Biology subjects

Gohlke, H.

Publications and source records attributed to Gohlke, H..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Long-read transcriptome sequencing of CLL and MDS patients uncovers molecular effects of SF3B1 mutations

BackgroundMutations in splicing factor 3B subunit 1 (SF3B1) frequently occur in patients with chronic lymphocytic leukemia (CLL) and myelodysplastic syndromes (MDS). These mutations have a different effect on the disease prognosis with beneficial effect in MDS and worse prognosis in CLL patients. A full-length transcriptome approach can expand our knowledge on SF3B1 mutation effects on RNA splicing and its contribution to patient survival and treatment options. ResultsWe applied long-read transcriptome sequencing to 44 MDS and CLL patients with and without SF3B1 mutations and found > 60% of novel isoforms. Splicing alterations were largely shared between cancer types and specifically affected the usage of introns and 3 splice sites. Our data highlighted a constrained window at canonical 3 splice sites in which dynamic splice site switches occurred in SF3B1-mutated patients. Using transcriptome-wide RNA binding maps and molecular dynamics simulations, we showed multimodal SF3B1 binding at 3 splice sites and predicted reduced RNA binding at the second binding pocket of SF3B1K700E. ConclusionsOur work presents the hitherto most complete long-read transcriptome sequencing study in CLL and MDS and provides a resource to study aberrant splicing in cancer. Moreover, we showed that different disease prognosis results most likely from the different cell types expanded during cancerogenesis rather than different mechanism of action of the mutated SF3B1. These results have important implications for understanding the role of SF3B1 mutations in hematological malignancies and other related diseases. HighlightsO_LILong-read transcriptome sequencing data enables the identification of > 60% of novel isoforms in the transcriptomes of CLL and MDS patients and isogenic cell lines. C_LIO_LISF3B1 mutations trigger common splicing alterations upon SF3B1 mutations across patient cohorts, most frequently decreased intron retention and increased alternative 3 splice site usage. C_LIO_LIMutation effect depends on alternative 3 splice site and branch point positioning that coincide with bimodal SF3B1 binding at these sites C_LIO_LIMolecular dynamics simulations predict reduced binding of SF3B1K700E to mRNA at the second binding pocket harboring the polypyrimidine tract. C_LI

cancer biology↗

bioRxiv2024

Molecular mechanisms underlying single nucleotidepolymorphism-induced reactivity decrease in CYP2D6

Cytochrome P450 2D6 (CYP2D6) is one of the most important enzymes involved in drug metabolism. Genetic polymorphism can influence drug metabolism by CYP2D6 such that a therapy is seriously affected by under- or overdosing of drugs. However, a general explanation at the atomistic level for poor activity is missing so far. Here we show for the 20 most common single nucleotide polymorphisms (SNPs) of CYP2D6 that poor metabolism is driven by four mechanisms. We found in extensive all-atom molecular dynamics simulations that the rigidity of the I-helix (central helix), distance between central phenylalanines (stabilizing bound substrate), availability of basic residues on the surface of CYP2D6 (binding of Cytochrome P450 reductase), and position of arginine 132 (electron transfer to heme) are essential for an extensive function of the enzyme. These results were applied to SNPs with unknown effects and potential SNPs that may lead to poor drug metabolism were identified. The revealed molecular mechanisms might be important for other drug-metabolizing Cytochrome P450 enzymes. Table of Contents Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/574226v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@517961org.highwire.dtl.DTLVardef@f7ded3org.highwire.dtl.DTLVardef@1462baaorg.highwire.dtl.DTLVardef@1dcabae_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

Identification of ligands binding to MB327-PAM-1, a binding pocket relevant for resensitization of nAChRs

Desensitization of nicotinic acetylcholine receptors (nAChRs) can be induced by overstimulation with acetylcholine (ACh) caused by an insufficient degradation of ACh after poisoning with organophosphorus compounds (OPCs). Currently, there is no generally applicable treatment for OPC poisoning that directly targets the desensitized nAChR. The bispyridinium compound MB327, an allosteric modulator of nAChR, has been shown to act as a resensitizer of nAChRs, indicating that drugs binding directly to nAChRs can have beneficial effects after OPC poisoning. However, MB327 also acts as an inhibitor of nAChRs at higher concentrations and can thus not be used for OPC poisoning treatment. Consequently, novel, more potent resensitizers are required. To successfully design novel ligands, the knowledge of the binding site is of utmost importance. Recently, we performed in silico studies to identify a new potential binding site of MB327, MB327-PAM-1, for which a more affine ligand, UNC0646, has been described. In this work, we performed ligand-based screening approaches to identify novel analogs of UNC0646 to help further understand the structure-affinity relationship of this compound class. Furthermore, we used structure-based screenings and identified compounds representing four new chemotypes binding to MB327-PAM-1. One of these compounds, cycloguanil, is the active metabolite of the antimalaria drug proguanil and shows a higher affinity towards MB327-PAM-1 than MB327. Furthermore, cycloguanil can reestablish the muscle force in soman-inhibited rat muscles. These results can act as a starting point to develop more potent resensitizers of nAChR and to close the gap in the treatment after OPC poisoning.

