Gohlke H

References (18)

Title : MS Binding Assays with UNC0642 as reporter ligand for the MB327 binding site of the nicotinic acetylcholine receptor - Nitsche_2024_Toxicol.Lett_392_94
Author(s) : Nitsche V , Hofner G , Kaiser J , Gertzen CGW , Seeger T , Niessen KV , Steinritz D , Worek F , Gohlke H , Paintner FF , Wanner KT
Ref : Toxicol Lett , 392 :94 , 2024
Abstract : 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 this study, UNC0642 has been utilized as a reporter ligand to establish new Binding Assays for this target. These assays follow the concept of MS Binding Assays for which by assessing the amount of bound reporter ligand by mass spectrometry no radiolabeled material is required. According to the results of the performed MS Binding Assays comprising saturation and competition experiments it can be concluded, that UNC0642 used as a reporter ligand addresses the MB327 binding site of the Torpedo-nAChR. This is further supported by the outcome of ex vivo studies carried out with poisoned rat diaphragm muscles as well as by in silico studies predicting the binding mode of UNC0646, an analog of UNC0642 with the highest binding affinity, in the recently proposed binding site of MB327 (MB327-PAM-1). With UNC0642 addressing the MB327 binding site of the Torpedo-nAChR, this and related quinazoline derivatives represent a promising starting point for the development of novel ligands of the nAChR as antidotes for the treatment of intoxications with organophosphorus compounds. Further, the new MS Binding Assays are a potent alternative to established assays and of particular value, as they do not require the use of radiolabeled material and are based on a commercially available compound as reporter ligand, UNC0642, exhibiting one of the highest binding affinities for the MB327 binding site known so far.
ESTHER : Nitsche_2024_Toxicol.Lett_392_94
PubMedSearch : Nitsche_2024_Toxicol.Lett_392_94
PubMedID: 38216073

Title : Synthesis and Biological Evaluation of Novel MB327 Analogs as Resensitizers for Desensitized Nicotinic Acetylcholine Receptors after Intoxication with Nerve Agents - Bernauer_2024_Toxicol.Lett__
Author(s) : Bernauer T , Nitsche V , Kaiser J , Gertzen CGW , Hofner G , Niessen KV , Seeger T , Steinritz D , Worek F , Gohlke H , Wanner KT , Paintner FF
Ref : Toxicol Lett , : , 2024
Abstract : Poisoning with organophosphorus compounds, which can lead to a cholinergic crisis due to the inhibition of acetylcholinesterase and the subsequent accumulation of acetylcholine (ACh) in the synaptic cleft, is a serious problem for which treatment options are currently insufficient. Our approach to broadening the therapeutic spectrum is to use agents that interact directly with desensitized nicotinic acetylcholine receptors (nAChRs) in order to induce functional recovery after ACh overstimulation. Although MB327, one of the most prominent compounds investigated in this context, has already shown positive properties in terms of muscle force recovery, this compound is not suitable for use as a therapeutic agent due to its insufficient potency. By means of in silico studies based on our recently presented allosteric binding pocket at the nAChR, i.e. the MB327-PAM-1 binding site, three promising MB327 analogs with a 4-aminopyridinium ion partial structure (PTM0056, PTM0062 and PTM0063) were identified. In this study, we present the synthesis and biological evaluation of a series of new analogs of the aforementioned compounds with a 4-aminopyridinium ion partial structure (PTM0064-PTM0072), as well as hydroxy-substituted analogs of MB327 (PTMD90-0012 and PTMD90-0015) designed to substitute entropically unfavorable water clusters identified during molecular dynamics simulations. The compounds were characterized in terms of their binding affinity towards the aforementioned binding site by applying the UNC0642 MS Binding Assays and in terms of their muscle force reactivation in rat diaphragm myography. More potent compounds were identified compared to MB327, as some of them showed a higher affinity towards MB327-PAM-1 and also a higher recovery of neuromuscular transmission at lower compound concentrations. To improve the treatment of organophosphate poisoning, direct targeting of nAChRs with appropriate compounds is a key step, and this study is an important contribution to this research.
ESTHER : Bernauer_2024_Toxicol.Lett__
PubMedSearch : Bernauer_2024_Toxicol.Lett__
PubMedID: 38759939

