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.

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Publication Details

Journal
Communications Biology
Published
2025-09-25
DOI
https://doi.org/10.1038/s42003-025-08676-7
Primary Topic
RNA and protein synthesis mechanisms
Type
article
Field-Weighted Citation Impact
0.00

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article

Elucidation of the structure and molecular mechanisms of the aspartate antiporter

Takeshi Murata, Takeshi Kawabata, Lan Guan, Keietsu Abe et al.
Communications Biology
RNA and protein synthesis mechanisms
article

Elucidation of the structure and molecular mechanisms of the aspartate antiporter

Takeshi Murata, Takeshi Kawabata, Lan Guan, Keietsu Abe, Kenji Inaba, Takashi Fujiki, Kei Nanatani, Kaoru Mitsuoka, S. Koshiba, Masafumi Hidaka, Parameswaran Hariharan, Fumika Chiba, Kota Kunii, Satoshi Ogasawara, Satoshi Watanabe, Satoshi Katsube, Ryo Kanno, Masayuki Yamamoto, K Toda, Takashi Yamanaka, Akari Miyamoto
article en

Abstract

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.

Communications BiologyVol. 8(1)
Texas Tech University (US), Kyushu University (JP), Okinawa Institute of Science and Technology Graduate University (JP), Chiba University (JP), Tohoku University (JP), SPring-8 (JP), Tohoku Medical Megabank Organization (JP), Texas Tech University Health Sciences Center (US), The University of Osaka (JP)
Japan Agency for Medical Research and Development, Noda Institute for Scientific Research, Kato Memorial Bioscience Foundation, Okinawa Institute of Science and Technology Graduate University, National Institutes of Health, Japan Society for the Promotion of Science
Openalex Percentile: Top 77%
RNA and protein synthesis mechanisms
DOIOpen Access PDFOpenAlex
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