Biology subjects

Einsle, O.

Publications and source records attributed to Einsle, O..

9 recordsLinked to original sources

bioRxiv2026

Metabolic regulation and structural mechanism of glutamine synthetase AMPylation

In bacteria, glutamine synthetase (GS) is the main ammonium assimilation enzyme. Its activity is tightly regulated according to cellular energy status and carbon/nitrogen balance through reversible AMPylation catalyzed by the bifunctional enzyme GlnE, which is controlled by the signal transducer GlnB protein. Although GS AMPylation has been extensively studied, the GlnB:GlnE:GS regulatory pathway exhibits substantial plasticity among bacterial groups, and the structural basis of GS inhibition by AMPylation remains unclear. Here, we describe how carbon, nitrogen, and energy signals regulate GS AMPylation in Herbaspirillum seropedicae and uncover the structural mechanism underlying enzyme inhibition. Our data reveal that GS AMPylation is independent of unmodified GlnB, whereas uridylylated GlnB (GlnB-UMP) inhibits AMPylation under nitrogen-limiting conditions through a GlnB-GlnE complex modulated by 2-oxoglutarate. We further show that GlnE directly senses glutamine under nitrogen-sufficient conditions, with signal integration depending primarily on energy availability. To elucidate the mechanism of AMPylation GS inhibition, we solved Cryo-EM structures of unmodified and AMPylated GS in complex with MgATP and MnADP. Structural comparisons revealed that AMPylation increases the flexibility of the AMP-loop, disrupting a hydrogen-bond network that stabilizes Arg342 in the orientation required to position the ATP {gamma}-phosphate in its catalytic conformation for efficient phosphoryl transfer to glutamate. These findings reveal how metabolic signals are integrated to regulate GS AMPylation and provide the first structural insights into the mechanism underlying bacterial GS inhibition by AMPylation.

microbiology↗

bioRxiv2026

Powering methanogenesis from fatty acids by a twin-heme-mediated reverse redox-loop

The conversion of organic matter into methane is central to the global carbon cycle and engineered biogas production. In this process, syntrophic bacteria oxidize fatty acid fermentation products to acetate, coupled to the generation of H2 or formate, which are subsequently utilized by methanogenic archaea. During fatty acid {beta}-oxidation, a membrane-bound electron-transferring flavoprotein (ETF):methylmenaquinone (MMK) oxidoreductase complex (EMO) has been proposed to drive endergonic electron transfer from reduced ETF to CO2 through a reverse redox loop, with its mechanistic basis remaining unresolved. Here we report cryo-electron microscopy structures of EMO and the EMO-ETF complex from Syntrophus aciditrophicus at 2.0 and 3.0 [A] resolution, respectively. Complex formation induces substantial conformational rearrangements in ETF, positioning its flavin for efficient electron transfer to non-cubane [4Fe:4S] and [4Fe:5S] clusters. The membrane-integral domain harbors three heme b cofactors, including a specialized twin-heme unit that mediates proton-motive-force-driven MMK reduction. The structural and functional similarity of EMOs to heterodisulfide reductases, together with their broad distribution across bacteria and archaea, suggests an evolutionary link between methanogenesis and fatty acid {beta}-oxidation, illustrating how ancient redox systems were repurposed for new metabolic functions.

microbiology↗

bioRxiv2026

Stabilizing Plasmodium falciparum Proteins for Small Molecule Drug Discovery

Early-stage drug discovery relies on the availability of stable protein for reliable biophysical characterization of ligand binding. However, many Plasmodium falciparum proteins are challenging to produce in heterologous systems, which limits their experimental utility. To address this, we tested whether ProteinMPNN-guided sequence design could generate stabilized surrogate constructs that retain wild-type-like structure and binding thermodynamics. Designs were generated with constraints to maintain conserved and binding-site residues for three therapeutically relevant targets: PfBDP1-BRD, PfBDP4-BRD, and PfK13-KREP. The resulting constructs showed markedly increased thermal stability. Using PfBDP1-BRD as a benchmark, isothermal titration calorimetry confirmed that the stabilized variants retained wild-type-like binding thermodynamics with a known ligand. Extending this approach to other targets, a PfK13-KREP construct led to an apo structure with a binding pocket closely matching the wild type, and a stabilized PfBDP4-BRD surrogate - a previously unstable target - enabled the identification of PfBDP4-BRD binders and a 1.25 [A] co-crystal structure with a newly found inhibitor. Our findings demonstrate that computationally stabilized surrogates are practical and effective tools for robust biophysics and structure-enabled drug discovery against otherwise challenging malaria proteins.