Title : A novel binding site in the nicotinic acetylcholine receptor for MB327 can explain its allosteric modulation relevant for organophosphorus-poisoning treatment - Kaiser_2023_Toxicol.Lett_373_160
Author(s) : Kaiser J , Gertzen CGW , Bernauer T , Hofner G , Niessen KV , Seeger T , Paintner FF , Wanner KT , Worek F , Thiermann H , Gohlke H
Ref : Toxicol Lett , 373 :160 , 2023
Abstract : Organophosphorus compounds (OPCs) are highly toxic compounds that can block acetylcholine esterase (AChE) and thereby indirectly lead to an overstimulation of muscarinic and nicotinic acetylcholine receptors (nAChRs). The current treatment with atropine and AChE reactivators (oximes) is insufficient to prevent toxic effects, such as respiratory paralysis, after poisonings with various OPCs. Thus, alternative treatment options are required to increase treatment efficacy. Novel therapeutics, such as the bispyridinium non-oxime MB327, have been found to reestablish neuromuscular transmission by interacting directly with nAChR, probably via allosteric mechanisms. To rationally design new, more potent drugs addressing nAChR, knowledge of the binding mode of MB327 is fundamental. However, the binding pocket of MB327 has remained elusive. Here, we identify a new potential allosteric binding pocket (MB327-PAM-1) of MB327 located at the transition of the extracellular to the transmembrane region using blind docking experiments and molecular dynamics simulations. MB327 forms striking interactions with the receptor at this site. The interacting amino acids are highly conserved among different subunits and different species. Correspondingly, MB327 can interact with several nAChR subtypes from different species. We predict by rigidity analysis that MB327 exerts an allosteric effect on the orthosteric binding pocket and the transmembrane domain after binding to MB327-PAM-1. Furthermore, free ligand diffusion MD simulations reveal that MB327 also has an affinity to the orthosteric binding pocket, which agrees with recently published results that related bispyridinium compounds show inhibitory effects via the orthosteric binding site. The newly identified binding site allowed us to predict structural modifications of MB327, resulting in the more potent resensitizers PTM0062 and PTM0063.
ESTHER : Kaiser_2023_Toxicol.Lett_373_160
PubMedSearch : Kaiser_2023_Toxicol.Lett_373_160
PubMedID: 36503818

Title : Resolution of Maximum Entropy Method-Derived Posterior Conformational Ensembles of a Flexible System Probed by FRET and Molecular Dynamics Simulations - Dittrich_2023_J.Chem.Theory.Comput__
Author(s) : Dittrich J , Popara M , Kubiak J , Dimura M , Schepers B , Verma N , Schmitz B , Dollinger P , Kovacic F , Jaeger KE , Seidel CAM , Peulen TO , Gohlke H
Ref : J Chem Theory Comput , : , 2023
Abstract : Maximum entropy methods (MEMs) determine posterior distributions by combining experimental data with prior information. MEMs are frequently used to reconstruct conformational ensembles of molecular systems for experimental information and initial molecular ensembles. We performed time-resolved Forster resonance energy transfer (FRET) experiments to probe the interdye distance distributions of the lipase-specific foldase Lif in the apo state, which likely has highly flexible, disordered, and/or ordered structural elements. Distance distributions estimated from ensembles of molecular dynamics (MD) simulations serve as prior information, and FRET experiments, analyzed within a Bayesian framework to recover distance distributions, are used for optimization. We tested priors obtained by MD with different force fields (FFs) tailored to ordered (FF99SB, FF14SB, and FF19SB) and disordered proteins (IDPSFF and FF99SBdisp). We obtained five substantially different posterior ensembles. As in our FRET experiments the noise is characterized by photon counting statistics, for a validated dye model, MEM can quantify consistencies between experiment and prior or posterior ensembles. However, posterior populations of conformations are uncorrelated to structural similarities for individual structures selected from different prior ensembles. Therefore, we assessed MEM simulating varying priors in synthetic experiments with known target ensembles. We found that (i) the prior and experimental information must be carefully balanced for optimal posterior ensembles to minimize perturbations of populations by overfitting and (ii) only ensemble-integrated quantities like inter-residue distance distributions or density maps can be reliably obtained but not ensembles of atomistic structures. This is because MEM optimizes ensembles but not individual structures. This result for a highly flexible system suggests that structurally varying priors calculated from varying prior ensembles, e.g., generated with different FFs, may serve as an ad hoc estimate for MEM reconstruction robustness.
ESTHER : Dittrich_2023_J.Chem.Theory.Comput__
PubMedSearch : Dittrich_2023_J.Chem.Theory.Comput__
PubMedID: 37023001

