Mechanism of LolCDE as a molecular extruder of bacterial triacylated lipoproteins

Abstract

Present in all bacteria, lipoproteins are central in bacterial growth and antibiotic resistance. These proteins use lipid acyl chains attached to the N-terminal cysteine residue to anchor on the outer surface of cytoplasmic membrane. In Gram-negative bacteria, many lipoproteins are transported to the outer membrane (OM), a process dependent on the ATP-binding cassette (ABC) transporter LolCDE which extracts the OM-targeted lipoproteins from the cytoplasmic membrane for subsequent trafficking across the periplasm. Lipid-anchored proteins pose a unique challenge for transport machinery as they have both hydrophobic lipid moieties and soluble protein component, and the underlying mechanism is poorly understood. Here we determined the cryo-EM structures of nanodisc-embedded LolCDE in the nucleotide-free and nucleotide-bound states at 3.8-Å and 3.5-Å resolution, respectively. The structural analyses, together with biochemical and mutagenesis studies, uncover how LolCDE specifically recognizes its substrate by establishing multiple interactions with the lipid and N-terminal peptide moieties of the lipoprotein, and identify the amide-linked acyl chain as the key element for LolCDE interaction. Upon nucleotide binding, the transmembrane helices and the periplasmic domains of LolCDE undergo large-scale, asymmetric movements, resulting in extrusion of the captured lipoprotein. Comparison of LolCDE and MacB reveals the conserved mechanism of type VII ABC transporters and emphasizes the unique properties of LolCDE as a molecule extruder of triacylated lipoproteins.

Cryo-EM structures of LolCDE reveal the molecular mechanism of bacterial lipoprotein sorting in Escherichia coli

“…In the V-shaped cavity, the 3 acyl chains of RcsF straddles the TMD interface of LolC and LolE, with 1 acyl chain, R1, residing on the Loop LolC side, and the other two, R2 and R3, extending to the Loop LolE side ( Fig 1E ). This is in line with the 2 recently reported cryo-EM structures of the lipoprotein-bound LolCDE complexes [ 33 , 34 ]. Importantly, the proteinaceous part of RcsF extends from the V-shaped cavity and appears only on the Loop LolC side, suggesting that RcsF might enter the cavity laterally via the Loop LolC -TM2 LolE interface.…”

“…In summary, while our RcsF-LolCDE and AMPPNP-LolCDE structures overlay well with the recently published structures [ 33 , 34 ] ( S9A and S9B Fig ), our apo-LolCDE structure is strikingly different from the apo-LolCDE structure (denoted as apo-LolCDE*, PDB code: 7ARI) by Tang and colleagues [ 33 ] ( S9C Fig ). This prompted us to further verify the apo-LolCDE structure using biochemical approaches.…”

“…Collectively, here, we report 3 LolCDE structures, apo, RcsF-, and AMPPNP-bound states. While the latter two correlate well with the previously published structures reported by Tang and colleagues and Sharma and colleagues, the former deviates significantly from the apo-LolCDE � structure reported by Tang and colleagues [33,34]. The functional analysis confirms our apo-LolCDE structure, highlighting that lipoproteins enter and bind in the cavity of apo-LolCDE devoid of apparent conformational changes in an ATP-independent manner.…”

“…In stark contrast to lipoprotein binding, AMPPNP binding causes significant conformational changes of LolCDE (S8B In summary, while our RcsF-LolCDE and AMPPNP-LolCDE structures overlay well with the recently published structures [33,34]…”

“…Recently, Kaplan and colleagues reported the crystal structure of LolA in complex with the periplasmic domain of the LolC [ 25 ]. Tang and colleagues and Sharma and colleagues determined cryo-EM structures of LolCDE in different conformational states [ 33 , 34 ]. High-resolution structures of the Lol components promise to greatly advance our understanding of the molecular mechanism of lipoprotein biogenesis.…”