Molecular Mechanisms Underlying Medium-Chain Free Fatty Acid-Regulated Activity of the Phospholipase PlaF from Pseudomonas aeruginosa.

PlaF is a membrane-bound phospholipase A1 from Pseudomonas aeruginosa that is involved in remodeling membrane glycerophospholipids (GPLs) and modulating virulence-associated signaling and metabolic pathways. Previously, we identified the role of medium-chain free fatty acids (FFAs) 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 has been 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 for 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 gas chromatography-mass spectrometry 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 FFAs may regulate the activity of PlaF, a potential bacterial drug target.

The ISG15-Protease USP18 Is a Pleiotropic Enhancer of HIV-1 Replication.

The innate immune response to viruses is formed in part by interferon (IFN)-induced restriction factors, including ISG15, p21, and SAMHD1. IFN production can be blocked by the ISG15-specific protease USP18. HIV-1 has evolved to circumvent host immune surveillance. This mechanism might involve USP18. In our recent studies, we demonstrate that HIV-1 infection induces USP18, which dramatically enhances HIV-1 replication by abrogating the antiviral function of p21. USP18 downregulates p21 by accumulating misfolded dominant negative p53, which inactivates wild-type p53 transactivation, leading to the upregulation of key enzymes involved in de novo dNTP biosynthesis pathways and inactivated SAMHD1. Despite the USP18-mediated increase in HIV-1 DNA in infected cells, it is intriguing to note that the cGAS-STING-mediated sensing of the viral DNA is abrogated. Indeed, the expression of USP18 or knockout of ISG15 inhibits the sensing of HIV-1. We demonstrate that STING is ISGylated at residues K224, K236, K289, K347, K338, and K370. The inhibition of STING K289-linked ISGylation suppresses its oligomerization and IFN induction. We propose that human USP18 is a novel factor that potentially contributes in multiple ways to HIV-1 replication.

Extracting binding energies and binding modes from biomolecular simulations of fragment binding to endothiapepsin.

Fragment-based drug discovery (FBDD) aims to discover a set of small binding fragments that may be subsequently linked together. Therefore, in-depth knowledge of the individual fragments' structural and energetic binding properties is essential. In addition to experimental techniques, the direct simulation of fragment binding by molecular dynamics (MD) simulations became popular to characterize fragment binding. However, former studies showed that long simulation times and high computational demands per fragment are needed, which limits applicability in FBDD. Here, we performed short, unbiased MD simulations of direct fragment binding to endothiapepsin, a well-characterized model system of pepsin-like aspartic proteases. To evaluate the strengths and limitations of short MD simulations for the structural and energetic characterization of fragment binding, we predicted the fragments' absolute free energies and binding poses based on the direct simulations of fragment binding and compared the predictions to experimental data. The predicted absolute free energies are in fair agreement with the experiment. Combining the MD data with binding mode predictions from molecular docking approaches helped to correctly identify the most promising fragments for further chemical optimization. Importantly, all computations and predictions were done within 5 days, suggesting that MD simulations may become a viable tool in FBDD projects.

Application of the C3-Methyltransferase StspM1 for the Synthesis of the Natural Pyrroloindole Motif.

Even though pyrroloindoles are widely present in natural products with different kinds of biological activities, their selective synthesis remains challenging with existing tools in organic chemistry, and there is furthermore a demand for stereoselective and mild methods to access this structural motif. Nature uses C3-methyltransferases to form the pyrroloindole framework, starting from the amino acid tryptophan. In the present study, the SAM-dependent methyltransferase StspM1 from Streptomyces sp. HPH0547 is used to build the pyrroloindole structural motif in tryptophan-based diketopiperazines (DKP). The substrate scope of the enzyme regarding different Trp-Trp-DKP isomers was investigated on an experimental and computational level. After further characterization and optimization of the methylation reaction with a design of experiment approach, a preparative scale reaction with the immobilized enzyme including a SAM regeneration system was performed to show the synthetic use of this biocatalytic tool to access the pyrroloindole structural motif.

