Biology subjects

Jaeger, K.-E.

Publications and source records attributed to Jaeger, K.-E..

12 recordsLinked to original sources

bioRxiv2026

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

The lipase LipA from Pseudomonas aeruginosa is an extracellular enzyme that plays an important role in bacterial infections. Prior its export via the type II secretion system, LipA requires the cognate membrane-anchored foldase LipH for maturation in the periplasm. Though structural studies elucidated the architecture of the LipH:lipase complex, how the full-length, membrane-tethered foldase recognizes, folds, and releases its client has remained barely understood. Here, we combine in silico and in vitro analysis to resolve 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. The chaperoning cavity is transiently closed or occluded by both the proximal membrane and the linker polypeptide, which is further confirmed by structural analysis. Despite the steric hindrance, the full-length LipH reconstituted into lipid-based nanodiscs and amphipols 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 secretion.

biochemistry↗

bioRxiv2025

Dimer asymmetry in signaling of blue-light sensor histidine kinases

Photoreceptor sensory histidine kinases (SHKs) couple light absorption to conformational changes regulating two-component signaling. Despite their importance and widespread use in optogenetics, the underlying structural signaling mechanisms remain poorly understood. We here engineered dimeric SHKs based on Pseudomonas putida short light-oxygen-voltage (LOV) proteins and determined their crystal structures. Regardless of illumination, the structures adopted a light-state like LOV-LOV dimer with symmetric/straight kinase modules. In contrast, small-angle X-ray scattering together with functional assays revealed pronounced light-dependent rearrangements in solution and allowed the assignment of the kinase-ON dark state to an asymmetric/kinked conformation, whereas the light state adopts a symmetric/straight structure. Comparative analyses of natural and engineered SHKs identified conserved motifs linking LOV domain rotation to kinase activity. The findings highlight the central role of dimer asymmetry and flexibility in SHK signaling, thereby not least informing the engineering of new light-responsive signaling systems.

biochemistry↗

bioRxiv2025

A Novel Approach to Combat Pseudomonas aeruginosa: Repurposing Pharmaceuticals for Inhibition of Phospholipase A

Phospholipases A (PLAs) play critical roles in cellular physiology, making human PLAs established drug targets. On the other hand, the potential of bacterial PLAs as targets for antimicrobial drug development remains underexplored. In this study, we curated a library of 23 approved and investigational pharmaceuticals, some of which inhibit human PLA-like enzymes, through a combination of ligand structure-based searches and textual mining in literature and compound databases. Experimental screening identified that compounds GW4869, darapladib, and rilapladib significantly inhibit Pseudomonas aeruginosa growth by more than 50 %. While these compounds did not reduce biofilm formation, GW4869 increased the proportion of dead cells in established biofilms, suggesting its role in compromising biofilm cell viability. Biochemical assays revealed that all three compounds inhibited the enzymatic activity of PlaF, a PLA virulence factor of P. aeruginosa, by decreasing the affinity of a model substrate. Molecular dynamics simulations and binding free energy analyses indicate that GW4869 binds to the substrate-binding and product-release tunnels of PlaF, suggesting GW4869 as a non-covalent competitive inhibitor. Notably, the mutant strain P. aeruginosa {Delta}plaF proved to be GW4869 resistant and did not display differential growth upon GW4869 treatment, further indicating PlaF as the primary GW4869 target. Furthermore, GW4869 and rilapladib significantly enhanced the efficacy of the last-resort antibiotic imipenem in combination treatments. These findings highlight the potential of GW4869, darapladib, and rilapladib to act as repurposed inhibitors of PlaF or PLA-dependent mechanisms in P. aeruginosa and underscore the promise of combination therapies against intracellular PLAs to combat antimicrobial resistance.

molecular biology↗

bioRxiv2024

Magnetic Protein Aggregates Generated by Supramolecular Assembly of Ferritin Cages - A Modular Strategy for the Immobilization of Enzymes

