Isocitrate binds to the itaconic acid–responsive LysR-type transcriptional regulator RipR in Salmonella pathogenesis

• DOI: 10.1016/j.jbc.2022.102562
• Publication Date: 2022-10-02T21:40:54Z
• Authors: Nayeon Ki; Jinshil Kim; Inseong Jo; Yongseong Hyun; Sangryeol Ryu; Nam-Chul Ha
• Keywords: Itaconic acid

Macrophages produce itaconic acid in phagosomes response to bacterial cell wall component lipopolysaccharide eliminate invading pathogenic bacteria. Itaconic competitively inhibits the first enzyme of glyoxylate cycle. To overcome stress, bacteria employ LysR-type transcriptional regulator RipR. However, it remains unknown which molecule activates RipR pathogenesis. In this study, we determined crystal structure regulatory domain from intracellular pathogen Salmonella. The exhibited typical dimeric arrangement with putative ligand-binding site between two subdomains. Our isothermal titration calorimetry experiments identified isocitrate as physiological ligand RipR, whose level is increased stress. We further found that 3-phenylpropionic significantly decreased resistance an challenge. Consistently, complex revealed compound antagonistically bound site. This study provides molecular basis survival stress our immune systems. 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Tumescheit Mirdita Söding Steinegger Foldseek: fast search.bioRxiv. ([preprint])https://doi.org/10.1101/2022.02.07.479398Crossref (0) superposition ligand-bound structures suggested 2). seemed be ligand-free structure, since no extra electron density map pocket RD.Table 1Results Foldseek structurePDB IDScoreE-valuesSequence identity (%)Gene IDDescriptionOligomer state2H9B7874.292e-2025.4BenM Acinetobacter baylyi ADP1 (R156H/T157S)LysR-type regulatorHomodimer2F787759.231e-2025.7BenM baylyiLysR-type regulatorHomodimer2F7A7721.118e-1925.7BenM cis, cis-muconate regulatorHomodimer2H997691.354e-1925.7BenM (R156H, T157S)LysR-type regulatorHomodimer2F977641.863e-1925.7BenM (high pH)LysR-type regulatorHomodimer2F6G7553.308e-1924.7BenM ADP1LysR-type regulatorHomodimer3GLB7533.758e-1924.7CatM (R156H)LysR-type regulatorHomodimer2H987427.584e-1925.7CatM (V158M)LysR-type regulatorHomodimer3K1N7418.084e-1925.7BenM (full-length)LysR-type regulatorHomodimer3K1P7331.347e-1824.6BenM (E226K) regulatorHomodimer2F7C7271.975e-1824.7CatM regulatorHomodimer1IXC6892.233e-1721.6CbnR Cupriavidus necatorLysR-type regulatorHomotetramer6G1D6882.380e-1724.1OxyR Corynebacterium glutamicumLysR-type regulatorHomotetramer2HXR6803.966e-1723CynR K-12Probable regulatorHomodimer5TED6784.505e-1721.9QuiR shikimate Listeria monocytogenes EGD-eLysR-type regulatorHomotrimer Open table tab sites were lined Ser100, Arg148, Leu202 2), mostly conserved among (31Craven Ezezika O.C. Haddad Hall R.A. Momany Neidle E.L. Inducer BenM, ADP1.Mol. 72: 881-894Crossref particular, Arg residue corresponding Arg148 makes ionic carboxylic cis-cis-muconate (PDB code: 2F7A; Fig. Thus, these findings suggest contains moieties cis-cis-muconate. whether responsive treatment, treated investigated ripCBA, quantitative real-time PCR (qRT-PCR) showed treatment levels over 200-fold, confirming 3A). Since plays significant Typhimurium, analogs RD. seek selected together analogs, isocitrate, succinic malic oxaloacetate, cis-aconitate, measured binding affinities compounds proteins (ITC) 20 mM Hepes (pH 7.0). ITC thermograms strongly submicromolar Kd value (0.31 μM) stoichiometry 0.5 per monomer 3B). heat 3B), unusually high indicated interactions newly binding. contrast, did not give compelling S3).Figure 5Binding A, acid–bound (pale green) (gray). = 0.603 Å) shown stick (yellow) represented sticks. black rectangle indicates close-up view. B, growth curves WT SL1344 LB medium various concentrations (0, 0.1, 0.5, mM) A600. mean SEM values calculated replicate experiments. C, ripA analyzed (qRT-PCR). strain cultured 4 h 5 acid. normalized samples containing without Transcription target gyrB. Error bars represent SD experiments, p Student's t test. ∗p < 0.05. D, presence both macromolecule displayed injection (raw data; top) heat/enthalpy (bottom). added then sample titrated isocitrate. ITC, calorimetry; domain.View Large Image Figure ViewerDownload Hi-res image Download (PPT) investigate attempted solve Unfortunately, hamper cocrystallization allow soaking crystals. instead docked silico AutoDock Vina PyRx (32Dallakyan Olson A.J. Small-molecule library screening docking PyRx.Methods Mol. 1263: 243-250Crossref (1390) S4A). Gromacs minimized energy depicted isocitrate-bound 4A). side-chains Glu127, Ile200, Thr98, Ala99, Pro226 solvent-accessible S4B). moiety predicted comparison cis-cis-muconate–bound (Figs. 2B When in-silico compared substantial observed, because minimization change receptor elution volumes buffer size-exclusion chromatography, volume S5). These indicate conformation more compact, RDs. analyze dimer binding, performed dynamics (MD) simulation program, used MD biomolecules (33Pronk Pall Schulz Larsson Bjelkmar Apostolov al.Gromacs 4.5: high-throughput parallel open toolkit.Bioinformatics. 2013; 845-854Crossref (5230) motions largely stopped 200 ns during process. According result, subunit (chain B) moved interior 8 4B), while A) remained pocket. 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Menendez Rivas Alfonso al.3-Hydroxyphenylpropionate phenylpropionate synergistic activators MhpR 284: 21218-21228Abstract (24) 1A Although show responses, common (36Manso Moreover, 2–predicted site–lining S2 S6). tested experiment (Kd 5.22 less than S7 2C). binds extensive examine 2.8 resolution. intersubdomain 5A). near phenyl ring surrounded Leu204 Comparing benzoic positions similar. captured interacted S8). substantially