Journal Article

PUBDB-2025-05268

http://join2-wiki.gsi.de/foswiki/pub/Main/Artwork/join2_logo100x88.png The dynamic lateral gate of the mitochondrial β-barrel biogenesis machinery is blocked by darobactin A

Diederichs, K. A. ; Botos, I. ; Hayashi, S. ; Gutishvili, G. ; Kotov, V.Extern*CSSB*DESY* ; Kuo, K. ; Iinishi, A. ; Cooper, G. ; Schwarz, B. ; Celia, H. ; Marlovits, T.CSSB* ; Lewis, K. ; Gumbart, J. C. ; Mindell, J. A. (Corresponding author) ; Buchanan, S. K. (Corresponding author)

2025
Springer Nature [London]

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Please use a persistent id in citations: doi:10.1038/s41467-025-66417-0  doi:10.3204/PUBDB-2025-05268

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.

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Contributing Institute(s):

1. CSSB-UKE-TM (CSSB-UKE-TM)

Research Program(s):

1. 633 - Life Sciences – Building Blocks of Life: Structure and Function (POF4-633) (POF4-633)

Experiment(s):

1. (Sample Preparation and Characterisation Facility)

Appears in the scientific report 2025

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