The transmembrane α‐helix of LptC participates in LPS extraction by the LptB₂FGC transporter

Abstract

The cell envelope of Escherichia coli has four major structural components: the inner membrane (IM) surrounding the cytoplasm; the outer membrane (OM), the aqueous compartment between these two membranes known as the periplasm; and the peptidoglycan cell wall that resides in the periplasm (Figure 1a) (Silhavy et al., 2010).Unlike the IM, which is a phospholipid bilayer, the OM of most Gram-negative bacteria contains phospholipids in the inner leaflet and glycolipids known as lipopolysaccharide (LPS) in the outer leaflet (Kamio & Nikaido, 1976). LPS, which is essential in at least some Gram-negative bacteria including E. coli, is an amphipathic molecule with three major structural components: Lipid A, an oligosaccharide core, and the O-antigen polysaccharide Raetz & Whitfield, 2002;Zhang et al., 2013). Tight packing of LPS molecules at the cell surface together with its hydrophilic components create a permeability barrier against hydrophobic molecules including many antimicrobials (Nikaido, 2003). This inherent resistance provided by LPS contributes to the difficulty in treating and developing new antibiotics for Gram-negative bacterial infections (Zgurskaya & Rybenkov, 2020).LPS is synthesized in the IM and then transported to the OM by the lipopolysaccharide transport (Lpt) system, which is comprised of LptA-G in E. coli (Figure 1a) (Wilson & Ruiz, 2021). The ATP-binding

A Borrelia burgdorferi LptD homolog is required for flipping of surface lipoproteins through the spirochetal outer membrane

“…BB0464 (LptC) intriguingly was predicted to be a lipoprotein with a relatively short 14‐amino acid N‐terminal signal II peptide (SignalP6.0 probability = 0.99). Thus, LptC would be anchored in the IM via a different mechanism than gram‐negative LptC, which uses an N‐terminal α‐helix membrane anchor (Wilson & Ruiz, 2022). The top model showed the expected overall β‐jellyroll fold (pLDDT 90.3), with a short N‐terminal α‐helix following four disordered tether residues after the predicted N‐terminal cysteine.…”

“…Yet, a requirement for LptC lipidation may represent an additional early and intrinsic quality control checkpoint at the IM that prevents assembly of a functional Lpt complex if its own maturation – and that of its lipoprotein cargo – is disturbed. At the same time, a lipidated LptC would lack the recently discovered rate‐modulating function of the anchoring transmembrane α‐helix of gram‐negative LptC (Wilson & Ruiz, 2022). Our future studies will test these hypotheses and determine structure–function relationships of the B. burgdorferi Lpt and Lol pathway components, define their individual cargo specificities and potential interactions, and reassess previously identified Borrelia lipoprotein sorting determinants.…”

Dynamic basis of lipopolysaccharide export by LptB2FGC

“…The LILBID-MS data show that LPS is released upon ATP hydrolysis by LptB 2 FG ( Figure 6H-J, Figure 7 steps 1-2 ). Thus, LptC is required for a productive transport cycle in the wild-type protein 24,57,58 . Our observations suggest that the TM-LptC dynamically associates and dissociates at the lateral gate-2, even in the apo-state while its β-jellyroll domain is firmly bound to LptF β-jellyroll ( Figure 7, step 3 ).…”

ABC Transporters in Bacterial Nanomachineries

“…ATP, which can bind before ejection of LptC TM , as new data suggest, is hydrolyzed to ADP and released from the NBDs. This reopens the cavity, and LptC TM can bind again between TMH5 of LptF (THM5 F ) and TMH1 of LptG (TMH1 G ) to allow the next transport cycle (see Figure 7 , state v) [ 82 ].…”

“…Two observations were made: (i) The presence of the LptC TM increases the stability of the protein, possibly by benefiting from a complex formation with LptB 2 FG, and ii) the phenotype of LptB and LptF/G mutants with defects in ATP binding and NBD-TMD coupling were affected, indicating that the LptC TM plays a role in these steps. This new data hints that i) binding of ATP to LptB can occur with the LptC TM associated with LptFG (although it is not obligatory) and (ii) the coupling helices in LptFG, as well as the corresponding groove region in LptB, take part in LptC TM displacement from the transporters cavity, meaning that the sole entry of LPS into the cavity is not sufficient for this process [ 82 ]. The proposal that ATP can bind to LptB 2 FG with LptC TM still present is contrary to former models, in which LPS binding and formation of tighter contacts to LptFG is displacing LptC TM with ATP binding afterwards, particularly as structures of LptB 2 FG with bound LPS and displaced LptC TM were lacking nucleotides so far [ 79 ].…”