Efficient and cost-effective immobilization methods are crucial for advancing the utilization of enzymes in industrial biocatalysis. To this end, in vivo immobilization methods relying on the completely biological production of immobilizates represent an interesting alternative to conventional carrier-based immobilization methods. In this contribution, we present a novel immobilization strategy utilizing in vivo produced, magnetic protein aggregates (MPAs). MPA production is facilitated by the expression of gene fusions consisting of genes encoding for the yellow fluorescent protein variant citrine and variants of the iron storage protein ferritin, including a magnetically enhanced ferritin mutant from Escherichia coli. Expression of the gene fusions allows supramolecular assembly of the fusion proteins in vivo, which is driven by citrine-dependent dimerization of ferritin cages. Upon cell lysis, the assemblies coalesce in solution to form MPAs. The fusion of the mutant E. coli ferritin to citrine yields fluorescent, insoluble protein aggregates that display magnetic properties, verified by their attraction to neodymium magnets. We further demonstrate that these novel, fully in vivo produced protein aggregates can be magnetically purified without the need for ex vivo iron-loading. Utilizing a bait/prey strategy, MPAs were functionalized by the post-translational attachment of an alcohol dehydrogenase to the MPA particles to enable proof-of-concept for enzyme immobilization, giving rise to catalytically-active magnetic protein aggregates (CatMPAs). The resulting (Cat)MPAs could easily be obtained from crude cell extracts via centrifugation, or purified using magnetic columns, and exhibited superior stability. The strategy presented here therefore represents a highly modular method to produce magnetic enzyme immobilizates which can be obtained with high purity.

biochemistry↗

bioRxiv2023

Molecular Mechanisms Underlying Medium-Chain Length Free Fatty Acid-regulated Activity of the Phospholipase PlaF from P. aeruginosa

PlaF is a membrane-bound phospholipase A1 from P. aeruginosa that is involved in remodeling membrane glycerophospholipids (GPLs) and modulation of virulence-associated signaling and metabolic pathways. Previously, we identified the role of medium-chain free fatty acids (FFA) in inhibiting PlaF activity and promoting homodimerization, yet the underlying molecular mechanism remained elusive. Here, we used unbiased and biased molecular dynamics simulations and free energy computations to assess how PlaF interacts with FFAs localized in the water milieu surrounding the bilayer or within the bilayer, and how these interactions regulate PlaF activity. Medium-chain FFAs localized in the upper bilayer leaflet can stabilize inactive dimeric PlaF, likely through interactions with charged surface residues as experimentally validated. Potential of mean force (PMF) computations indicate that membrane-bound FFAs may facilitate the activation of monomeric PlaF by lowering the activation barrier of changing into a tilted, active configuration. We estimated that the coupled equilibria of PlaF monomerization-dimerization and tilting at the physiological concentration of PlaF lead to the majority of PlaF forming inactive dimers when in a cell membrane loaded with decanoic acid (C10). This is in agreement with a suggested in vivo product feedback loop and GC-MS profiling results indicating that PlaF catalyzes the release of C10 from P. aeruginosa membranes. Additionally, we found that C10 in the water milieu can access the catalytic site of active monomeric PlaF, contributing to the competitive component of C10-mediated PlaF inhibition. Our study provides mechanistic insights into how medium-chain FFA may regulate the activity of PlaF, a potential bacterial drug target.

biophysics↗

bioRxiv2022

Virulence adaptation of Pseudomonas aeruginosa phospholipase mutant with altered membrane phospholipid composition

Membrane protein and phospholipid (PL) composition changes in response to environmental cues and during infections. Covalent modification and remodelling of the acyl chain length of PLs is an important bacterial adaptation mechanism. However, little is known about which bacterial pathways are regulated in response to altered PL composition. Here, we showed that P. aeruginosa phospholipase A, PlaF, which modulates membrane PL composition, is important for biofilm biogenesis, and we performed whole-cell quantitative proteomics of P. aeruginosa wild-type and {Delta}plaF biofilms to identify pathways regulated by PlaF. The results revealed profound alterations in the abundance of several two-component systems (TCSs), including accumulation of PprAB, which controls the transition to biofilm. Furthermore, a unique phosphorylation pattern of transcriptional regulators, transporters and metabolic enzymes, as well as differential production of seven proteases, in {Delta}plaF, indicate that PlaF-mediated virulence adaptation involves complex transcriptional and posttranscriptional regulation. Moreover, proteomics revealed the depletion of pyoverdine-mediated iron uptake pathway proteins in {Delta}plaF, which agrees with the decreased concentrations of extracellular pyoverdine and intracellular iron and is likely responsible for its prolonged lag growth phase, presumably due to reduced iron uptake. Conversely, the accumulation of proteins from alternative iron-uptake systems in {Delta}plaF suggests that PlaF may function as a switch between different iron-acquisition pathways. The observation that {Delta}plaF accumulates PL-acyl chain modifying and PL synthesis enzymes reveals novel insights into the role of PlaF for membrane PL homeostasis. Although the precise mechanism by which PlaF simultaneously affects multiple pathways remains to be elucidated, we suggest that PlaF-catalyses the degradation of PLs which then serve as a signal that is amplified by proteins of two-component, phosphorylation and proteolytic degradation systems to elicit the global adaptive response in P. aeruginosa.

