Search Results
Effects of proline substitutions on the thermostable LOV domain from Chloroflexus aggregans
2020, Remeeva, Alina, Nazarenko, Vera V., Goncharov, Ivan M., Yudenko, Anna, Smolentseva, Anastasia, Semenov, Oleg, Kovalev, Kirill, GĂĽlbahar, Cansu, Schwaneberg, Ulrich, Davari, Mehdi D., Gordeliy, Valentin, Gushchin, Ivan
Light-oxygen-voltage (LOV) domains are ubiquitous photosensory modules found in proteins from bacteria, archaea and eukaryotes. Engineered versions of LOV domains have found widespread use in fluorescence microscopy and optogenetics, with improved versions being continuously developed. Many of the engineering efforts focused on the thermal stabilization of LOV domains. Recently, we described a naturally thermostable LOV domain from Chloroflexus aggregans. Here we show that the discovered protein can be further stabilized using proline substitution. We tested the effects of three mutations, and found that the melting temperature of the A95P mutant is raised by approximately 2◦ C, whereas mutations A56P and A58P are neutral. To further evaluate the effects of mutations, we crystallized the variants A56P and A95P, while the variant A58P did not crystallize. The obtained crystal structures do not reveal any alterations in the proteins other than the introduced mutations. Molecular dynamics simulations showed that mutation A58P alters the structure of the respective loop (Aβ-Bβ), but does not change the general structure of the protein. We conclude that proline substitution is a viable strategy for the stabilization of the Chloroflexus aggregans LOV domain. Since the sequences and structures of the LOV domains are overall well-conserved, the effects of the reported mutations may be transferable to other proteins belonging to this family. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.
Glycolytic flux control by drugging phosphoglycolate phosphatase
2022, Jeanclos, Elisabeth, Schlötzer, Jan, Hadamek, Kerstin, Yuan-Chen, Natalia, Alwahsh, Mohammad, Hollmann, Robert, Fratz, Stefanie, Yesilyurt-Gerhards, Dilan, Frankenbach, Tina, Engelmann, Daria, Keller, Angelika, Kaestner, Alexandra, Schmitz, Werner, Neuenschwander, Martin, Hergenröder, Roland, Sotriffer, Christoph, von Kries, Jens Peter, Schindelin, Hermann, Gohla, Antje
Targeting the intrinsic metabolism of immune or tumor cells is a therapeutic strategy in autoimmunity, chronic inflammation or cancer. Metabolite repair enzymes may represent an alternative target class for selective metabolic inhibition, but pharmacological tools to test this concept are needed. Here, we demonstrate that phosphoglycolate phosphatase (PGP), a prototypical metabolite repair enzyme in glycolysis, is a pharmacologically actionable target. Using a combination of small molecule screening, protein crystallography, molecular dynamics simulations and NMR metabolomics, we discover and analyze a compound (CP1) that inhibits PGP with high selectivity and submicromolar potency. CP1 locks the phosphatase in a catalytically inactive conformation, dampens glycolytic flux, and phenocopies effects of cellular PGP-deficiency. This study provides key insights into effective and precise PGP targeting, at the same time validating an allosteric approach to control glycolysis that could advance discoveries of innovative therapeutic candidates.
Three in One: The Versatility of Hydrogen Bonding Interaction in Halide Salts with Hydroxy-Functionalized Pyridinium Cations
2021, Al Sheakh, Loai, Niemann, Thomas, Villinger, Alexander, Stange, Peter, Zaitsau, Dzmitry H., Strate, Anne, Ludwig, Ralf
The paradigm of supramolecular chemistry relies on the delicate balance of noncovalent forces. Here we present a systematic approach for controlling the structural versatility of halide salts by the nature of hydrogen bonding interactions. We synthesized halide salts with hydroxy-functionalized pyridinium cations [HOCn Py]+ (n=2, 3, 4) and chloride, bromide and iodide anions, which are typically used as precursor material for synthesizing ionic liquids by anion metathesis reaction. The X-ray structures of these omnium halides show two types of hydrogen bonding: 'intra-ionic' H-bonds, wherein the anion interacts with the hydroxy group and the positively charged ring at the same cation, and 'inter-ionic' H-bonds, wherein the anion also interacts with the hydroxy group and the ring system but of different cations. We show that hydrogen bonding is controllable by the length of the hydroxyalkyl chain and the interaction strength of the anion. Some molten halide salts exhibit a third type of hydrogen bonding. IR spectra reveal elusive H-bonds between the OH groups of cations, showing interaction between ions of like charge. They are formed despite the repulsive interaction between the like-charged ions and compete with the favored cation-anion H-bonds. All types of H-bonding are analyzed by quantum chemical methods and the natural bond orbital approach, emphasizing the importance of charge transfer in these interactions. For simple omnium salts, we evidenced three distinct types of hydrogen bonds: Three in one!