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 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.

The metagenome-derived esterase PET40 is highly promiscuous and hydrolyses polyethylene terephthalate (PET).

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. Besides acting on PET, PET40 has a versatile substrate spectrum, hydrolyzing δ-lactones, ß-lactam antibiotics, the polyester-polyurethane Impranil® DLN, and various para-nitrophenyl 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, bis(2-hydroxyethyl) terephthalate, and a PET trimer. We also solved the crystal structure of the inactive PET40 variant S178A to 1.60 Å resolution. PET40 is active throughout a wide pH (pH 4-10) and temperature range (4-65 °C) and remarkably stable in the presence of 5% SDS, making it a promising enzyme as a starting point for further investigations and optimization approaches.

The cyclophilin A-binding loop of the capsid regulates the human TRIM5α sensitivity of nonpandemic HIV-1.

The rather few cases of humans infected by HIV-1 N, O, or P raise the question of their incomplete adaptation to humans. We hypothesized that early postentry restrictions may be relevant for the impaired spread of these HIVs. One of the best-characterized species-specific restriction factors is TRIM5α. HIV-1 M can escape human (hu) TRIM5α restriction by binding cyclophilin A (CYPA, also known as PPIA, peptidylprolyl isomerase A) to the so-called CYPA-binding loop of its capsid protein. How non-M HIV-1s interact with huTRIM5α is ill-defined. By testing full-length reporter viruses (Δ env) of HIV-1 N, O, P, and SIVgor (simian IV of gorillas), we found that in contrast to HIV-1 M, the nonpandemic HIVs and SIVgor showed restriction by huTRIM5α. Work to identify capsid residues that mediate susceptibility to huTRIM5α revealed that residue 88 in the capsid CYPA-binding loop was important for such differences. There, HIV-1 M uses alanine to resist, while non-M HIV-1s have either valine or methionine, which avail them for huTRIM5α. Capsid residue 88 determines the sensitivity to TRIM5α in an unknown way. Molecular simulations indicated that capsid residue 88 can affect trans-to-cis isomerization patterns on the capsids of the viruses we tested. These differential CYPA usages by pandemic and nonpandemic HIV-1 suggest that the enzymatic activity of CYPA on the viral core might be important for its protective function against huTRIM5α.

AmberTools.

AmberTools is a free and open-source collection of programs used to set up, run, and analyze molecular simulations. The newer features contained within AmberTools23 are briefly described in this Application note.

Regulation of STING activity in DNA sensing by ISG15 modification.

Sensing of human immunodeficiency virus type 1 (HIV-1) DNA is mediated by the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) signaling axis. Signal transduction and regulation of this cascade is achieved by post-translational modifications. Here we show that cGAS-STING-dependent HIV-1 sensing requires interferon-stimulated gene 15 (ISG15). ISG15 deficiency inhibits STING-dependent sensing of HIV-1 and STING agonist-induced antiviral response. Upon external stimuli, STING undergoes ISGylation at residues K224, K236, K289, K347, K338, and K370. Inhibition of STING ISGylation at K289 suppresses STING-mediated type Ⅰ interferon induction by inhibiting its oligomerization. Of note, removal of STING ISGylation alleviates gain-of-function phenotype in STING-associated vasculopathy with onset in infancy (SAVI). Molecular modeling suggests that ISGylation of K289 is an important regulator of oligomerization. Taken together, our data demonstrate that ISGylation at K289 is crucial for STING activation and represents an important regulatory step in DNA sensing of viruses and autoimmune responses.

LISTER: Semiautomatic 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 be created by the corresponding researchers, which takes effort and time. Here, we developed LISTER as a methodological and algorithmic solution to create and extract metadata from annotated, template-based experimental 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. LISTER consists of four components: annotation language to support metadata extraction; customized eLabFTW entries using specific hierarchies, templates, and tags to structure reusable scientific documentation; a "container" concept in eLabFTW, making metadata of a particular container content extractable along with its underlying, related experiments via a single click; a Python-based app to enable easy-to-use, semiautomated metadata extraction from eLabFTW entries. LISTER outputs metadata in machine-readable .json and human-readable .xlsx formats, and Material and Methods (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. DSpace is used as a data cataloging platform for hosting the extracted metadata and research data. We applied LISTER to computational biophysical chemistry, protein biochemistry, and molecular biology, and our concept should be extendable to other life science areas.