pharmacology and toxicology↗

bioRxiv2023

MS Binding Assays with UNC0642 as reporter ligand for the MB327 binding site of the nicotinic acetylcholine receptor

Intoxications with organophosphorus compounds (OPCs) based chemical warfare agents and insecticides may result in a detrimental overstimulation of muscarinic and nicotinic acetylcholine receptors evolving into a cholinergic crisis leading to death due to respiratory failure. In the case of the nicotinic acetylcholine receptor (nAChR), overstimulation leads to a desensitization of the receptor, which cannot be pharmacologically treated so far. Still, compounds interacting with the MB327 binding site of the nAChR like the bispyridinium salt MB327 have been found to re-establish the functional activity of the desensitized receptor. Only recently, a series of quinazoline derivatives with UNC0642 as one of the most prominent representatives has been identified to address the MB327 binding site of the nAChR as well. In the present study, MS Binding Assays utilizing UNC0642 as a reporter ligand have been established. Thus, the binding of UNC0642 towards Torpedo-nAChR has been characterized in MS saturation and competition experiments. According to the results, UNC0642 addresses the MB327 binding site of the Torpedo-nAChR. This conclusion is further supported by the outcome of ex vivo studies performed with poisoned rat diaphragm muscles as well as by in silico studies predicting the binding mode of the most affine analog UNC0646 in the recently proposed binding site of MB327 (MB327-PAM-1). The new MS Binding Assays based on the commercially available reporter ligand UNC0642 as one of the most affine ligands for the MB327 binding site are a potent and valuable alternative to established assays.

pharmacology and toxicology↗

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↗

bioRxiv2023

LISTER: Semi-automatic metadata extraction from annotated experiment documentation in eLabFTW

The availability of scientific methods, code, and data is key for reproducing an experiment. Research data should be made available following the FAIR principle (findable, accessible, interoperable, and reusable). For that, the annotation of research data with metadata is central. However, existing research data management workflows often require that metadata should be created by the corresponding researchers, which takes effort and time. Here, we developed LISTER as a methodological and algorithmic solution to disentangle the creation of metadata from ontology alignment and extract metadata from annotated template-based experiment documentation using minimum effort. We focused on tailoring the integration between existing platforms by using eLabFTW as the electronic lab notebook and adopting the ISA (investigation, study, assay) model as the abstract data model framework; DSpace is used as a data cataloging platform. LISTER consists of three components: customized eLabFTW entries using specific hierarchies, templates, and tags; a container concept in eLabFTW, making metadata of a particular container content extractable along with its underlying, related containers; a Python-based app to enable easy-to-use, semi-automated metadata extraction from eLabFTW entries. LISTER outputs metadata as machine-readable .json and human-readable .csv formats, and MM descriptions in .docx format that could be used in a thesis or manuscript. The metadata can be used as a basis to create or extend ontologies, which, when applied to the published research data, will significantly enhance its value due to a more complete and holistic understanding of the data, but might also enable scientists to identify new connections and insights in their field. We applied LISTER to the fields of computational biophysical chemistry as well as protein biochemistry and molecular biology, and our concept should be extendable to other life science areas.

bioinformatics↗

bioRxiv2022

The first archaeal PET-degrading enzyme belongs to the feruloyl-esterase family

Polyethylene terephthalate (PET) is a commodity polymer known to globally contaminate marine and terrestrial environments. Today, around 40 bacterial and fungal PET-active enzymes (PETases) are known, originating from four bacterial and two fungal phyla. In contrast, no archaeal enzyme has been identified to degrade PET. Here we report on the structural and biochemical characterization of PET46, an archaeal promiscuous feruloyl esterase exhibiting degradation activitiy on PET, bis-, and mono-(2-hydroxyethyl) terephthalate (BHET and MHET). The enzyme, found by a sequence-based metagenome search, was derived from a non-cultivated, deep-sea Candidatus Bathyarchaeota archaeon. Biochemical characterization demonstrated that PET46 is a promiscuous, heat-adapted hydrolase. Its crystal structure was solved at a resolution of 1.71 [A]. It shares the core alpha/beta-hydrolase fold with bacterial PETases, but contains a unique lid common in feruloyl esterases, which is involved in substrate binding. Thus, our study significantly widens the currently known diversity of PET-hydrolyzing enzymes, by demonstrating PET depolymerization by a lignin-degrading esterase.

