Abstract
Lipopolysaccharides (LPS) of Gram-negative bacteria are critical for the defence against cytotoxic substances and must be transported from the inner membrane (IM) to the outer membrane (OM) through a bridge formed by seven membrane proteins (LptBFGCADE). The IM component LptB2FG powers the process through a yet unclarified mechanism. Here we report three high-resolution cryo-EM structures of LptB2FG alone and complexed with LptC (LptB2FGC), trapped in either the LPS- or AMP-PNP-bound state. The structures reveal conformational changes between these states and substrate binding with or without LptC. We identify two functional transmembrane arginine-containing loops interacting with the bound AMP-PNP and elucidate allosteric communications between the domains. AMP-PNP binding induces an inward rotation and shift of the transmembrane helices of LptFG and LptC to tighten the cavity, with the closure of two lateral gates, to eventually expel LPS into the bridge. Functional assays reveal the functionality of the LptF and LptG periplasmic domains. Our findings shed light on the LPS transport mechanism.
Highlights
Lipopolysaccharides (LPS) of Gram-negative bacteria are critical for the defence against cytotoxic substances and must be transported from the inner membrane (IM) to the outer membrane (OM) through a bridge formed by seven membrane proteins (LptBFGCADE)
LptB2 constitutes the cytoplasmic nucleotide-binding domains (NBDs) dimer and the six TM helices of LptF and LptG (F_TM1-TM6 and G_TM1TM6) constitute two transmembrane domains (TMDs) that are arranged to form a central cavity with two surface gaps between TM1F and TM5G and TM1G and TM5F termed as lateral gates (Fig. 1c, d)
In the sfLptB2FGC LPS-bound structure, the TM helix of LptC located between TM1G and TM5F opens up the lateral gate and enlarges the central cavity (Fig. 3a–d)
Summary
Lipopolysaccharides (LPS) of Gram-negative bacteria are critical for the defence against cytotoxic substances and must be transported from the inner membrane (IM) to the outer membrane (OM) through a bridge formed by seven membrane proteins (LptBFGCADE). We report three high-resolution cryo-EM structures of LptB2FG alone and complexed with LptC (LptB2FGC), trapped in either the LPS- or AMP-PNP-bound state. To understand the mechanisms of how LptB2FGC recognises and acts to transport LPS, we obtained high-resolution cryoelectron microscopy (cryo-EM) structures of LptB2FGC complexed with substrate LPS or ATP analogue β-γ-imidoadenosine 5′-triphosphate (AMP-PNP). Our high-resolution cryo-EM structures reveal atomic details in the LPS-binding and ATP-binding cavities, and mutagenic assays allowed us further to identify functional residues in the TM cavity and two periplasmic domains of LptF and LptG involved in LPS recognition and transport process including two essential arginine residues LptF R292 and LptG R301 in the cytoplasmic loop 2 of LptF or LptG. Conformational changes and molecular shifts between domains upon nucleotide binding reveal working mechanism of the transporter
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