The conserved lysine residue in transmembrane helix 5 is pivotal for the cytoplasmic gating of the <scp>L-</scp>amino acid transporters
L-Amino acid transporters (LATs) play a key role in a wide range of physiological processes. Defects in LATs can lead to neurological disorders and aminoacidurias, while the overexpression of these transporters is related to cancer. BasC is a bacterial LAT transporter with an APC fold. In this study, to monitor the cytoplasmic motion of BasC, we developed a single-molecule Förster resonance energy transfer assay that can characterize the conformational states of the intracellular gate in solution at room temperature. Based on combined biochemical and biophysical data and molecular dynamics simulations, we propose a model in which the conserved lysine residue in TM5 supports TM1a to explore both open and closed states within the cytoplasmic gate under apo conditions. This equilibrium can be altered by substrates, mutation of conserved lysine 154 in TM5, or a transport-blocking nanobody interacting with TM1a. Overall, these findings provide insights into the transport mechanism of BasC and highlight the significance of the lysine residue in TM5 in the cytoplasmic gating of LATs.
Authors
- Jan Steyaert (ORCID: https://orcid.org/0000-0002-3825-874X)
- Els Pardon (ORCID: https://orcid.org/0000-0002-2466-0172)
- Modesto Orozco (ORCID: https://orcid.org/0000-0002-8608-3278)
- Adrià Nicolàs-Aragó (ORCID: https://orcid.org/0000-0001-5233-529X)
- Manuel Palacı́n (ORCID: https://orcid.org/0000-0002-8670-293X)
- Joana Fort (ORCID: https://orcid.org/0000-0003-0399-2116)
- Óscar Llorca (ORCID: https://orcid.org/0000-0001-5705-0699)
- Susanna Bodoy (ORCID: https://orcid.org/0000-0001-9261-4373)
- Niels Zijlstra
- Maria Martinez Molledo (ORCID: https://orcid.org/0000-0002-2948-0891)
- Thorben Cordes (ORCID: https://orcid.org/0000-0002-8598-5499)
- Despoina Kapiki
- Patricia Gómez-Gejo (ORCID: https://orcid.org/0009-0002-8597-3401)
- Luca Maggi (ORCID: https://orcid.org/0000-0003-3579-5942)
- Paula González-Novoa (ORCID: https://orcid.org/0000-0001-5304-1568)
Institutions
- Universitat de Vic - Universitat Central de Catalunya (ES)
- Vrije Universiteit Brussel (BE)
- TU Dortmund University (DE)
- Instituto de Salud Carlos III (ES)
- Spanish National Cancer Research Centre (ES)
- Centre for Biomedical Network Research on Rare Diseases (ES)
- Institute for Research in Biomedicine (ES)
- VIB-VUB Center for Structural Biology (BE)
- Instituto de Investigación de Enfermedades Raras (ES)
- Universitat de Barcelona (ES)
- Ludwig-Maximilians-Universität München (DE)
Publication Details
- Journal
- PNAS Nexus
- Published
- 2024-12-23
- DOI
- https://doi.org/10.1093/pnasnexus/pgae584
- Citations
- 1
- Primary Topic
- Amino Acid Enzymes and Metabolism
- Type
- article
- Field-Weighted Citation Impact
- 0.29
Funders
- Alexander von Humboldt-Stiftung
- Institute for Research in Biomedicine
- Centres de Recerca de Catalunya
- European Commission
- Deutsche Forschungsgemeinschaft
- Generalitat de Catalunya
- Ministerio de Ciencia e Innovación
- Agència de Gestió d'Ajuts Universitaris i de Recerca
- Instituto de Salud Carlos III
- European Regional Development Fund
- Agencia Estatal de Investigación