9D6A

Human excitatory amino acid transporter 3 (EAAT3) with bound L-Cysteine in an intermediate outward facing state

Summary for 9D6A

• Entry DOI: 10.2210/pdb9d6a/pdb
• EMDB information: 46591
• Descriptor: Excitatory amino acid transporter 3, CYSTEINE, SODIUM ION, ... (5 entities in total)
• Functional Keywords: human eaat3, cysteine, iofs-star, protein transport
• Biological source: Homo sapiens (human)
• Total number of polymer chains: 1
• Total formula weight: 57511.58

Authors

Qiu, B.,Boudker, O. (deposition date: 2024-08-14, release date: 2025-01-22)

Primary citation

Qiu, B.,Boudker, O.
Structural basis of the excitatory amino acid transporter 3 substrate recognition.
Biorxiv, 2024

PubMed Abstract: Excitatory amino acid transporters (EAATs) reside on cell surfaces and uptake substrates, including L-glutamate, L-aspartate, and D-aspartate, using ion gradients. Among five EAATs, EAAT3 is the only isoform that can efficiently transport L-cysteine, a substrate for glutathione synthesis. Recent work suggests that EAAT3 also transports the oncometabolite R-2-hydroxyglutarate (R-2HG). Here, we examined the structural basis of substrate promiscuity by determining the cryo-EM structures of EAAT3 bound to different substrates. We found that L-cysteine binds to EAAT3 in thiolate form, and EAAT3 recognizes different substrates by fine-tuning local conformations of the coordinating residues. However, using purified human EAAT3, we could not observe R-2HG binding or transport. Imaging of EAAT3 bound to L-cysteine revealed several conformational states, including an outward-facing state with a semi-open gate and a disrupted sodium-binding site. These structures illustrate that the full gate closure, coupled with the binding of the last sodium ion, occurs after substrate binding. Furthermore, we observed that different substrates affect how the transporter distributes between a fully outward-facing conformation and intermediate occluded states on a path to the inward-facing conformation, suggesting that translocation rates are substrate-dependent.

PubMed: 39282329
DOI: 10.1101/2024.09.05.611541

Experimental method

ELECTRON MICROSCOPY (2.6 Å)