Title : An archaeal lid-containing feruloyl esterase degrades polyethylene terephthalate - Perez-Garcia_2023_Commun.Chem_6_193
Author(s) : Perez-Garcia P , Chow J , Costanzi E , Gurschke M , Dittrich J , Dierkes RF , Molitor R , Applegate V , Feuerriegel G , Tete P , Danso D , Thies S , Schumacher J , Pfleger C , Jaeger KE , Gohlke H , Smits SHJ , Schmitz RA , Streit WR
Ref : Commun Chem , 6 :193 , 2023
Abstract : Polyethylene terephthalate (PET) is a commodity polymer known to globally contaminate marine and terrestrial environments. Today, around 80 bacterial and fungal PET-active enzymes (PETases) are known, originating from four bacterial and two fungal phyla. In contrast, no archaeal enzyme had been identified to degrade PET. Here we report on the structural and biochemical characterization of PET46 (RLI42440.1), an archaeal promiscuous feruloyl esterase exhibiting degradation activity on semi-crystalline PET powder comparable to IsPETase and LCC (wildtypes), and higher activity on bis-, and mono-(2-hydroxyethyl) terephthalate (BHET and MHET). The enzyme, found by a sequence-based metagenome search, is 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 widens the currently known diversity of PET-hydrolyzing enzymes, by demonstrating PET depolymerization by a plant cell wall-degrading esterase.
ESTHER : Perez-Garcia_2023_Commun.Chem_6_193
PubMedSearch : Perez-Garcia_2023_Commun.Chem_6_193
PubMedID: 37697032
Gene_locus related to this paper: 9arch-PETcan211 , 9cren-PETcan204 , 9arch-PET46

Title : The metagenome-derived esterase PET40 is highly promiscuous and hydrolyses polyethylene terephthalate (PET) - Zhang_2023_Febs.j__
Author(s) : Zhang H , Dierkes RF , Perez-Garcia P , Costanzi E , Dittrich J , Cea PA , Gurschke M , Applegate V , Partus K , Schmeisser C , Pfleger C , Gohlke H , Smits SHJ , Chow J , Streit WR
Ref : Febs J , : , 2023
Abstract : Polyethylene terephthalate (PET) is a widely used synthetic polymer and known to contaminate marine and terrestrial ecosystems. Only few PET-active microorganisms and enzymes (PETases) are currently known and it is debated whether degradation activity for PET originates from promiscuous enzymes with broad substrate spectra that primarily act on natural polymers or other bulky substrates, or whether microorganisms evolved their genetic makeup to accepting PET as a carbon source. Here, we present a predicted diene lactone hydrolase designated PET40, which acts on a broad spectrum of substrates, including PET. It is the first esterase with activity on PET from a GC-rich Gram-positive Amycolatopsis species belonging to the Pseudonocardiaceae (Actinobacteria). It is highly conserved within the genera Amycolatopsis and Streptomyces. PET40 was identified by sequence-based metagenome search using a PETase-specific Hidden Markov Model (HMM). Besides acting on PET, PET40 has a versatile substrate spectrum, hydrolyzing delta-lactones, beta-lactam antibiotics, the polyester-polyurethane Impranil(a) DLN, and various para-nitrophenyl (pNP) ester substrates. Molecular docking suggests that the PET degradative activity is likely a result of the promiscuity of PET40, as potential binding modes were found for substrates encompassing mono(2-hydroxyethyl) terephthalate (MHET), bis(2-hydroxyethyl) terephthalate (BHET), and a PET trimer (PET(3) ). We also solved the crystal structure of the inactive PET40 variant S178A to 1.60 A resolution. PET40 is active throughout a wide pH (pH 4-10) and temperature range (4-65 degreesC) and remarkably stable in the presence of 5% sodium dodecyl sulfate (SDS), making it a promising enzyme as a starting point for further investigations and optimization approaches.
ESTHER : Zhang_2023_Febs.j__
PubMedSearch : Zhang_2023_Febs.j__
PubMedID: 37549040
Gene_locus related to this paper: 9pseu-PET40

