Mechanistic basis of LolDF-mediated lipoprotein transport in Acinetobacter.

Lipoprotein trafficking is essential for Gram-negative bacteria to maintain the outer membrane, a permeability barrier critical for survival and antibiotic resistance. While most bacteria employ the canonical LolCDE transporter, Acinetobacter utilizes a noncanonical LolDF system whose mechanism has remained unclear. Here we determine cryo-EM structures of Acinetobacter baylyi LolDF in substrate-bound, AMP-PNP-bound, and LolA-bound states. LolDF forms a symmetric homodimer with a central cavity that accommodates three acyl chains at the membrane level, suggesting lateral extraction of lipoproteins directly from the inner membrane. AMP-PNP-driven conformational changes induce a coordinated symmetric constriction of the cavity that promotes substrate expulsion, distinct from the asymmetric mechanism observed in LolCDE. Functional studies further identify key door-bar residues required for transport and reveal that charged residues at the +3 position function as an inner membrane retention signal. Together, these findings reveal a mechanistically distinct lipoprotein trafficking system and provide a framework for targeting multidrug-resistant Gram-negative pathogens.