Crystal Structure of a Trimeric Sodium/Aspartate Symporter with Protomers in Mixed Outward- and Inward-Facing States
GltPH is a bacterial trimeric sodium-coupled aspartate transporter homologous to human glutamate transporters that catalyze the uptake of the neurotransmitter from the synaptic space. Crystal structures of GltPH in the outward- and inward-facing conformations suggest that a “transport” domain carries ions and aspartate across the membrane by sliding along an immobile “trimerization” domain. To explore the range of motions of the transport domain, we have designed pairs of cysteine mutations, which upon cross-linking capture the transport domain in distinct positions relative to the membrane and the trimerization regions. Remarkably, cross-linking results suggest that the transport domain is able to move significantly further towards the cytoplasm compared to what could be inferred from the previously determined structure of the inward-facing state. To further understand the structural changes in the transporter, we have crystallized and determined a low-resolution structure of one of these cross-linked mutants. Strikingly, we found that the cross-link broke and that the three protomers relaxed to an assortment of inward- and outward-facing conformations. Two monomers show conformations nearly identical to that of the published inward-facing state. The third protomer assumes an outward-facing conformation. Remarkably, this position of the transport domain is significantly more inward compared to the previously reported outward-facing conformation. Combined, our data suggest a highly dynamic nature of the transport domain, which explores a range of orientations relative to the trimerization domain. Furthermore, we provide the structural evidence supporting the hypothesis that the three protomers function independently within the trimeric transporter. Finally, because the two inward-facing protomers are not constrained by cross-links, we conclude that the observed position of the transport domain likely corresponds to an energetic minimum.