Expanding the clinical and immunological phenotypes of PAX1-deficient SCID and CID patients.

Paired box 1 (PAX1) deficiency has been reported in a small number of patients diagnosed with otofaciocervical syndrome type 2 (OFCS2). We described six new patients who demonstrated variable clinical penetrance. Reduced transcriptional activity of pathogenic variants confirmed partial or complete PAX1 deficiency. Thymic aplasia and hypoplasia were associated with impaired T cell immunity. Corrective treatment was required in 4/6 patients. Hematopoietic stem cell transplantation resulted in poor immune reconstitution with absent naïve T cells, contrasting with the superior recovery of T cell immunity after thymus transplantation. Normal ex vivo differentiation of PAX1-deficient CD34+ cells into mature T cells demonstrated the absence of a hematopoietic cell-intrinsic defect. New overlapping features with DiGeorge syndrome included primary hypoparathyroidism (n = 5) and congenital heart defects (n = 2), in line with PAX1 expression during early embryogenesis. Our results highlight new features of PAX1 deficiency, which are relevant to improving early diagnosis and identifying patients requiring corrective treatment.

An archaeal lid-containing feruloyl esterase degrades polyethylene terephthalate.

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 Å. 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.

Integrative dynamic structural biology unveils conformers essential for the oligomerization of a large GTPase.

Guanylate binding proteins (GBPs) are soluble dynamin-like proteins that undergo a conformational transition for GTP-controlled oligomerization and disrupt membranes of intracellular parasites to exert their function as part of the innate immune system of mammalian cells. We apply neutron spin echo, X-ray scattering, fluorescence, and EPR spectroscopy as techniques for integrative dynamic structural biology to study the structural basis and mechanism of conformational transitions in the human GBP1 (hGBP1). We mapped hGBP1's essential dynamics from nanoseconds to milliseconds by motional spectra of sub-domains. We find a GTP-independent flexibility of the C-terminal effector domain in the µs-regime and resolve structures of two distinct conformers essential for an opening of hGBP1 like a pocket knife and for oligomerization. Our results on hGBP1's conformational heterogeneity and dynamics (intrinsic flexibility) deepen our molecular understanding relevant for its reversible oligomerization, GTP-triggered association of the GTPase-domains and assembly-dependent GTP-hydrolysis.

Combining structural and coevolution information to unveil allosteric sites.

Understanding allosteric regulation in biomolecules is of great interest to pharmaceutical research and computational methods emerged during the last decades to characterize allosteric coupling. However, the prediction of allosteric sites in a protein structure remains a challenging task. Here, we integrate local binding site information, coevolutionary information, and information on dynamic allostery into a structure-based three-parameter model to identify potentially hidden allosteric sites in ensembles of protein structures with orthosteric ligands. When tested on five allosteric proteins (LFA-1, p38-α, GR, MAT2A, and BCKDK), the model successfully ranked all known allosteric pockets in the top three positions. Finally, we identified a novel druggable site in MAT2A confirmed by X-ray crystallography and SPR and a hitherto unknown druggable allosteric site in BCKDK validated by biochemical and X-ray crystallography analyses. Our model can be applied in drug discovery to identify allosteric pockets.

Functional characterization of novel or yet uncharacterized ATP7B missense variants detected in patients with clinical Wilson's disease.

