Elucidating bacterial lipid transport mechanisms by the envelope-spanning LetAB complex.

Abstract

Gram-negative bacteria are surrounded by an outer membrane (OM) that protects them against environmental stressors and toxic compounds, and therefore contributes to antibiotic resistance. To generate this barrier, membrane lipids, which are poorly soluble in water, must be transported across the hydrophilic periplasmic space between the inner membrane (IM) and OM. Bacteria have evolved various mechanisms to achieve active transport across the cell envelope. The Let (Lipophilic Envelope-spanning Tunnel) system, comprising LetA and LetB, is one system thought to be involved in maintaining OM integrity. LetB forms a hydrophobic tunnel long enough to span the periplasm, potentially facilitating the sheltered movement of substrates directly between the two membranes. However, it is unclear how lipids are extracted from the IM and transported into LetB, or vice versa. The only known binding partner of LetB is the IM protein, LetA, which belongs to an unstudied family of proteins. We have obtained a ∼3.5 Å cryo-EM structure of the LetAB complex, which reveals that LetA is a pseudodimeric integral inner membrane protein with two cytoplasmic zinc finger domains that interact with each other to stabilize the protein. LetA binds at one end of LetB, where it interacts with the central tunnel of LetB to form a substrate translocation pathway between the two subunits. Our structural and biochemical data reveal a possible substrate-binding site in LetA. LetA also contains a putative proton/ion permeation pathway, lined with ionizable residues that are essential for function, which we hypothesize may play a role in energizing transport. We will discuss how the LetA and LetB subunits come together to facilitate lipid transport, and provide a model for how proteins in the LetA protein family may function.