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000641981 1001_ $$00000-0001-9444-9797$$aDiederichs, Kathryn A.$$b0
000641981 245__ $$aThe dynamic lateral gate of the mitochondrial β-barrel biogenesis machinery is blocked by darobactin A
000641981 260__ $$a[London]$$bSpringer Nature$$c2025
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000641981 520__ $$aThe 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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000641981 7001_ $$00000-0002-7702-6749$$aBotos, Istvan$$b1
000641981 7001_ $$00000-0002-6376-3860$$aHayashi, Scout$$b2
000641981 7001_ $$00000-0001-5681-8539$$aGutishvili, Gvantsa$$b3
000641981 7001_ $$0P:(DE-H253)PIP1082976$$aKotov, Vadim$$b4
000641981 7001_ $$00009-0001-5682-6173$$aKuo, Katie$$b5
000641981 7001_ $$aIinishi, Akira$$b6
000641981 7001_ $$00000-0003-3548-3551$$aCooper, Gwendolyn$$b7
000641981 7001_ $$00000-0002-5894-953X$$aSchwarz, Benjamin$$b8
000641981 7001_ $$00000-0003-1203-8586$$aCelia, Herve$$b9
000641981 7001_ $$0P:(DE-H253)PIP1021412$$aMarlovits, Thomas$$b10
000641981 7001_ $$00000-0002-1335-9982$$aLewis, Kim$$b11
000641981 7001_ $$00000-0002-1510-7842$$aGumbart, James C.$$b12
000641981 7001_ $$0P:(DE-HGF)0$$aMindell, Joseph A.$$b13$$eCorresponding author
000641981 7001_ $$0P:(DE-HGF)0$$aBuchanan, Susan K.$$b14$$eCorresponding author
000641981 773__ $$0PERI:(DE-600)2553671-0$$a10.1038/s41467-025-66417-0$$gVol. 16, no. 1, p. 11349$$n1$$p11349$$tNature Communications$$v16$$x2041-1723$$y2025
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