Wilson's disease (WD, MIM#277900) is an autosomal recessive disorder resulting in copper excess caused by biallelic variants in the ATP7B gene (MIM#606882) encoding a copper transporting P-type ATPase. ATP7B variants of unknown significance (VUS) are detected frequently, sometimes impeding a clear diagnosis. Functional analyses can help to classify these variants as benign or pathogenic. Additionally, variants already classified as (likely) pathogenic benefit from functional analyses to understand their pathomechanism, thus contribute to the development of personalized treatment approaches in the future. We described clinical features of six WD patients and functionally characterized five ATP7B missense variants (two VUS, three yet uncharacterized likely pathogenic variants), detected in these patients. We determined the protein level, copper export capacity, and cellular localization in an in vitro model and potential structural consequences using an ATP7B protein model based on AlphaFold. Our analyses give insight into the pathomechanism and allowed reclassification for the two VUS to likely pathogenic and for two of the three likely pathogenic variants to pathogenic.

Resolution of Maximum Entropy Method-Derived Posterior Conformational Ensembles of a Flexible System Probed by FRET and Molecular Dynamics Simulations.

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 Förster 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.

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

MOTIVATION: TopEnzyme is a database of structural enzyme models created with TopModel and is linked to the SWISS-MODEL repository 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. RESULTS: 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 within Swiss-Prot. AVAILABILITY AND IMPLEMENTATION: We provide a full web interface to the database at https://cpclab.uni-duesseldorf.de/topenzyme/.

Enzyme adaptation to habitat thermal legacy shapes the thermal plasticity of marine microbiomes.

Microbial communities respond to temperature with physiological adaptation and compositional turnover. Whether thermal selection of enzymes explains marine microbiome plasticity in response to temperature remains unresolved. By quantifying the thermal behaviour of seven functionally-independent enzyme classes (esterase, extradiol dioxygenase, phosphatase, beta-galactosidase, nuclease, transaminase, and aldo-keto reductase) in native proteomes of marine sediment microbiomes from the Irish Sea to the southern Red Sea, we record a significant effect of the mean annual temperature (MAT) on enzyme response in all cases. Activity and stability profiles of 228 esterases and 5 extradiol dioxygenases from sediment and seawater across 70 locations worldwide validate this thermal pattern. Modelling the esterase phase transition temperature as a measure of structural flexibility confirms the observed relationship with MAT. Furthermore, when considering temperature variability in sites with non-significantly different MATs, the broadest range of enzyme thermal behaviour and the highest growth plasticity of the enriched heterotrophic bacteria occur in samples with the widest annual thermal variability. These results indicate that temperature-driven enzyme selection shapes microbiome thermal plasticity and that thermal variability finely tunes such processes and should be considered alongside MAT in forecasting microbial community thermal response.

Atomic Resolution Insights into pH Shift Induced Deprotonation Events in LS-Shaped Aß(1-42) Amyloid Fibrils.

Alzheimer's disease is a neurodegenerative disorder associated with the deposition of misfolded aggregates of the amyloid-ß protein (Aß). Aß(1-42) is one of the most aggregation-prone components in senile plaques of AD patients. We demonstrated that relatively homogeneous Aß(1-42) fibrils with one predominant fold visible in solid-state NMR spectra can be obtained at acidic pH. The structure of these fibrils differs remarkably from some other polymorphs obtained at neutral pH. In particular, the entire N-terminal region is part of the rigid fibril core. Here, we investigate the effects of a pH shift on the stability and the fold of these fibrils at higher pH values. Fibril bundling at neutral pH values renders cryo-EM studies impractical, but solid-state NMR spectroscopy, molecular dynamics simulations, and biophysical methods provide residue-specific structural information under these conditions. The LS-fold of the Aß(1-42) fibrils does not change over the complete pH range from pH 2 to pH 7; in particular, the N-terminus remains part of the fibril core. We observe changes in the protonation state of charged residues starting from pH 5 on a residue-specific level. The deprotonation of the C-terminal carboxyl group of A42 in the intermolecular salt bridge with D1 and K28 is slow on the NMR time scale, with a local pKa of 5.4, and local conformations of the involved residues are affected by deprotonation of A42. Thus, we demonstrate that this fibril form is stable at physiological pH values.