molecular biology↗

bioRxiv2022

The periplasmic chaperone Skp prevents misfolding of the secretory lipase A from Pseudomonas aeruginosa

Pseudomonas aeruginosa is a wide-spread opportunistic human pathogen and a high-risk factor for immunodeficient people and patients with cystic fibrosis. The extracellular lipase A belongs to the virulence factors of P. aeruginosa. The lipase undergoes folding and activation in the periplasm prior the secretion. Here, we demonstrate that the ubiquitous periplasmic chaperone Skp of P. aeruginosa, but not SurA, FkpA, PpiD or YfgM, efficiently prevents misfolding of the aggregation-prone lipase A and facilitates its activation by a specific foldase LipH. Small-angle X-ray scattering visualizes the trimeric architecture of P. aeruginosa Skp and identifies two primary conformations of the chaperone, a compact and a widely open. We describe two binding modes of Skp to the lipase, with affinities of 20 nM and 2 M, which correspond to 1:1 and 1:2 stoichiometry of the lipase:Skp complex. Two Skp trimers are required to stabilize the lipase via the apolar interactions, which are not affected by high salt concentrations typical for the sputum of cystic fibrosis patients. The chaperoning effect of Skp points to its potent role in maturation and secretion of the lipase in Pseudomonas species.

biochemistry↗

bioRxiv2021

Substrate access mechanism in a novel membrane-bound phospholipase A of Pseudomonas aeruginosa concordant with specificity and regioselectivity

PlaF is a cytoplasmic membrane-bound phospholipase A1 from Pseudomonas aeruginosa that alters the membrane glycerophospholipid (GPL) composition and fosters the virulence of this human pathogen. PlaF activity is regulated by a dimer-to-monomer transition followed by tilting of the monomer in the membrane. However, how substrates reach the active site and how the characteristics of the active site tunnels determine the activity, specificity, and regioselectivity of PlaF for natural GPL substrates has remained elusive. Here, we combined unbiased and biased all-atom molecular dynamics (MD) simulations and configurational free energy computations to identify access pathways of GPL substrates to the catalytic center of PlaF. Our results map out a distinct tunnel through which substrates access the catalytic center. PlaF variants with bulky tryptophan residues in this tunnel revealed decreased catalysis rates due to tunnel blockage. The MD simulations suggest that GPLs preferably enter the active site with the sn-1 acyl chain first, which agrees with the experimentally demonstrated PLA1 activity of PlaF. We propose that the acyl chain-length specificity of PlaF is determined by the structural features of the access tunnel, which results in favorable free energy of binding of medium-chain GPLs. The suggested egress route conveys fatty acid products to the dimerization interface and, thus, contributes to understanding the product feedback regulation of PlaF by fatty acid-triggered dimerization. These findings open up opportunities for developing potential PlaF inhibitors, which may act as antibiotics against P. aeruginosa.

biochemistry↗

bioRxiv2021

Evidence for a bacterial Lands cycle phospholipase A: Structural and mechanistic insights into membrane phospholipid remodeling

Cells steadily adapt their membrane glycerophospholipid (GPL) composition to changing environmental and developmental conditions. While the regulation of membrane homeostasis via GPL synthesis in bacteria has been studied in detail, the mechanisms underlying the controlled degradation of endogenous GPLs remain unknown. Thus far, the function of intracellular phospholipases A (PLAs) in GPL remodeling (Lands cycle) in bacteria is not clearly established. Here, we identified the first cytoplasmic membrane-bound phospholipase A1 (PlaF) from Pseudomonas aeruginosa involved in the Lands cycle. PlaF is an important virulence factor, as the P. aeruginosa {Delta}plaF mutant showed strongly attenuated virulence in Galleria mellonella and macrophages. We present a 2.0-[A]-resolution crystal structure of PlaF, the first structure that reveals homodimerization of a single-pass transmembrane (TM) full-length protein. PlaF dimerization, mediated solely through the intermolecular interactions of TM and juxtamembrane regions, inhibits its activity. A dimerization site and the catalytic sites are linked by an intricate ligand-mediated interaction network which likely explains the product (fatty acid) feedback inhibition observed with the purified PlaF protein. We used molecular dynamics simulations and configurational free energy computations to suggest a model of PlaF activation through a coupled monomerization and tilting of the monomer in the membrane, which constrains the active site cavity into contact with the GPL substrates. Thus, these data show the importance of the GPL remodeling pathway for virulence and pave the way for the development of a novel therapeutic class of antibiotics targeting PlaF-mediated membrane GPL remodeling. SynopsisMembrane homeostasis can be regulated by phospholipase-controlled deacylation of endogenous glycerophospholipids (GPLs) followed by reacylation of products, known as the Lands cycle in eukaryotes. Here we show that the human pathogen Pseudomonas aeruginosa uses intracellular phospholipase A1 (PlaF) to modulate membrane GPL composition, which is the first example in bacteria. This newly identified PLA1 indirectly regulates the bacterial virulence properties by hydrolyzing a specific set of membrane GPLs. The crystal structure of full-length PlaF dimers bound to natural ligands, MD simulations, and biochemical approaches provide insights into the molecular mechanism of dimerization-mediated inactivation of this single-pass transmembrane PLA1. Our findings shed light on a mechanism by which bacterial intracellular PLAs might regulate membrane homeostasis what showcases these enzymes as a promising target for a new class of antibiotics.

