Fig. 6: Proposed functional mechanism of darobactin A inhibition of SAM complex in vitro.

A In the absence of darobactin A, the Sam50 lateral gate dynamically opens and closes (i, ii). Mitochondrial outer membrane β-barrel precursor proteins containing a C-terminal β-signal are targeted to the SAM complex (i). The Sam50 lateral gate is stabilized in the open conformation by the β-signal binding to Sam50 β1 (iii). The precursor protein is hypothesized to fold by sequential β-hairpin insertions (iv). Sam37 stabilizes the precursor protein as it folds (v). After the final precursor protein β-strands are incorporated, the first pairs with the last, and the mature β-barrel is released laterally into the membrane (vi). B In contrast, the addition of darobactin A stabilizes the Sam50 lateral gate in the open conformation by binding to Sam50 β1 (iii). Darobactin A remains stably associated with Sam50 β1 which blocks the binding of precursor protein β-signal (iii) and therefore prevents assembly of the β-barrel protein into the outer membrane (iv). C BamA lateral gate remains closed in the absence of substrate (i). The BamA lateral gate opens to accommodate the β-signal binding to BamA β1 (ii). The precursor protein is hypothesized to fold by the β-hairpin insertions, and remains associated with the BAM complex until folding is complete (iii, iv). After the final precursor protein β-strand is incorporated, the mature β-barrel is released laterally into the membrane (v). D Darobactin A binds to BamA β1, however the BamA lateral gate remains closed through interactions between BamA β1 and β16 (i, ii). Darobactin A remains stably associated with BamA β1, blocking precursor β-signal binding and therefore inhibiting β-barrel biogenesis (iii). N = N-terminus, C = C-terminus.