Cryo-EM structures of lipopolysaccharide transporter LptB2FGC in lipopolysaccharide or AMP-PNP-bound states reveal its transport mechanism

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.

Structural insight into outer membrane asymmetry maintenance of Gram-negative bacteria by the phospholipid transporter MlaFEDB

“…In our current MlaFEDB structure, NBD dimerization is not visualized (Figure 5B). It is worth noting that the densities of the bound nucleotides in these MlaFEDB structures are larger than those we obtained for LptBFGC complex 27 . It may be that the nucleotides in these MlaFEDB structures are not tightly bound in the NBDs, a state before or after dimerization of the NBDs occurred, thus no conformational changes of the complex observed (Figures 5A and 5B).…”

“…The catalytic mutant MlaF(Glu170Ala)EDB showed less effect on anterograde transport of PLs (Figure S11D). MlaF as the NBD of the complex shares folds with other ABC transporters 27,[51][52][53] . MlaF shows 24.54% amino acid sequence identity to LptB, the NBD of LPS transport complex LptBFGC (Figure S11F), and structural similarity with an RMSD of 3.81 Å over 358 aligned Cα residues (Figure S11G).…”

“…ATPase activity assay was performed using ATPase/GTPase Activity Assay Kit (Bioassay systems) as previously reported 27 . The phosphate standards and blank control for colorimetric detection was prepared according to the manufacturer's instructions (ATPase/GTPase Assay Kit, Bioassay systems).…”

“…Progress has been made in understanding the pathways for transporting and assembling OM components including the studies of β-barrel assembly machine (Bam) and LPS transport (Lpt) pathways 13,14,23,[15][16][17][18][19][20][21][22] . The reported high-resolution structures and in vitro transport assays of seven Lpt transport proteins (LptA-G) allowed advanced understanding of the mechanisms how LPS is transported and assembled onto the outer leaflet of the OM 5,8,[32][33][34][24][25][26][27][28][29][30][31] . On the other hand, the molecular mechanism of how PLs are trafficked between the IM and the OM to maintain the OM asymmetry remains unclear.…”

Structure of bacterial phospholipid transporter MlaFEDB with substrate bound

“…We modeled these as the most abundant PL species in E. coli, phosphatidylethanolamine, and these lipids make extensive contacts with both MlaE subunits as well as MlaD (Figure 4 -figure supplement 2a; see Methods for additional details). Unlike recent structures of the LPS exporter bound to LPS, where all of the acyl chains project downward into the hydrophobic pocket of LptFG (Li, Orlando and Liao, 2019b;Tang et al, 2019), the lipids bound to MlaFEDB appear to be trapped in different conformations (Figure 4c), perhaps intermediates in the process of being transferred between MlaE and the MlaD pore. In one lipid molecule (lipid 1), both acyl chains are bound in the pocket of MlaE (Figure 4c, d).…”