Cryo-EM structures of LolCDE reveal the molecular mechanism of bacterial lipoprotein sorting in Escherichia coli
Fig 2
Verification of the apo-LolCDE structure.
(A) Comparison of our apo-LolCDE structure (left) to the apo-LolCDE* structure (right, PDB code:7ARI). Zoom-in view showing 2 amino acids in 2 PLDs, Ala106 and Ser173, which were replaced with cysteines in (B to E). Leu256LolC shown in purple spheres was substituted with pBPA for in vitro photo-crosslinking in (C). (B) Coomassie-stained SDS–PAGE gel assessing disulfide bond formation of LolCA106CDES173C and RcsF-LolCA106CDES173C. The samples of lanes 2 through 4 were supplemented with SDS loading dye without β-ME, and the samples of lanes 6 through 8 were supplemented with SDS loading dye with β-ME. Note that RcsF migrates slower after addition of reducing agent. (C) In vitro photo-crosslinking. LolCL256pBPADE proteins with or without 2 cysteine mutations were reconstituted with RcsF in nanodisc. The LolC×RcsF and the LolE-LolC×RcsF adducts were detected by immunoblotting. (D) The in vitro lipoprotein transport assays. To break intermolecular disulfide bond, the nanodisc-embedded RcsF-LolCA106CDES173C protein was incubated with TCEP prior to the addition of LolA (W70pBPA). (E) Complementation assays. The dilutions were spotted on LB plates with (right) or without (left) TCEP. Protein leaky expression levels of the LolC and LolE proteins were detected by western blotting (bottom). Data shown in (B to E) are representatives of 3 replicates.