Elucidation of the structure and molecular mechanisms of the aspartate antiporter.

The transport of compounds across the cell membrane is essential for maintaining cellular homeostasis. Secondary exchange transporters mediate the movement of a wide range of substrates against their concentration gradients by harnessing the energy stored in electrochemical gradients. However, the molecular mechanism of substrate exchange by secondary transporters remains unclear. Here, we determined the structures of the aspartate exchanger AspT from Tetragenococcus halophilus using cryo-EM single-particle analysis and X-ray crystallography. We captured AspT in two distinct conformations: the apo outward-facing state and the substrate (L-Aspartate)-bound partially-open inward-facing intermediate state. AspT functions as a homodimer and comprises three domains: a dimerization domain, a substrate transport domain, and a soluble domain. Within each monomer, two hairpin loops in the transport domain form a single substrate-binding pocket. Upon L-aspartate binding, the transport domain carrying the substrate translocates toward the cytoplasmic side of the membrane, forming an outer barrier that blocks the periplasmic access to the binding pocket. These structural insights reveal that AspT mediates substrate translocation via an elevator-type alternating-access mechanism involving a stable partially-open inward-facing intermediate. By elucidating the mechanism of substrate exchange in secondary transporters, this study advances our understanding of membrane transport leading to translational applications in biotechnology.