Structures of the Pseudomonas aeruginosa MlaC-MlaD complexes reveal a conformational switch mediated by the C-terminal helix of MlaC

Gram-negative bacteria maintain an asymmetric outer membrane that protects cells from environmental stresses and antibiotics. The maintenance of lipid asymmetry (Mla) pathway contributes to outer membrane lipid homeostasis through phospholipid transport between the outer and inner membranes. Although the periplasmic lipid carrier MlaC is thought to transfer phospholipids to the inner membrane MlaFEDB transporter via the hexameric protein MlaD, the molecular mechanism underlying this process remains unclear. Here we show crystal structures of two distinct MlaC-MlaD complexes from Pseudomonas aeruginosa that reveal distinct conformational states of MlaC. In these structures, an ordered conformation of the C-terminal 8 helix of MlaC positions MlaC distally from the central pore of the MlaD hexamer and limits accessibility of the lipid-binding cavity, whereas partial disordering of the 8 helix allows closer association with the MlaD hexamer and increased exposure of the cavity. Structure-based biochemical analyses further demonstrate that the C-terminal region negatively regulates MlaC- MlaD interaction while stabilizing phospholipid binding. These findings identify the C-terminal 8 helix as a conformational switch that couples MlaC positioning with lipid cavity accessibility, providing structural insight into phospholipid transfer at the MlaC-MlaD interface.