Title : A phospholipase B from Pseudomonas aeruginosa with activity towards endogenous phospholipids affects biofilm assembly - Weiler_2022_Biochim.Biophys.Acta.Mol.Cell.Biol.Lipids_1867_159101
Author(s) : Weiler AJ , Spitz O , Gudzuhn M , Schott-Verdugo SN , Kamel M , Thiele B , Streit WR , Kedrov A , Schmitt L , Gohlk H , Kovacic F , Gohlke H
Ref : Biochimica & Biophysica Acta Molecular & Cellular Biology Lipids , 1867 :159101 , 2022
Abstract : Pseudomonas aeruginosa is a severe threat to immunocompromised patients due to its numerous virulence factors and biofilm-mediated multidrug resistance. It produces and secretes various toxins with hydrolytic activities including phospholipases. 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. At reaction equilibrium, PaPlaB purified from detergent-solubilized membranes of E. coli released fatty acids (FAs) from sn-1 and sn-2 positions of phospholipids at the molar ratio of 51:49. PaPlaB in vitro hydrolyzed P. aeruginosa phospholipids reconstituted in detergent micelles and phospholipids reconstituted in vesicles. Cellular localization studies indicate that PaPlaB is a cell-bound PLA of P. aeruginosa and that it is peripherally bound to both membranes in E. coli, yet the active form was predominantly associated with the cytoplasmic membrane of E. coli. Decreasing the concentration of purified and detergent-stabilized PaPlaB leads to increased enzymatic activity, and at the same time triggers oligomer dissociation. We showed that the free FA profile, biofilm amount and architecture of the wild type and deltaplaB differ. However, it remains to be established how the PLB activity of PaPlaB is regulated by homooligomerisation and how it relates to the phenotype of the P. aeruginosa deltaplaB. This novel putative virulence factor contributes to our understanding of phospholipid degrading enzymes and might provide a target for new therapeutics against P. aeruginosa biofilms.
ESTHER : Weiler_2022_Biochim.Biophys.Acta.Mol.Cell.Biol.Lipids_1867_159101
PubMedSearch : Weiler_2022_Biochim.Biophys.Acta.Mol.Cell.Biol.Lipids_1867_159101
PubMedID: 35063652
Gene_locus related to this paper: pseae-PA2927

Title : Structural, mechanistic and physiological insights into phospholipase A-mediated membrane phospholipid degradation in Pseudomonas aeruginosa - Bleffert_2022_Elife_11_e72824
Author(s) : Bleffert F , Granzin J , Caliskan M , Schott-Verdugo SN , Siebers M , Thiele B , Rahme LG , Felgner S , Dormann P , Gohlke H , Batra-Safferling R , Erich-Jager K , Kovacic F
Ref : Elife , 11 : , 2022
Abstract : 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 A(1) (PlaF) from Pseudomonas aeruginosa, which might be involved in the Lands cycle. PlaF is an important virulence factor, as the P. aeruginosa deltaplaF 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. The dimerization site and the catalytic sites are linked by an intricate ligand-mediated interaction network, which might explain 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 PlaF mediated GPL remodeling pathway for virulence and could pave the way for the development of novel therapeutics targeting PlaF.
ESTHER : Bleffert_2022_Elife_11_e72824
PubMedSearch : Bleffert_2022_Elife_11_e72824
PubMedID: 35536643
Gene_locus related to this paper: pseae-PA2949

