Binding and transport of D-aspartate by the glutamate transporter homolog GltTk

Abstract

Mammalian glutamate transporters are crucial players in neuronal communication as they perform neurotransmitter reuptake from the synaptic cleft. Besides L-glutamate and L-aspartate, they also recognize D-aspartate, which might participate in mammalian neurotransmission and/or neuromodulation. Much of the mechanistic insight in glutamate transport comes from studies of the archeal homologs GltPh from Pyrococcus horikoshii and GltTk from Thermococcus kodakarensis. Here, we show that GltTk transports D-aspartate with identical Na+: substrate coupling stoichiometry as L-aspartate, and that the affinities (Kd and Km) for the two substrates are similar. We determined a crystal structure of GltTk with bound D-aspartate at 2.8 Å resolution. Comparison of the L- and D-aspartate bound GltTk structures revealed that D-aspartate is accommodated with only minor rearrangements in the structure of the binding site. The structure explains how the geometrically different molecules L- and D-aspartate are recognized and transported by the protein in the same way.

Structural ensemble of a glutamate transporter homologue in lipid nanodisc environment

“…Mutagenesis studies [18][19][20] , single molecule fluorescence resonance energy transfer (smFRET) 21,22 , high-speed atomic force microscopy studies (HS-AFM) 23 , and molecular dynamics(MD) simulations 24,25 strongly indicate that the transport domains of the three protomers move independently, hence should frequently visit asymmetric states during turnover conditions. However, extensive structural studies of glutamate transporters have revealed almost exclusively symmetrical arrangements of transport domains either in outward or inward states 9,10,12,17,[26][27][28] . The only asymmetric state observed to date is in the crystal structure of a Glt Ph mutant 29 .…”

Glutamate transporters: a broad review of the most recent archaeal and human structures

“…The residues in the substrate-binding site of eukaryotic glutamate transporters and archaeal transporter homologues are mostly conserved [12,15,16,23] (Figure 3A,B). The aspartate, situated in the centre of the membrane plane in the OFC, is coordinated by the tips of HP1 and HP2, and side chains on TM7 and TM8 [15].…”

“…The best structurally characterized members of SLC1 transporters are archaeal homologues Glt Ph and Glt Tk [12][13][14][15][16][17][18][19][20][21] (Table 1). They share ∼36% sequence identity with eukaryotic EAATs [22] with many conserved residues involved in substrate and ion binding and transport [13].…”

Glutamate transporters contain a conserved chloride channel with two hydrophobic gates

“…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 .…”