing structure of GltPh. In the present study, we use this recently resolved structure to simulate the release of substrate to the cytoplasm and the roles of HP1 and HP2 in this process. The highly flexible HP2 loop is observed to serve as an activator (or initiator) prompting the release of a gatekeeper Na+ to the cytoplasm and promoting the influx of water molecules from the cytoplasm, which effectively disrupt substrate–protein interactions and drive the dislodging of the substrate from its binding site. The completion of substrate release and exit, however, entails the opening of the highly stable HP1 loop as well. Overall, the unique conformational flexibility of the HP2 loop, the dissociation of a Na+, the hydration of binding pocket, and final yielding of the HP1 loop 3-Ser motif emerge as the successive events controlling the release of the bound substrate to the cell interior by glutamate transporters.

Coupled ion binding and structural transitions along the transport cycle of glutamate transporters

“…It was suggested previously that HP1 participates in intracellular gating in Glt Ph (Reyes et al, 2009; DeChancie et al, 2010). Indeed, the observed movement of HP1 generates a small opening, leading to the substrate and Ct sites (Figure 9—figure supplement 2), and it is reminiscent of the movement observed in molecular dynamics simulations (DeChancie et al, 2010; Zomot and Bahar, 2013). However, this conformational difference is too small to be interpreted unambiguously.

10.7554/eLife.02283.024Figure 9.Movements of the HP1-TM7a structural module.( A ) Superimposition of Glt Ph in transport domains when bound to Tl + in the apo-like conformation (grey) and when prepared in an alkali-free solution (colors).

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SLC1 glutamate transporters

“…In addition, molecular dynamics (MD) simulations based on these structures have been valuable in understanding transport mechanism [38,74,80-81,110,185,191]. This section will focus on evidence from functional studies, while structural insight will be discussed in the next section.…”

“…Once the fully loaded complex is formed with RL2 closed (outward occluded state, [20]), the elevator-like motion of the transport domain is facilitated, which, possibly through intermediates, results in formation of the inward occluded form [158,202]. Once the internal gate (RL1) opens, possibly driven by dissociation of Na + from the Na2 site, the substrate can dissociate, eventually also leading to the release of the remaining Na + ions [38]. It should be noted that a crystal structures in the inward-facing apo -form, or in the blocker-bound state are not available.…”

Low Affinity and Slow Na+ Binding Precedes High Affinity Aspartate Binding in the Secondary-active Transporter GltPh

“…The conformational change reported by F273W is significantly slower than the initial substrate binding (k on ), which is manifested by the difference between the K d values calculated from the rate constants by stopped-flow fluorescence measurements and thermodynamics using ITC measurements (18 M versus 2.7 nM, respectively). The conformation change could for example be associated with an opening of the inward-facing lid HP1 (37,38) or a reorientation of the whole transport domain after binding of both Na ϩ and aspartate (32). However, for the F273W mutant the k obs values of aspartate binding did not saturate at high aspartate concentration, in contrast to L130W (28).…”