Title : Critical assessment of structure-based approaches to improve protein resistance in aqueous ionic liquids by enzyme-wide saturation mutagenesis - El Harrar_2022_Comput.Struct.Biotechnol.J_20_399
Author(s) : El Harrar T , Davari MD , Jaeger KE , Schwaneberg U , Gohlke H
Ref : Comput Struct Biotechnol J , 20 :399 , 2022
Abstract : Ionic liquids (IL) and aqueous ionic liquids (aIL) are attractive (co-)solvents for green industrial processes involving biocatalysts, but often reduce enzyme activity. Experimental and computational methods are applied to predict favorable substitution sites and, most often, subsequent site-directed surface charge modifications are introduced to enhance enzyme resistance towards aIL. However, almost no studies evaluate the prediction precision with random mutagenesis or the application of simple data-driven filtering processes. Here, we systematically and rigorously evaluated the performance of 22 previously described structure-based approaches to increase enzyme resistance to aIL based on an experimental complete site-saturation mutagenesis library of Bacillus subtilis Lipase A (BsLipA) screened against four aIL. We show that, surprisingly, most of the approaches yield low gain-in-precision (GiP) values, particularly for predicting relevant positions: 14 approaches perform worse than random mutagenesis. Encouragingly, exploiting experimental information on the thermostability of BsLipA or structural weak spots of BsLipA predicted by rigidity theory yields GiP = 3.03 and 2.39 for relevant variants and GiP = 1.61 and 1.41 for relevant positions. Combining five simple-to-compute physicochemical and evolutionary properties substantially increases the precision of predicting relevant variants and positions, yielding GiP = 3.35 and 1.29. Finally, combining these properties with predictions of structural weak spots identified by rigidity theory additionally improves GiP for relevant variants up to 4-fold to -10 and sustains or increases GiP for relevant positions, resulting in a prediction precision of -90% compared to -9% in random mutagenesis. This combination should be applicable to other enzyme systems for guiding protein engineering approaches towards improved aIL resistance.
ESTHER : El Harrar_2022_Comput.Struct.Biotechnol.J_20_399
PubMedSearch : El Harrar_2022_Comput.Struct.Biotechnol.J_20_399
PubMedID: 35070165

Title : Evidence for a bacterial Lands cycle phospholipase A: Structural and mechanistic insights into membrane phospholipid remodeling - Bleffert_2021_Biorxiv__
Author(s) : Bleffert F , Granzin J , Caliskan M , Schott-Verdugo SN , Siebers M , Thiele B , Rahme L , Felgner S , Dormann P , Gohlke H , Batra-Safferling R , Jaeger KE , Kovacic F
Ref : Biorxiv , : , 2021
Abstract : 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 A 1 (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. Synopsis Membrane 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 A 1 (PlaF) to modulate membrane GPL composition, which is the first example in bacteria. This newly identified PLA 1 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 PLA 1 . 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.
ESTHER : Bleffert_2021_Biorxiv__
PubMedSearch : Bleffert_2021_Biorxiv__
Gene_locus related to this paper: pseae-PA2949

Title : Substrate access mechanism in a novel membrane-bound 1 phospholipase A of Pseudomonas aeruginosa concordant with specificity and regioselectivity - Ahmad_2021_Biorxiv__
Author(s) : Ahmad S , Strunk CH , Schott-Verdugo SN , Jaeger KE , Kovacic F , Gohlke H
Ref : Biorxiv , : , 2021
Abstract : 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.
ESTHER : Ahmad_2021_Biorxiv__
PubMedSearch : Ahmad_2021_Biorxiv__
Gene_locus related to this paper: pseae-PA2949

