Structural ensemble of a glutamate transporter homologue in lipid nanodisc environment

Arkhipova, Valentina; Guskov, Albert; Slotboom, Dirk Jan

doi:10.1038/s41467-020-14834-8

Cited by 64 publications

(123 citation statements)

References 57 publications

Supporting

Mentioning

112

Contrasting

Order By: Relevance

“…In this so-called "unlocked" conformation, there was sufficient space for HP2 to open. More recent studies of ASCT2 and of an archaeal GltTk further showed that HP2 could open, suggesting that it serves as a gate in both the OFS and IFS (Garaeva et al, 2019;Arkhipova et al, 2020). Here, we report a series of Cryo-EM structures of GltPh reconstituted into nanodiscs in the IFS and OFS.…”

mentioning

confidence: 68%

Large domain movements through lipid bilayer mediate substrate release and inhibition of glutamate transporters

Wang

Boudker

2020

Preprint

View full text Add to dashboard Cite

Glutamate transporters are essential players in glutamatergic neurotransmission in the brain, where they maintain extracellular glutamate below cytotoxic levels and allow for rounds of transmission. The structural bases of their function are well established, particularly within a model archaeal homologue, sodium and aspartate symporter GltPh. However, the mechanism of gating on the cytoplasmic side of the membrane remains ambiguous. We report Cryo-EM structures of GltPh reconstituted into nanodiscs, including those structurally constrained in the cytoplasm-facing state and either apo, bound to sodium ions only, substrate, or blockers. The structures show that both substrate translocation and release involve movements of the bulky transport domain through the lipid bilayer. They further reveal a novel mode of inhibitor binding and show how solutes release is coupled to protein conformational changes. Finally, we describe how domain movements are associated with the displacement of bound lipids and significant membrane deformations, highlighting the potential regulatory role of the bilayer. MAIN TEXTSodium and aspartate symporter GltPh is an archaeal homologue of human glutamate transporters, which clear the neurotransmitter glutamate from the synaptic cleft following rounds of neurotransmission (Danbolt, 2001). GltPh has served as a model system to uncover the structural and mechanistic features of glutamate transporters (Yernool et al., 2004;Boudker et al., 2007;Reyes et al., 2009;Akyuz et al., 2013;Erkens et al., 2013;Reyes et al., 2013;Verdon et al., 2014;Akyuz et al., 2015;Hanelt et al., 2015;Mcilwain et al., 2016;Scopelliti et al., 2018). Recently, structural studies on other members of the family, including human variants, have enriched the field and have been mostly consistent with earlier findings on GltPh (Canul-Tec et al., 2017;Garaeva et al., 2018;Yu et al., 2019).Collectively, these studies provide what appears to be a nearly complete picture of the structural changes that underlie transport. Briefly, the transporters are homotrimers with each protomer consisting of a centrally located scaffold or trimerization domain and a peripheral transport domain that harbors the L-aspartate (L-asp) and three sodium (Na + ) ions binding sites. The crucial conformational transition from the outward-facing state (OFS), in which L-asp binding site is near the extracellular solution, into the inward-facing

show abstract

mentioning

confidence: 68%

Large domain movements through lipid bilayer mediate substrate release and inhibition of glutamate transporters

Wang

Boudker

2020

Preprint

View full text Add to dashboard Cite

show abstract

“…These results suggest that in the absence of transmembrane electrochemical gradients and substrate, hEAAT3 g strongly favors the inward-facing conformation, similar to the archaeal homologue Glt Tk 34 . In this state, at least under our imaging conditions, the transporter has a remarkably low substrate affinity, perhaps in tens of mM.…”

Section: Resultsmentioning

confidence: 78%

“…of 1.3 Å, with the most significant conformational changes occurring in TM7 and HP2 (Figure 2e) . In further considering the coupling mechanism, we took advantage of the observations that the conformational changes underlying ion and substrate binding are confined to transport domains and similar in the outward- and inward-facing states of glutamate transporters 34,37,38 . We, therefore, compared the transport domains of IFS-Apo, IFS-Na + , and OFS-Asp to visualize structural events during binding.…”

