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Bradyrhizobium diazoefficiens, a bacterial symbiont of soybean and other leguminous plants, enters a nodulation‐promoting genetic programme in the presence of host‐produced flavonoids and related signalling compounds. Here, we describe the crystal structure of an isoflavonoid‐responsive regulator (FrrA) from Bradyrhizobium, as well as cocrystal structures with inducing and noninducing ligands (genistein and naringenin, respectively). The structures reveal a TetR‐like fold whose DNA‐binding domain is capable of adopting a range of orientations. A single molecule of either genistein or naringenin is asymmetrically bound in a central cavity of the FrrA homodimer, mainly via C–H contacts to the π‐system of the ligands. Strikingly, however, the interaction does not provoke any conformational changes in the repressor. Both the flexible positioning of the DNA‐binding domain and the absence of structural change upon ligand binding are corroborated by small‐angle X‐ray scattering (SAXS) experiments in solution. Together with a model of the promoter‐bound state of FrrA our results suggest that inducers act as a wedge, preventing the DNA‐binding domains from moving close enough together to interact with successive positions of the major groove of the palindromic operator.
The papain-like protease (PLpro) of SARS-CoV-2 is essential for viral propagation and, additionally, dysregulation of the host innate immune system. Using a library of 40 potential metal-chelating compounds we performed an X-ray crystallographic screening against PLpro. As outcome we identified six compounds binding to the target protein. Here we describe the interaction of one hydrazone (H1) and five thiosemicarbazone (T1-T5) compounds with the two distinct natural substrate binding sites of PLpro for ubiquitin and ISG15. H1 binds to a polar groove at the S1 binding site by forming several hydrogen bonds with PLpro. T1-T5 bind into a deep pocket close to the polyubiquitin and ISG15 binding site S2. Their interactions are mainly mediated by multiple hydrogen bonds and further hydrophobic interactions. In particular compound H1 interferes with natural substrate binding by sterical hindrance and induces conformational changes in protein residues involved in substrate binding, while compounds T1-T5 could have a more indirect effect. Fluorescence based enzyme activity assay and complementary thermal stability analysis reveal only weak inhibition properties in the high micromolar range thereby indicating the need for compound optimization. Nevertheless, the unique binding properties involving strong hydrogen bonding and the various options for structural optimization make the compounds ideal lead structures. In combination with the inexpensive and undemanding synthesis, the reported hydrazone and thiosemicarbazones represent an attractive scaffold for further structure-based development of novel PLpro inhibitors by interrupting protein-protein interactions at the S1 and S2 site.