Title : Discovery of new acetylcholinesterase inhibitors for Alzheimer's disease: virtual screening and in vitro characterisation - David_2021_J.Enzyme.Inhib.Med.Chem_36_491
Author(s) : David B , Schneider P , Schafer P , Pietruszka J , Gohlke H
Ref : J Enzyme Inhib Med Chem , 36 :491 , 2021
Abstract : For more than two decades, the development of potent acetylcholinesterase (AChE) inhibitors has been an ongoing task to treat dementia associated with Alzheimer's disease and improve the pharmacokinetic properties of existing drugs. In the present study, we used three docking-based virtual screening approaches to screen both ZINC15 and MolPort databases for synthetic analogs of physostigmine and donepezil, two highly potent AChE inhibitors. We characterised the in vitro inhibitory concentration of 11 compounds, ranging from 14 to 985 microM. The most potent of these compounds, S-I 26, showed a fivefold improved inhibitory concentration in comparison to rivastigmine. Moderate inhibitors carrying novel scaffolds were identified and could be improved for the development of new classes of AChE inhibitors.
ESTHER : David_2021_J.Enzyme.Inhib.Med.Chem_36_491
PubMedSearch : David_2021_J.Enzyme.Inhib.Med.Chem_36_491
PubMedID: 33478277

Title : Promiscuous Esterases Counterintuitively Are Less Flexible than Specific Ones - Nutschel_2021_J.Chem.Inf.Model__
Author(s) : Nutschel C , Coscolin C , David B , Mulnaes D , Ferrer M , Jaeger KE , Gohlke H
Ref : J Chem Inf Model , : , 2021
Abstract : 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 an esterase sequence space can be screened by rigidity analyses for promiscuous esterases as starting points for further exploration in biotechnology and synthetic chemistry.
ESTHER : Nutschel_2021_J.Chem.Inf.Model__
PubMedSearch : Nutschel_2021_J.Chem.Inf.Model__
PubMedID: 33949194

Title : A Novel Polyester Hydrolase From the Marine Bacterium Pseudomonas aestusnigri - Structural and Functional Insights - Bollinger_2020_Front.Microbiol_11_114
Author(s) : Bollinger A , Thies S , Knieps-Grunhagen E , Gertzen C , Kobus S , Hoppner A , Ferrer M , Gohlke H , Smits SHJ , Jaeger KE
Ref : Front Microbiol , 11 :114 , 2020
Abstract : Biodegradation of synthetic polymers, in particular polyethylene terephthalate (PET), is of great importance, since environmental pollution with PET and other plastics has become a severe global problem. Here, we report on the polyester degrading ability of a novel carboxylic ester hydrolase identified in the genome of the marine hydrocarbonoclastic bacterium Pseudomonas aestusnigri VGXO14T. The enzyme, designated PE-H, belongs to the type IIa family of PET hydrolytic enzymes as indicated by amino acid sequence homology. It was produced in Escherichia coli, purified and its crystal structure was solved at 1.09 A resolution representing the first structure of a type IIa PET hydrolytic enzyme. The structure shows a typical alpha/beta-hydrolase fold and high structural homology to known polyester hydrolases. PET hydrolysis was detected at 30C with amorphous PET film (PETa), but not with PET film from a commercial PET bottle (PETb). A rational mutagenesis study to improve the PET degrading potential of PE-H yielded variant PE-H (Y250S) which showed improved activity, ultimately also allowing the hydrolysis of PETb. The crystal structure of this variant solved at 1.35 A resolution allowed to rationalize the improvement of enzymatic activity. A PET oligomer binding model was proposed by molecular docking computations. Our results indicate a significant potential of the marine bacterium P. aestusnigri for PET degradation.
ESTHER : Bollinger_2020_Front.Microbiol_11_114
PubMedSearch : Bollinger_2020_Front.Microbiol_11_114
PubMedID: 32117139
Gene_locus related to this paper: 9psed-peh