Section: Resultsmentioning

confidence: 99%

Transport mechanism of the neuronal excitatory amino acid transporter

Qiu

Matthies

Fortea

et al. 2020

Preprint

View full text Add to dashboard Cite

16Human excitatory amino acid transporter 3 (hEAAT3) mediates glutamate uptake in neurons, 17 intestine, and kidney. Here, we report Cryo-EM structures of hEAAT3 in several functional states 18 where the transporter is empty, bound to coupled sodium ions only, or fully loaded with three 19 sodium ions, a proton, and the substrate aspartate. The structures suggest that hEAAT3 operates 20 by an elevator mechanism involving three functionally independent subunits. When the substrate-21 binding site is near the cytoplasm, it has a remarkably low affinity for the substrate, perhaps 22 facilitating its release and allowing for the rapid transport turnover. The mechanism of the coupled 23 uptake of the sodium ions and the substrate is conserved across evolutionarily distant families and 24 is augmented by coupling to protons in EAATs. The structures further suggest a mechanism by 25 which conserved glutamate mediates proton symport. 26 27 antioxidant glutathione [15][16][17] . Thus, activators of hEAAT3 might be useful to boost cysteine uptake 47 into neurons under pathologic conditions. Due to the high concentration of glutamate in neurons, 48 hEAAT3 might also run in reverse during ischemia, spilling excitotoxic levels of glutamate into 49 the extracellular space. Therefore, specific inhibitors of hEAAT3 might be clinically helpful in 50 reducing glutamate-mediated damage following transient ischemic conditions and depolarization 51 of membranes. 52 53 A detailed understanding of the hEAAT3 structure and mechanism would be required to enable 54 pharmacologic manipulations. Toward this end, we have determined structures of hEAAT3 using 55Cryo-EM. We show that the transporter is a homotrimer. Each protomer consists of two domains, 56 a central trimerization scaffold domain and a peripheral transport domain containing the substrate-57 binding site. The individual hEAAT3 protomers undergo elevator-like transitions between the 58 outward-and inward-facing conformations, in which their transport domains localize closer to the 59 extracellular space and the cytoplasm, respectively. They appear to be independent of each other 60 so that we observe trimers with every possible arrangement of the outward-and inward-facing 61 protomers. Remarkably, when we imaged hEAAT3 in the presence of L-aspartate (L-asp) and 62 sodium (Na + ) ions, we observe that only the outward-facing protomers were bound to the amino 63 acid, while the inward-facing protomers remained substrate-free. We conclude that the substrate-64 free transporter has a strong preference for the inward-facing conformation while the outward-65 facing transporter has a significantly higher affinity for the amino acid. By comparing the 66 structures of the transport domain when free of ligands, bound to Na + ions only and bound to Na + 67 ions, protons, and L-asp, we provide mechanistic insights into symport of the amino acid, Na + and 68 protons in hEAAT3. 69 70 Results 71The elevator mechanism of hEAAT3. We heterologously expressed and purified full-length 72 hEAAT3 wit...

show abstract

“…X-ray crystal structures and, more recently, single-particle cryo-electron microscopy (cryo-EM) structures of several members of the SLC1A family have revealed distinct conformations within the transport cycle. Structures of EAAT1 11 , neutral amino acid transporter 2 (ASCT2) [12][13][14] , and the archaeal homologs, GltPh 2, 15-22 and GltTk [23][24][25] have shown that SLC1A transporters share a similar trimeric structure in which each protomer, comprising a transport and a scaffold domain, can function independently [25][26][27] . EAATs utilize a twisting elevator mechanism of transport, in which the transport domain, containing the substrate and co-transport ion binding sites, shuttles across the membrane during transport while the scaffold domain makes inter-subunit contacts and remains anchored to the membrane 2,13 .…”

Section: Introductionmentioning

confidence: 99%

Glutamate transporters contain a conserved chloride channel with two hydrophobic gates

Chen

Pant

et al. 2020

Preprint

View full text Add to dashboard Cite

Glutamate is the most abundant excitatory neurotransmitter in the central nervous system, therefore its precise control is vital for maintaining normal brain function and preventing excitotoxicity 1 . Removal of extracellular glutamate is achieved by plasma membrane-bound transporters, which couple glutamate transport to sodium, potassium and pH gradients using an elevator mechanism 2-5 . Glutamate transporters also conduct chloride ions via a channel-like process that is thermodynamically uncoupled from transport 6-8 . However, the molecular mechanisms that allow these dual-function transporters to carry out two seemingly contradictory roles are unknown. Here we report the cryo-electron microscopy structure of a glutamate transporter homologue in an open-channel state, revealing an aqueous cavity that is formed during the transport cycle. Using functional studies and molecular dynamics simulations, we show that this cavity is an aqueous-accessible chloride permeation pathway gated by two hydrophobic regions, and is conserved across mammalian and archaeal glutamate transporters. Our findings provide insight into the mechanism by which glutamate transporters support their dual function and add a crucial piece of information to aid mapping of the complete transport cycle shared by the SLC1A transporter family.

show abstract

Structural ensemble of a glutamate transporter homologue in lipid nanodisc environment

Cited by 64 publications

References 57 publications

Large domain movements through lipid bilayer mediate substrate release and inhibition of glutamate transporters

Large domain movements through lipid bilayer mediate substrate release and inhibition of glutamate transporters

Transport mechanism of the neuronal excitatory amino acid transporter

Glutamate transporters contain a conserved chloride channel with two hydrophobic gates

Contact Info

Product

Resources

About