biochemistry↗

bioRxiv2022

TopEnzyme: A framework and database for structural coverage of the functional enzyme space

TopEnzyme is a database of structural enzyme models created with TopModel and is linked to the SWISS-MODEL and AlphaFold Protein Structure Database to provide an overview of structural coverage of the functional enzyme space for over 200,000 enzyme models. It allows the user to quickly obtain representative structural models for 60% of all known enzyme functions. We assessed the models with TopScore and contributed 9039 good-quality and 1297 high-quality structures. Furthermore, we compared these models to AlphaFold2 models with TopScore and found that the TopScore differs only by 0.04 on average in favor of AlphaFold2. We tested TopModel and AlphaFold2 for targets not seen in the respective training databases and found that both methods create qualitatively similar structures. When no experimental structures are available, this database will facilitate quick access to structural models across the currently most extensive structural coverage of the functional enzyme space.

bioinformatics↗

bioRxiv2022

Spectroscopic and QM/MM studies of the Cu(I) binding site of the plant ethylene receptor ETR1

Herein, we present the first spectroscopic characterization of the Cu(I) active site of the plant ethylene receptor ETR1. The X-ray absorption (XAS) and extended X-ray absorption fine structure (EXAFS) spectroscopy presented here establish that ETR1 has a low-coordinate Cu(I) site. The EXAFS resolves a mixed first coordination sphere of N/O and S scatterers at distances consistent with potential histidine and cysteine residues. This finding agrees with the coordination of residues C65 and H69 to the Cu(I) site, which are critical for ethylene activity and well-conserved. Further, the Cu K-edge XAS and EXAFS of ETR1 exhibit spectroscopic changes upon addition of ethylene that are attributed to modifications in the Cu(I) coordination environment, suggestive of ethylene binding. Results from umbrella sampling simulations of the proposed ethylene binding helix of ETR1 at a mixed quantum mechanics/molecular mechanics (QM/MM) level agree with the EXAFS fit distance changes upon ethylene binding, particularly in the increase of the distance between H69 and Cu(I), and yield binding energetics comparable to experimental dissociation constants. The observed changes in the copper coordination environment might be the triggering signal for the transmission of the ethylene response.

biophysics↗

bioRxiv2022

Vasor: Accurate prediction of variant effects for amino acid substitutions in MDR3

Background / RationaleThe phosphatidylcholine floppase MDR3 is an essential hepatobiliary transport protein. MDR3 dysfunction is associated with various liver diseases, ranging from severe progressive familial intrahepatic cholestasis to transient forms of intrahepatic cholestasis of pregnancy and familial gallstone disease. Single amino acid substitutions are often found as causative of dysfunction, but identifying the substitution effect in in vitro studies is time- and cost-intensive. Main resultsWe developed Vasor (Variant assessor of MDR3), a machine learning-based model to classify novel MDR3 missense variants into the categories benign or pathogenic. Vasor was trained on the, to date, largest dataset specific for MDR3 of benign and pathogenic variants and uses general predictors, namely EVE, EVmutation, PolyPhen-2, I-Mutant2.0, MUpro, MAESTRO, PON-P2, and other variant properties such as half-sphere exposure, PTM site, and secondary structure disruption as input. Vasor consistently outperformed the integrated general predictors and the external prediction tool MutPred2, leading to the current best prediction performance for MDR3 single-site missense variants (on an external test set: F1-score: 0.90, MCC: 0.80). Furthermore, Vasor predictions cover the entire sequence space of MDR3. Vasor is accessible as a webserver at https://cpclab.uni-duesseldorf.de/mdr3_predictor/ for users to rapidly obtain prediction results and a visualization of the substitution site within the MDR3 structure. ConclusionThe MDR3-specific prediction tool Vasor can provide reliable predictions of single site amino acid substitutions, giving users a fast way to assess initially whether a variant is benign or pathogenic.

bioinformatics↗

bioRxiv2022

Rational design yields molecular insights on leaf binding of the anchor peptide Macaque Histatin