Title : The Membrane-Integrated Steric Chaperone Lif Facilitates Active Site Opening of Pseudomonas aeruginosa Lipase A - Verma_2020_J.Comput.Chem_41_500
Author(s) : Verma N , Dollinger P , Kovacic F , Jaeger KE , Gohlke H
Ref : J Comput Chem , 41 :500 , 2020
Abstract : Lipases are essential and widely used biocatalysts. Hence, the production of lipases requires a detailed understanding of the molecular mechanism of its folding and secretion. Lipase A from Pseudomonas aeruginosa, PaLipA, constitutes a prominent example that has additional relevance because of its role as a virulence factor in many diseases. PaLipA requires the assistance of a membrane-integrated steric chaperone, the lipase-specific foldase Lif, to achieve its enzymatically active state. However, the molecular mechanism of how Lif activates its cognate lipase has remained elusive. Here, we show by molecular dynamics simulations at the atomistic level and potential of mean force computations that Lif catalyzes the activation process of PaLipA by structurally stabilizing an intermediate PaLipA conformation, particularly a beta-sheet in the region of residues 17-30, such that the opening of PaLipA's lid domain is facilitated. This opening allows substrate access to PaLipA's catalytic site. A surprising and so far not fully understood aspect of our study is that the open state of PaLipA is unstable compared to the closed one according to our computational and in vitro biochemical results. We thus speculate that further interactions of PaLipA with the Xcp secretion machinery and/or components of the extracellular matrix contribute to the remaining activity of secreted PaLipA. (c) 2019 Wiley Periodicals, Inc.
ESTHER : Verma_2020_J.Comput.Chem_41_500
PubMedSearch : Verma_2020_J.Comput.Chem_41_500
PubMedID: 31618459
Gene_locus related to this paper: pseae-llipa

Title : Structural and dynamic insights revealing how lipase binding domain MD1 of Pseudomonas aeruginosa foldase affects lipase activation - Viegas_2020_Sci.Rep_10_3578
Author(s) : Viegas A , Dollinger P , Verma N , Kubiak J , Viennet T , Seidel CAM , Gohlke H , Etzkorn M , Kovacic F , Jaeger KE
Ref : Sci Rep , 10 :3578 , 2020
Abstract : Folding and cellular localization of many proteins of Gram-negative bacteria rely on a network of chaperones and secretion systems. Among them is the lipase-specific foldase Lif, a membrane-bound steric chaperone that tightly binds (K(D) = 29 nM) and mediates folding of the lipase LipA, a virulence factor of the pathogenic bacterium P. aeruginosa. Lif consists of five-domains, including a mini domain MD1 essential for LipA folding. However, the molecular mechanism of Lif-assisted LipA folding remains elusive. Here, we show in in vitro experiments using a soluble form of Lif (sLif) that isolated MD1 inhibits sLif-assisted LipA activation. Furthermore, the ability to activate LipA is lost in the variant sLif(Y99A), in which the evolutionary conserved amino acid Y99 from helix alpha1 of MD1 is mutated to alanine. This coincides with an approximately three-fold reduced affinity of the variant to LipA together with increased flexibility of sLif(Y99A) in the complex as determined by polarization-resolved fluorescence spectroscopy. We have solved the NMR solution structures of P. aeruginosa MD1 and variant MD1(Y99A) revealing a similar fold indicating that a structural modification is likely not the reason for the impaired activity of variant sLif(Y99A). Molecular dynamics simulations of the sLif:LipA complex in connection with rigidity analyses suggest a long-range network of interactions spanning from Y99 of sLif to the active site of LipA, which might be essential for LipA activation. These findings provide important details about the putative mechanism for LipA activation and point to a general mechanism of protein folding by multi-domain steric chaperones.
ESTHER : Viegas_2020_Sci.Rep_10_3578
PubMedSearch : Viegas_2020_Sci.Rep_10_3578
PubMedID: 32107397
Gene_locus related to this paper: pseae-llipa

