Structural insights into inhibitory mechanism of human excitatory amino acid transporter EAAT2

Abstract

Glutamate is a pivotal excitatory neurotransmitter in mammalian brains, but excessive glutamate causes numerous neural disorders. Almost all extracellular glutamate is retrieved by the glial transporter, Excitatory Amino Acid Transporter 2 (EAAT2), belonging to the SLC1A family. However, in some cancers, EAAT2 expression is enhanced and causes resistance to therapies by metabolic disturbance. Despite its crucial roles, the detailed structural information about EAAT2 has not been available. Here, we report cryo-EM structures of human EAAT2 in substrate-free and selective inhibitor WAY213613-bound states at 3.2 Å and 2.8 Å, respectively. EAAT2 forms a trimer, with each protomer consisting of transport and scaffold domains. Along with a glutamate-binding site, the transport domain possesses a cavity that could be disrupted during the transport cycle. WAY213613 occupies both the glutamate-binding site and cavity of EAAT2 to interfere with its alternating access, where the sensitivity is defined by the inner environment of the cavity. We provide the characterization of the molecular features of EAAT2 and its selective inhibition mechanism that may facilitate structure-based drug design for EAAT2.

A Medicinal Chemistry Perspective on Excitatory Amino Acid Transporter 2 Dysfunction in Neurodegenerative Diseases

“…Kato et al using point mutations showed that these residues forming the cavities are closely related to the sensitivities of the EAAT subtypes to inhibitors. 51 Indeed, the addition of an aromatic group (red in Figure 5 ) via amide bond ( 19 , IC 50 = 59 nM) increased the activity by ∼100-fold vs EAAT2. 19 was slightly more selective to EAAT2 than EAAT1(∼2-fold) and EAAT3 (and 40-fold).…”

“…Noteworthy, 26 , which is commercially available as WAY213613, remains the most selective and potent EAAT2 inhibitor to date. , Cryo-EM analysis has recently shown that WAY213613 inhibits glutamate uptake via EAAT2 by either interaction at the glutamate-binding site and sterically preventing HP2 loop movement (Figure ). The l -asparagine (Figure , blue and black squares) and 4-(2-bromo-4,5-difluorophenoxy) phenyl (Figure , green and red squares) moieties of 26 played distinct roles in the EAAT2 inhibition, by competing with the glutamate binding and sterically preventing the HP2 loop gating suspending the transport cycle of glutamate, respectively.…”

“… 47 − 49 Two independent research groups have releveled the cryo-electron microscopy (cryo-EM) structural information on human EAAT2 in complex at BS1 with glutamate 50 ( Figure 2 ) and a selective inhibitor WAY-213613. 50 , 51 BS2 is formed by the H71 amino acid residue in the TM2, L295 and K299 in TM5, and W472 in TM8 ( Figure 2 ). 52 More recently, BS3 defined by M477 on TM8, and F345, F348, F352, W355 on HP1 was identified as a binding pocket for negative allosteric modulators ( Figure 2 ).…”

“…BS1 is formed in the C-terminal portion of each EAAT2 monomer, which includes HP1, HP2, TM7, and TM8 (Figures and ). In fact, the D475 and R478 amino acid residues in TM8 are suggested to be the main players in the formation of this binding pocket. − Two independent research groups have releveled the cryo-electron microscopy (cryo-EM) structural information on human EAAT2 in complex at BS1 with glutamate (Figure ) and a selective inhibitor WAY-213613. , BS2 is formed by the H71 amino acid residue in the TM2, L295 and K299 in TM5, and W472 in TM8 (Figure ). More recently, BS3 defined by M477 on TM8, and F345, F348, F352, W355 on HP1 was identified as a binding pocket for negative allosteric modulators (Figure ).…”

“… More recently, BS3 defined by M477 on TM8, and F345, F348, F352, W355 on HP1 was identified as a binding pocket for negative allosteric modulators (Figure ). , …”