1In times of a constantly growing world population and increasing demand for food, sustainable agriculture is crucial. To reduce the amount of applied nutrients, herbicides, and fungicides, the rainfastness of plant protection agents is of pivotal importance. As a result of protective agent wash-off, plant protection is lost, and soils and groundwater are severely polluted. To date, rainfastness of plant protection products is achieved by adding polymeric adjuvants to the agrochemicals. However, polymeric adjuvants will be regarded as microplastics in the future, and environmentally friendly alternatives are needed. Anchor peptides (APs) are promising biobased and biodegradable adhesion promoters. While the adhesion of anchor peptides to artificial surfaces, such as polymers, has already been investigated in theory and experimentally, exploiting the adhesion to biological surfaces remains challenging. The complex nature and composition of biological surfaces such as plant leaf and fruit surfaces complicate the generation of accurate models. Here, we present the first detailed three-layered atomistic model of the surface of apple leaves and use it to compute free energy profiles of the adhesion and desorption of APs to and from that surface. Our model is validated by a novel fluorescence-based MTP assay that mimicks these complex processes and allows quantifying them. For the AP Macaque Histatin, we demonstrate that aromatic and positively charged amino acids are essential for binding to the waxy apple leaf surface. The established protocols should generally be applicable for tailoring the binding properties of APs to biological interfaces.

bioengineering↗

bioRxiv2021

Biophysical and pharmacokinetic characterization of a small-molecule inhibitor of RUNX1/ETO tetramerization with anti-leukemic effects

Acute myeloid leukemia (AML) is a malignant disease of immature myeloid cells and the most prevalent acute leukemia among adults. The oncogenic homo-tetrameric fusion protein RUNX1/ETO results from the chromosomal translocation t(8;21) and is found in AML patients. The nervy homology region 2 (NHR2) domain of ETO mediates tetramerization; this oligomerization is essential for oncogenic activity. Previously, we identified the first-in-class small-molecule inhibitor of NHR2 tetramer formation, 7.44, which was shown to specifically interfere with NHR2, restore gene expression down-regulated by RUNX1/ETO, inhibit the proliferation of RUNX1/ETO-depending SKNO-1 cells, and reduce the RUNX1/ETO-related tumor growth in a mouse model. However, no biophysical and structural characterization of 7.44 binding to the NHR2 domain has been reported. Likewise, the compound has not been characterized as to physicochemical, pharmacokinetic, and toxicological properties. Here, we characterize the interaction between the NHR2 domain of RUNX1/ETO and 7.44 by biophysical assays and show that 7.44 interferes with NHR2 tetramer stability and leads to an increase in the dimer population of NHR2. The affinity of 7.44 with respect to binding to NHR2 is Klig = 3.95 {+/-} 1.28 M. By NMR spectroscopy combined with molecular dynamics simulations, we show that 7.44 binds with both heteroaromatic moieties to NHR2 and interacts with or leads to conformational changes in the N-termini of the NHR2 tetramer. Finally, we demonstrate that 7.44 has favorable physicochemical, pharmacokinetic, and toxicological properties. Together with biochemical, cellular, and in vivo assessments, the results reveal 7.44 as a lead for further optimization towards targeted therapy of t(8;21) AML.

biophysics↗

bioRxiv2021

A MademoiseLLE domain binding platform links the key RNA transporter to endosomes

Spatiotemporal expression can be achieved by transport and translation of mRNAs at defined subcellular sites. An emerging mechanism mediating mRNA trafficking is microtubule- dependent co-transport on shuttling endosomes. Although progress has been made in identifying various components of the endosomal mRNA transport machinery, a mechanistic understanding of how these RNA-binding proteins are connected to endosomes is still lacking. Here, we demonstrate that a flexible MademoiseLLE (MLLE) domain platform within RNA- binding protein Rrm4 of Ustilago maydis is crucial for endosomal attachment. Our structure/function analysis uncovered three MLLE domains at the C-terminus of Rrm4 with a functionally defined hierarchy. MLLE3 recognises two PAM2-like sequences of the adaptor protein Upa1 and is essential for endosomal shuttling of Rrm4. MLLE1 and MLLE2 are most likely accessory domains exhibiting a variable binding mode for interaction with currently unknown partners. Thus, endosomal attachment of the mRNA transporter is orchestrated by a sophisticated MLLE domain binding platform.

