Characterizing the interactions between the LPS transport protein LptC and the ABC transporter LptB2FG.

Abstract

Synthesis of the essential glycolipid lipopolysaccharide (LPS) in Gram-negative bacteria is completed at the outer leaflet of the inner membrane (IM). Following this process, seven LPS transport (Lpt) proteins facilitate the movement of LPS to the outer membrane (OM). LPS transport to the OM is an essential process and disruption at any stage has detrimental effects on bacterial viability. LptB2FG, the first component of the Lpt system, is a type IV ABC transporter that provides the driving force for LPS extraction from the IM and subsequent transport across a stable protein bridge to the outer leaflet of the OM. LptC is a periplasmic protein anchored to the IM by a single transmembrane helix, which intercalates between the LptF and LptG transmembrane domains, and has been shown to regulate the ATPase activity of LptB2FG. Despite a high level of structural homology between the periplasmic domains of LptF and LptG, the crystal and cryo-EM structures of LptB2FG in complex with LptC show preferential binding of the periplasmic domain of LptC to the periplasmic domain of LptF. Therefore, the role of LptG in this transport process remains unknown. Additionally, we identified six partial loss of function LptC mutations during an in vivo exploratory mutagenesis study. To further investigate the LptB2FGC complex, site-directed spin labeling electron paramagnetic resonance (EPR) spectroscopy, microscale thermophoresis (MST), and an in vitro ATPase assay were employed to characterize differences between the binding partners of periplasmic LptF and LptG along with the mechanistic effects of the LptC loss of function mutants on partner binding and ATPase function or regulation. This work provides evidence suggesting a previously undefined interaction between LptC and LptG while also exploring the role of partial loss of function LptC mutants.