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Conformational dynamics of the membrane-anchored foldase LipH from Pseudomonas aeruginosa facilitates recognition and release of the client lipase

ABSTRACT: The lipase LipA from Pseudomonas aeruginosa is a multifunctional enzyme that plays an important role as a virulence factor in bacterial infections, but is also useful for a variety of biotechnological applications. This enzyme is secreted via the type II secretion system (T2SS) and strictly depends on its cognate membrane-anchored foldase LipH for maturation. While structural studies have elucidated the architecture of the assembled LipH:lipase complex, how the full-length, membrane-tethered foldase dynamically recognizes, folds, and releases its client has remained barely understood. Here, we combine all-atom molecular dynamics simulations, small-angle X-ray scattering (SAXS), hydrogen/deuterium exchange mass spectrometry, and reconstitution into membrane mimetics to investigate the conformational dynamics and function of the full-length LipH in a membrane context. Simulations reveal that the membrane-anchored LipH is highly dynamic, sampling a broad ensemble of conformations, in which the chaperoning cavity is transiently closed or occluded by both the proximal membrane and the linker polypeptide, which is further confirmed by SAXS analysis. Despite the steric hindrance, the full-length LipH reconstituted into membrane mimetics efficiently activates LipA, though it displays substantially reduced affinity for the client. We propose that the negatively charged membrane promotes release of the folded client, enabling multiple chaperoning cycles. Hydrogen/deuterium exchange analysis reveals that the MD2 domain of LipH in engaged in stable interactions with the lipase, whereas MD1 contacts are transient. Consistently, LipH readily captures N-terminal fragments of LipA, indicating that initial recognition relies on local interactions via the MD2 domain. Together, our results show how membrane coupling and intrinsic conformational plasticity modulate the function of the steric chaperone, and suggest that the membrane-anchored LipH balances capture, folding, and release of the client LipA to enable its efficient maturation and secretion.

INSTRUMENT(S):

ORGANISM(S): Pseudomonas Aeruginosa Pao1

SUBMITTER: Wieland Steinchen

LAB HEAD: Gert Bange

PROVIDER: PXD074746 | Pride | 2026-07-21

REPOSITORIES: Pride

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			Action	DRS
	231211_HX_532_Ba.DnX	Other
	231211_HX_532_Ba_A1_LipA.zip	Other
	231211_HX_532_Ba_A2_LipH.zip	Other
	231211_HX_532_Ba_A3_LipA-LipH.zip	Other
	231211_HX_532_Ba_PLGS.zip	Other

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Project description:Lysosomal acid lipase (Lipa), which is primarily produced in the liver, has been identified as one of the earliest and most impactful genetic factors linked to human coronary artery diseases. Despite its original source, it is also found to be highly expressed in macrophages. However, the functional importance of Lipa in macrophages has been largely unknown. Notably, individuals with certain risk alleles demonstrate elevated Lipa expression in monocytes and macrophages, but lower levels of hepatic and plasma Lipa, indicating a potential regulatory role of Lipa on atherosclerosis through its impact on macrophages. The development of atherosclerosis in genetic models lacking Lipa has presented challenges. Our hypothesis is that Lipa deficiency may influence atherosclerosis development by affecting macrophages. In order to investigate this, we established Lipa-deficient mice on an ApoE-knockout background, providing a model that enables us to evaluate the influence of Lipa on atherosclerosis. In our experiments, we observed hepatosplenomegaly and increased myeloid cell differentiation in Lipa−/−ApoE−/− mice fed a high-fat diet, which aligns with observations in humans with LIPA deficiency. Surprisingly, we also discovered that Lipa deficiency led to a significant inhibition of atherosclerosis in both Lipa+/−ApoE−/− and Lipa−/−ApoE−/− mice. Mechanistically, we found that such inhibition was linked to a notable reduction in foam cell formation and macrophage accumulation within atherosclerotic plaques. Notably, reduced Lipa expression in Lipa+/−ApoE−/− mice influenced foam cell formation and macrophage accumulation, but did not impact hepatic lipid metabolism or macrophage differentiation, as evidenced by flow cytometry and single-cell RNA-sequencing. These findings highlight a novel role for Lipa in modulating macrophage behavior during atherosclerosis, suggesting that Lipa may serve as a promising therapeutic target for the treatment of atherosclerosis.

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Conformational dynamics of the membrane-anchored foldase LipH from Pseudomonas aeruginosa facilitates recognition and release of the client lipase

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