biochemistry↗

bioRxiv2025

A novel inhibitor against the bromodomain PfBDP1 of the malaria pathogen Plasmodium falciparum

The rise of drug resistances in malaria necessitates the exploration of novel therapeutic strategies. Targeting epigenetic pathways could open new, promising treatment avenues. In this study, we focus on PfBDP1, an essential bromodomain protein in P. falciparum. Utilizing the pan-selective bromodomain inhibitor MPM6, we identified a potent initial hit and subsequently developed it into a nanomolar binder. Through a combination of virtual docking, isothermal titration calorimetry, and X-ray crystallography, we elucidated the molecular interactions of the new inhibitors with PfBD1. Our findings include the first cocrystallized structures of PfBD1 and PvBD1 with these inhibitors, providing insights into their binding mechanisms. Further validation using conditional knockdown of PfBDP1 in P. falciparum demonstrated parasite sensitivity to the inhibitor, underscoring its potential as a targeted therapeutic approach against malaria.

biochemistry↗

bioRxiv2024

Structural Insights into Broad-Range Polyphosphate Kinase 2 II Enzymes Applicable for Pyrimidine Nucleoside Diphosphate Synthesis

Polyphosphate kinases (PPK) play crucial roles in various biological processes, including energy storage and stress responses, through their interaction with inorganic polyphosphate (polyP) and the intracellular nucleotide pool. Members of the PPK family 2 (PPK2s) catalyse polyP-consuming phosphorylation of nucleotides. In this study, we characterised two PPK2 enzymes from Bacillus cereus (BcPPK2) and Lysinibacillus fusiformis (LfPPK2) to investigate their substrate specificity and potential for selective nucleotide synthesis. Both enzymes exhibited a broad substrate scope, selectively converting over 85% of pyrimidine nucleoside monophosphates (NMPs) to nucleoside diphosphates (NDPs), while nucleoside triphosphate (NTP) formation was observed only with purine NMPs. Preparative enzymatic synthesis of cytidine diphosphate (CDP) was applied to achieve an yield of 49%. Finally, structural analysis of five crystal structures of BcPPK2 and LfPPK2 provided insights into their active sites and substrate interactions. This study highlights PPK2-II enzymes as promising biocatalysts for the efficient and selective synthesis of pyrimidine NDPs.

biochemistry↗

bioRxiv2024

Nitrogenase structural evolution across Earth s history

Life on Earth is about 4 billion years old--nearly as old as the planet itself. Over this immense timespan, living systems and their biomolecules have both adapted to and driven profound changes in the Earths environment. Among these, certain critical enzymes emerged early and have persisted through planetary extremes. Here, we implement an integrated approach to investigate the structural evolution of nitrogenase, an ancient and globally essential enzyme responsible for biological nitrogen fixation. Despite the ecological diversity of its host microbes, nitrogenase retains strict functional constraints, including extreme oxygen sensitivity, high energy demands, and substrate availability. We combined phylogenetics, ancestral sequence reconstruction, protein crystallography and deep-learning based structural prediction to resurrect nearly three billion years of nitrogenase structural history. This effort represents the first effort to predict the full set of extant and ancestral structures along the evolutionary tree of a single enzyme, yielding over 5000 structural models. Our framework lays a foundation for reconstructing key structural constraints that shape protein evolution and examining ancient enzymes within the broader context of phylogenetic relationships and environmental transitions across geological timescales.

evolutionary biology↗

bioRxiv2022

Architecture of the NADH:ferredoxin oxidoreductase RNF that drives Biological Nitrogen Fixation

