The dynamic lateral gate of the mitochondrial β-barrel biogenesis machinery is blocked by darobactin A

The folding and insertion of β-barrel proteins into the mitochondrial outer membrane is facilitated by the sorting and assembly machinery (SAM) complex. Here we report two 2.8 Å cryo-EM structures of the Thermothelomyces thermophilus SAM complex in the absence of substrate in which the Sam50 lateral gate adopts two different conformations: the first is a closed lateral gate as observed in previously published structures, while the second contains a Sam50 with the first four β-strands rotated outwards by approximately 45°, resulting in an open lateral gate. The observed monomeric open conformation contrasts our previous work where the open conformation was adopted by non-physiological up-down dimers. To understand how these lateral gate dynamics are influenced by substrate, we studied the interaction of the SAM complex with a β-signal peptide mimic, darobactin A. Darobactin A binds to the SAM complex with nanomolar affinity and inhibits the import and assembly of mitochondrial β-barrel proteins in vitro. Lastly, we solved a 3.0 Å cryo-EM structure of the Thermothelomyces thermophilus SAM complex bound to darobactin A, which reveals that darobactin A stabilizes the Sam50 lateral gate similar to the open conformation by binding to strand β1, therefore blocking β-barrel biogenesis.

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Publication Details

Journal
Nature Communications
Published
2025-11-20
DOI
https://doi.org/10.1038/s41467-025-66417-0
Primary Topic
Mitochondrial Function and Pathology
Type
article
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article

The dynamic lateral gate of the mitochondrial β-barrel biogenesis machinery is blocked by darobactin A

Katie M. Kuo, Akira Iinishi, Kathryn A. Diederichs, Gwendolyn Cooper et al.
Nature Communications
Mitochondrial Function and Pathology
article

The dynamic lateral gate of the mitochondrial β-barrel biogenesis machinery is blocked by darobactin A

Katie M. Kuo, Akira Iinishi, Kathryn A. Diederichs, Gwendolyn Cooper, Susan K. Buchanan, Benjamin Schwarz, Gvantsa Gutishvili, Vadim Kotov, Hervé Celia, Istvan Botos, Joseph A. Mindell, Thomas C. Marlovits, James C. Gumbart, Kim Lewis, Scout Hayashi
article en

Abstract

The folding and insertion of β-barrel proteins into the mitochondrial outer membrane is facilitated by the sorting and assembly machinery (SAM) complex. Here we report two 2.8 Å cryo-EM structures of the Thermothelomyces thermophilus SAM complex in the absence of substrate in which the Sam50 lateral gate adopts two different conformations: the first is a closed lateral gate as observed in previously published structures, while the second contains a Sam50 with the first four β-strands rotated outwards by approximately 45°, resulting in an open lateral gate. The observed monomeric open conformation contrasts our previous work where the open conformation was adopted by non-physiological up-down dimers. To understand how these lateral gate dynamics are influenced by substrate, we studied the interaction of the SAM complex with a β-signal peptide mimic, darobactin A. Darobactin A binds to the SAM complex with nanomolar affinity and inhibits the import and assembly of mitochondrial β-barrel proteins in vitro. Lastly, we solved a 3.0 Å cryo-EM structure of the Thermothelomyces thermophilus SAM complex bound to darobactin A, which reveals that darobactin A stabilizes the Sam50 lateral gate similar to the open conformation by binding to strand β1, therefore blocking β-barrel biogenesis.

Nature CommunicationsVol. 16(1)
Northeastern University (US), National Institutes of Health (US), Georgia Institute of Technology (US), Universität Hamburg (DE), Deutsches Elektronen-Synchrotron DESY (DE), National Institute of Diabetes and Digestive and Kidney Diseases (US), University Medical Center Hamburg-Eppendorf (DE), National Institute of Neurological Disorders and Stroke (US), National Institute of Allergy and Infectious Diseases (US), Centre for Structural Systems Biology (DE)
Openalex Percentile: Top 68%
Mitochondrial Function and Pathology
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