Title : Pseudomonas aeruginosa esterase PA2949, a bacterial homolog of the human membrane esterase ABHD6: expression, purification and crystallization - Bleffert_2019_Acta.Crystallogr.F.Struct.Biol.Commun_75_270
Author(s) : Bleffert F , Granzin J , Gohlke H , Batra-Safferling R , Jaeger KE , Kovacic F
Ref : Acta Crystallographica F Struct Biol Commun , 75 :270 , 2019
Abstract : The human membrane-bound alpha/beta-hydrolase domain 6 (ABHD6) protein modulates endocannabinoid signaling, which controls appetite, pain and learning, as well as being linked to Alzheimer's and Parkinson's diseases, through the degradation of the key lipid messenger 2-arachidonylglycerol (2-AG). This makes ABHD6 an attractive therapeutic target that lacks structural information. In order to better understand the molecular mechanism of 2-AG-hydrolyzing enzymes, the PA2949 protein from Pseudomonas aeruginosa, which has 49% sequence similarity to the ABHD6 protein, was cloned, overexpressed, purified and crystallized. Overexpression of PA2949 in the homologous host yielded the membrane-bound enzyme, which was purified in milligram amounts. Besides their sequence similarity, the enzymes both show specificity for the hydrolysis of 2-AG and esters of medium-length fatty acids. PA2949 in the presence of n-octyl beta-D-glucoside showed a higher activity and stability at room temperature than those previously reported for PA2949 overexpressed and purified from Escherichia coli. A suitable expression host and stabilizing detergent were crucial for obtaining crystals, which belonged to the tetragonal space group I4122 and diffracted to a resolution of 2.54 A. This study provides hints on the functional similarity of ABHD6-like proteins in prokaryotes and eukaryotes, and might guide the structural study of these difficult-to-crystallize proteins.
ESTHER : Bleffert_2019_Acta.Crystallogr.F.Struct.Biol.Commun_75_270
PubMedSearch : Bleffert_2019_Acta.Crystallogr.F.Struct.Biol.Commun_75_270
PubMedID: 30950828
Gene_locus related to this paper: pseae-PA2949

Title : Structural Rigidity and Protein Thermostability in Variants of Lipase A from Bacillus subtilis - Rathi_2015_PLoS.One_10_e0130289
Author(s) : Rathi PC , Jaeger KE , Gohlke H
Ref : PLoS ONE , 10 :e0130289 , 2015
Abstract : Understanding the origin of thermostability is of fundamental importance in protein biochemistry. Opposing views on increased or decreased structural rigidity of the folded state have been put forward in this context. They have been related to differences in the temporal resolution of experiments and computations that probe atomic mobility. Here, we find a significant (p = 0.004) and fair (R2 = 0.46) correlation between the structural rigidity of a well-characterized set of 16 mutants of lipase A from Bacillus subtilis (BsLipA) and their thermodynamic thermostability. We apply the rigidity theory-based Constraint Network Analysis (CNA) approach, analyzing directly and in a time-independent manner the statics of the BsLipA mutants. We carefully validate the CNA results on macroscopic and microscopic experimental observables and probe for their sensitivity with respect to input structures. Furthermore, we introduce a robust, local stability measure for predicting thermodynamic thermostability. Our results complement work that showed for pairs of homologous proteins that raising the structural stability is the most common way to obtain a higher thermostability. Furthermore, they demonstrate that related series of mutants with only a small number of mutations can be successfully analyzed by CNA, which suggests that CNA can be applied prospectively in rational protein design aimed at higher thermodynamic thermostability.
ESTHER : Rathi_2015_PLoS.One_10_e0130289
PubMedSearch : Rathi_2015_PLoS.One_10_e0130289
PubMedID: 26147762
Gene_locus related to this paper: bacsu-lip