Biological nitrogen fixation requires substantial metabolic energy in form of ATP, as well as low-potential electrons that must derive from central metabolism. During aerobic growth, the free-living soil diazotroph Azotobacter vinelandii transfers electrons from the key metabolite NADH to the low-potential ferredoxin FdxA that serves as a direct electron donor to the dinitrogenase reductases. This process is mediated by the RNF complex that exploits the proton motive force over the cytoplasmic membrane to lower the midpoint potential of the transferred electron. Here we report the cryo-EM structure of the nitrogenase-associated RNF complex of A. vinelandii, a seven-subunit membrane protein assembly that contains four flavin cofactors and six iron-sulfur centers. Its function requires the strict coupling of electron and proton transfer but also involves major conformational changes within the assembly that can be traced with a combination of electron microscopy and modeling. One Sentence SummaryBiological nitrogen fixation requires electrons with a negative redox potential, and the diazotroph Azotobacter vinelandii can provide these through the evolutionarily ancient RNF complex that bridges the energy difference between NADH and a low-potential ferredoxin by exploiting the transmembrane proton gradient.

biochemistry↗

bioRxiv2022

Remorin proteins serve as membrane topology scaffolds in plants

Organization of membrane topologies in plants has so far been mainly attributed to the cell wall and the cytoskeleton. Taking rhizobial infections of legume root cells, where plasma membranes undergo dynamic and large-scale topology changes, as an initial model, we challenged this paradigm and tested whether additional scaffolds such as plant-specific remorins that accumulate on highly curved and often wall-less plasma membrane domains, control local membrane dynamics. Indeed, loss-of-function mutants of the remorin protein SYMREM1 failed to develop stabilized membrane tubes as found in colonized cells in wild-type plants, but released empty membrane spheres instead. Expression of this and other remorins in wall-less protoplasts allowed engineering different membrane topologies ranging from membrane blebs to long membrane tubes. Reciprocally, mechanically induced membrane indentations were equally stabilized by SYMREM1. This function is likely supported by remorin oligomerization into antiparallel dimers and the formation of higher order membrane scaffolding structures. Taken together we describe an evolutionary confined mechanism that allows the stabilization of large-scale membrane conformations and curvatures in plants. One-sentence summaryThe remorin SYMREM1 evolved as structural membrane scaffold that stabilizes membrane tubulation and curvature during symbiotic intracellular infections.

plant biology↗

bioRxiv2019

How thermophilic Gram-positive organisms perform extracellular electron transfer: characterization of the cell surface terminal reductase OcwA

Extracellular electron transfer is the key process underpinning the development of bioelectrochemical systems for the production of energy or added-value compounds. Thermincola potens JR is a promising Gram-positive bacterium to be used in these systems because it is thermophilic. In this paper we describe the structural and functional properties of the nonaheme cytochrome OcwA, which is the terminal reductase of this organism. The structure of OcwA, determined at 2.2[A] resolution shows that the overall-fold and organization of the hemes are not related to other metal reductases and instead are similar to that of multiheme cytochromes involved in the biogeochemical cycles of nitrogen and sulfur. We show that, in addition to solid electron acceptors, OcwA can also reduce soluble electron shuttles and oxyanions. These data reveal that OcwA can take the role of a respiratory swiss-army knife allowing this organism to grow in environments with rapidly changing availability of terminal electron acceptors without the need for transcriptional regulation and protein synthesis.\n\nImportanceThermophilic Gram-positive organisms were recently shown to be a promising class of organisms to be used in bioelectrochemical systems for the production of electrical energy. These organisms present a thick peptidoglycan layer that was thought to preclude them to perform extracellular electron transfer (i.e. exchange catabolic electrons with solid electron acceptors outside of the cell). In this manuscript we describe the structure and functional mechanisms of the multiheme cytochrome OcwA, the terminal reductase of the Gram-positive bacterium Thermincola potens JR found at the cell surface of this organism. The results presented here show that this protein is unrelated with terminal reductases found at the cell surface of other electroactive organisms. Instead, OcwA is similar to terminal reductases of soluble electron acceptors. Our data reveals that terminal oxidoreductases of soluble and insoluble substrates are evolutionarily related, providing novel insights into the evolutionary pathway of multiheme cytochromes.

biochemistry↗