Structure–function relationship of the <i>Pseudomonas aeruginosa</i><scp>AsmA</scp>‐like proteins <scp>YhdP</scp> and <scp>YdbH</scp> involved in outer membrane biogenesis

The outer membrane (OM) of Gram-negative bacteria is an asymmetric bilayer composed of glycerophospholipids (GPLs) in the inner leaflet and lipopolysaccharide in the outer leaflet, which is critical for viability and antibiotic resistance. While the mechanisms for lipopolysaccharide and OM protein transport across the periplasm are well characterized, it has only recently been proposed that AsmA-like proteins are likely involved in the anterograde transport of GPLs. Here, we investigated the structural and functional features of Pseudomonas aeruginosa YhdP, an AsmA-like protein that exhibits higher activity in maintaining OM integrity than its paralogs. Through structural predictions, molecular dynamics simulations, and genetic complementation assays, we demonstrated that two hydrophobic α-helices at the C-terminal region of YhdP are crucial for its function. Moreover, we found that fusing the YhdP C-terminal domain to YdbH enhances the functionality of this shorter and lower-activity AsmA-like protein. Specifically, a YdbH-YhdP fusion protein long enough to potentially span the periplasmic space was more active than the wild-type YdbH protein in promoting growth and OM integrity. Furthermore, while YdbH activity requires the OM protein partner YnbE, the functionality of the YdbH-YhdP fusion protein is YnbE-independent. These findings support the hypothesis that the YhdP C-terminus provides OM anchoring and likely assists GPL insertion, and prove that it can be used as a modular element for improving the activity of other AsmA-like proteins. Our work sheds light on the molecular determinants of GPL transport and offers a framework for studying the mode of action of AsmA-like proteins in OM biogenesis.