microbiology↗

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

Respiratory and C4-photosynthetic NAD-malic enzyme coexist in bundle sheath cells mitochondria and evolved via association of differentially adapted subunits

In different lineages of Cleomaceae, NAD-malic enzyme (NAD-ME) was independently co-opted to participate in C4 photosynthesis. In the C4 Cleome species Gynandropsis gynandra and Cleome angustifolia, all NAD-ME genes (NAD-ME, NAD-ME{beta}1, and NAD-ME{beta}2) were affected by C4 evolution and are expressed at higher levels than their orthologs in the C3 Cleome species Tarenaya hassleriana. In the latter C3 species, the NAD-ME housekeeping function is performed by two heteromers, NAD-ME/{beta}1 and NAD-ME/{beta}2, with similar biochemical properties. In both C4 species analyzed, this role is restricted the NAD-ME/{beta}2 heteromer. In the C4 species, NAD-ME/{beta}1 is exclusively present in the leaves, where it accounts for most of the enzymatic activity. GgNAD-ME/{beta}1 exhibits high catalytic efficiency and is differentially activated by the C4 intermediate aspartate, confirming its role as the C4-decarboxylase. During C4 evolution, GgNAD-ME{beta}1and CaNAD-ME{beta}1 lost their catalytic activity; their contribution to enzymatic activity results from a stabilizing effect on the associated -subunit. We conclude that in bundle sheath cell mitochondria of C4 Cleome species, the functions of NAD-ME as C4 photosynthetic decarboxylase and as a tricarboxylic acid cycle-associated housekeeping enzyme coexist and are performed by isoforms that combine the same subunit with differentially adapted {beta} subunits.

plant biology↗

bioRxiv2021

Novel intracellular phospholipase B from Pseudomonas aeruginosa with activity towards endogenous phospholipids affects biofilm assembly

Pseudomonas aeruginosa is a severe threat to immunocompromised patients due to its numerous virulence factors and multiresistance against antibiotics. This bacterium produces and secretes various toxins with hydrolytic activities including phospholipases A, C and D. However, the function of intracellular phospholipases for bacterial virulence has still not been established. Here we demonstrate that the hypothetical gene pa2927 of P. aeruginosa encodes a novel phospholipase B named PaPlaB. PaPlaB isolated from detergent-solubilized membranes of E. coli rapidly degraded various GPLs including endogenous GPLs isolated from P. aeruginosa cells. Cellular localization studies suggest that PaPlaB is peripherally bound to the inner and outer membrane of E. coli, yet the active form was predominantly associated with the cytoplasmic membrane. In vitro activity of purified and detergent-stabilized PaPlaB increases at lower protein concentrations. The size distribution profile of PaPlaB oligomers revealed that decreasing protein concentration triggers oligomer dissociation. These results indicate that homooligomerisation regulates PaPlaB activity by a yet unknown mechanism, which might be required for preventing bacteria from self-disrupting the membrane. We demonstrated that PaPlaB is an important determinant of the biofilm lifestyle of P. aeruginosa, as shown by biofilm quantification assay and confocal laser scanning microscopic analysis of biofilm architecture. This novel intracellular phospholipase B with a putative virulence role contributes to our understanding of membrane GPL degrading enzymes and may provide a target for new therapeutics against P. aeruginosa biofilms.

biochemistry↗

bioRxiv2021

Unsaturated fatty acids augment protein transport via the SecA:SecYEG translocon

The translocon SecYEG forms the primary protein-conducting channel in the cytoplasmic membrane of bacteria, and the associated ATPase SecA provides the energy for the transport of secretory and cell envelope protein precursors. The translocation requires negative charge at the lipid membrane surface, but its dependence on the properties of the membrane hydrophobic core is not known. Here, we demonstrate that SecA:SecYEG-mediated protein transport is immensely stimulated by unsaturated fatty acids (UFAs). Furthermore, UFA-rich tetraoleoyl-cardiolipin, but not bis(palmitoyloleoyl)-cardiolipin, facilitate the translocation via the monomeric translocon. Biophysical analysis and molecular dynamics simulations show that UFAs determine the loosely packed membrane interface, where the N-terminal amphipathic helix of SecA docks. While UFAs do not affect the translocon folding, they promote SecA binding to the membrane, and the effect is enhanced manifold at elevated ionic strength. Tight SecA:lipid interactions convert into the augmented translocation. As bacterial cells actively change their membrane composition in response to their habitat, the modulation of SecA:SecYEG activity via the fatty acids may be crucial for protein secretion over a broad range of environmental conditions.

biophysics↗