Abstract:The transport cycle in the glutamate transporter (GlT) is catalyzed by the cotransport of three Na(+) ions. However, the positions of only two of these ions (Na1 and Na2 sites) along with the substrate have been captured in the crystal structures reported for both the outward-facing and the inward-facing states of Glt(ph). Characterizing the third ion binding site (Na3) is necessary for structure-function studies attempting to investigate the mechanism of transport in GlTs at an atomic level, particularly for … Show more
“…Thus, all known ligand-binding sites are distorted in the apo forms (Figure 4). 10.7554/eLife.02283.003Figure 4.Remodeling of L-asp and Na + binding sites in the apo conformations.Close-up views of the fully bound (left) and apo (right) transport domains at L-asp binding site (top), Na1 and Na2 sites (middle), and one of the proposed locations for the third Na + binding site (Huang and Tajkhorshid, 2010; Bastug et al, 2012) (dashed circle). DOI:
http://dx.doi.org/10.7554/eLife.02283.00310.7554/eLife.02283.004Figure 4—figure supplement 1.Transport domain remains compact.Surface representation of the transport domain in fully bound and apo forms. DOI:
http://dx.doi.org/10.7554/eLife.02283.004…”
Membrane transporters that clear the neurotransmitter glutamate from synapses are driven by symport of sodium ions and counter-transport of a potassium ion. Previous crystal structures of a homologous archaeal sodium and aspartate symporter showed that a dedicated transport domain carries the substrate and ions across the membrane. Here, we report new crystal structures of this homologue in ligand-free and ions-only bound outward- and inward-facing conformations. We show that after ligand release, the apo transport domain adopts a compact and occluded conformation that can traverse the membrane, completing the transport cycle. Sodium binding primes the transport domain to accept its substrate and triggers extracellular gate opening, which prevents inward domain translocation until substrate binding takes place. Furthermore, we describe a new cation-binding site ideally suited to bind a counter-transported ion. We suggest that potassium binding at this site stabilizes the translocation-competent conformation of the unloaded transport domain in mammalian homologues.DOI:
http://dx.doi.org/10.7554/eLife.02283.001
“…Thus, all known ligand-binding sites are distorted in the apo forms (Figure 4). 10.7554/eLife.02283.003Figure 4.Remodeling of L-asp and Na + binding sites in the apo conformations.Close-up views of the fully bound (left) and apo (right) transport domains at L-asp binding site (top), Na1 and Na2 sites (middle), and one of the proposed locations for the third Na + binding site (Huang and Tajkhorshid, 2010; Bastug et al, 2012) (dashed circle). DOI:
http://dx.doi.org/10.7554/eLife.02283.00310.7554/eLife.02283.004Figure 4—figure supplement 1.Transport domain remains compact.Surface representation of the transport domain in fully bound and apo forms. DOI:
http://dx.doi.org/10.7554/eLife.02283.004…”
Membrane transporters that clear the neurotransmitter glutamate from synapses are driven by symport of sodium ions and counter-transport of a potassium ion. Previous crystal structures of a homologous archaeal sodium and aspartate symporter showed that a dedicated transport domain carries the substrate and ions across the membrane. Here, we report new crystal structures of this homologue in ligand-free and ions-only bound outward- and inward-facing conformations. We show that after ligand release, the apo transport domain adopts a compact and occluded conformation that can traverse the membrane, completing the transport cycle. Sodium binding primes the transport domain to accept its substrate and triggers extracellular gate opening, which prevents inward domain translocation until substrate binding takes place. Furthermore, we describe a new cation-binding site ideally suited to bind a counter-transported ion. We suggest that potassium binding at this site stabilizes the translocation-competent conformation of the unloaded transport domain in mammalian homologues.DOI:
http://dx.doi.org/10.7554/eLife.02283.001
“…Using the trimeric structure of Glt ph (28) (32,33). During the simulation, numerous water permeation events occur along a continuous aqueous pathway formed at the interface between the transport domain [specifically, helical hairpin 1 (HP1)] and the trimerization domain, but only in the monomer, which is in the intermediate state (Fig.…”
Membrane transporters rely on highly coordinated structural transitions between major conformational states for their function, to prevent simultaneous access of the substrate binding site to both sides of the membrane-a mode of operation known as the alternating access model. Although this mechanism successfully accounts for the efficient exchange of the primary substrate across the membrane, accruing evidence on significant water transport and even uncoupled ion transport mediated by transporters has challenged the concept of perfect mechanical coupling and coordination of the gating mechanism in transporters, which might be expected from the alternating access model. Here, we present a large set of extended equilibrium molecular dynamics simulations performed on several classes of membrane transporters in different conformational states, to test the presence of the phenomenon in diverse transporter classes and to investigate the underlying molecular mechanism of water transport through membrane transporters. The simulations reveal spontaneous formation of transient water-conducting (channel-like) states allowing passive water diffusion through the lumen of the transporters. These channel-like states are permeable to water but occluded to substrate, thereby not hindering the uphill transport of the primary substrate, i.e., the alternating access model remains applicable to the substrate. The rise of such water-conducting states during the large-scale structural transitions of the transporter protein is indicative of imperfections in the coordinated closing and opening motions of the cytoplasmic and extracellular gates. We propose that the observed water-conducting states likely represent a universal phenomenon in membrane transporters, which is consistent with their reliance on large-scale motion for function.major facilitator superfamily | LeuT-fold transporters | ABC transporters | neurotransmitter transporters
“…Although three sodium ions are cotransported with one aspartate (11), the crystal structures revealed only two sodium ion binding sites (Na1 and Na2) within each protomer (9). The binding site for the third Na ϩ is still debated (12)(13)(14)(15)(16)(17)(18). Experiments and simulations on Glt Ph and EAAT3 have suggested that the third sodium ion (Na3) may be coordinated by Thr 314 and Asn 401 in Glt Ph (18).…”
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