molecular biology↗

bioRxiv2021

Structural and mechanistic insight into spectral tuning in flavin-binding fluorescent proteins

Determining the molecular origin of spectral tuning in photoactive biological systems is instrumental for understanding their function. Spectral-tuning efforts for flavin-binding fluorescent proteins (FbFPs), an emerging class of fluorescent reporters, are limited by their dependency on protein-bound flavins, whose structure and hence electronic properties, cannot be altered by mutation. To address those shortcomings, we here present the photophysical, computational and structural characterization of structurally uncharacterized blue-shifted FbFPs, carrying a previously described lysine substitution within their flavin-binding pocket. X-ray structures reveal displacement of the lysine away from the chromophore and opening up of the structure as cause for the blue shift. Site-saturation mutagenesis and high-throughput screening, yielded a red-shifted variant, in which the lysine side chain of the blue-shifted variant is stabilized in close distance to the flavin by a secondary mutation, mechanistically accounting for the red shift. Thus, a single secondary mutation in a blue-shifted variant is sufficient to generate a red-shifted FbFP. Using spectroscopy, X-ray crystallography and quantum mechanics molecular mechanics calculations, we provide a firm structural and functional understanding of spectral tuning in FbFPs. We also show that the identified blue- and red-shifted variants allow for two-color microscopy based on spectral separation. In summary, the generated blue- and red-shifted variants represent promising new tools that should find application in life sciences.

biophysics↗

bioRxiv2020

Promiscuous esterases counterintuitively are less flexible than specific ones

Understanding mechanisms of promiscuity is increasingly important from a fundamental and application point of view. As to enzyme structural dynamics, more promiscuous enzymes generally have been recognized to also be more flexible. However, examples for the opposite received much less attention. Here, we exploit comprehensive experimental information on the substrate promiscuity of 147 esterases tested against 96 esters together with computationally efficient rigidity analyses to understand the molecular origin of the observed promiscuity range. Unexpectedly, our data reveal that promiscuous esterases are significantly less flexible than specific ones, are significantly more thermostable, and have a significantly increased specific activity. These results may be reconciled with a model according to which structural flexibility in the case of specific esterases serves for conformational proofreading. Our results signify that esterase sequence space can be screened by rigidity analyses for promiscuous esterases as starting points for further exploration in biotechnology and synthetic chemistry.

biophysics↗

bioRxiv2020

Ustilago maydis serves as a novel production host for the synthesis of plant and fungal sesquiterpenoids

AbtractSesquiterpenoids are important secondary metabolites with various pharma- and nutraceutical properties. In particular, higher basidiomycetes possess a versatile biosynthetic repertoire for these bioactive compounds. To date, only a few microbial production systems for fungal sesquiterpenoids have been established. Here, we introduce Ustilago maydis as a novel production host. This model fungus is a close relative of higher basidiomycetes. It offers the advantage of metabolic compatibility and potential tolerance for substances toxic to other microorganisms. We successfully implemented a heterologous pathway to produce the carotenoid lycopene that served as a straightforward read-out for precursor pathway engineering. Overexpressing genes encoding enzymes of the mevalonate pathway resulted in increased lycopene levels. Verifying the subcellular localisation of the relevant enzymes revealed that initial metabolic reactions might take place in peroxisomes: despite the absence of a canonical peroxisomal targeting sequence, acetyl-CoA C-acetyltransferase Aat1 localised to peroxisomes. By expressing the plant (+)-valencene synthase CnVS and the basidiomycete sesquiterpenoid synthase Cop6, we succeeded in producing (+)-valencene and -cuprenene, respectively. Importantly, the fungal compound yielded about tenfold higher titres in comparison to the plant substance. This proof of principle demonstrates that U. maydis can serve as promising novel chassis for the production of terpenoids.

microbiology↗