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Diadenosine tetraphosphate regulates biosynthesis of GTP in Bacillus subtilis

Nature Microbiology volume 7, pages 1442–1452 (2022)Cite this article

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Abstract

Diadenosine tetraphosphate (Ap4A) is a putative second messenger molecule that is conserved from bacteria to humans. Nevertheless, its physiological role and the underlying molecular mechanisms are poorly characterized. We investigated the molecular mechanism by which Ap4A regulates inosine-5′-monophosphate dehydrogenase (IMPDH, a key branching point enzyme for the biosynthesis of adenosine or guanosine nucleotides) in Bacillus subtilis. We solved the crystal structure of BsIMPDH bound to Ap4A at a resolution of 2.45 Å to show that Ap4A binds to the interface between two IMPDH subunits, acting as the glue that switches active IMPDH tetramers into less active octamers. Guided by these insights, we engineered mutant strains of B. subtilis that bypass Ap4A-dependent IMPDH regulation without perturbing intracellular Ap4A pools themselves. We used metabolomics, which suggests that these mutants have a dysregulated purine, and in particular GTP, metabolome and phenotypic analysis, which shows increased sensitivity of B. subtilis IMPDH mutant strains to heat compared with wild-type strains. Our study identifies a central role for IMPDH in remodelling metabolism and heat resistance, and provides evidence that Ap4A can function as an alarmone.

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Fig. 1: Ap4A inhibits IMPDH in a non-competitive manner.
Fig. 2: Mechanism of the Ap4A-dependent inhibition of IMPDH.
Fig. 3: Crystal structure and conformational changes in BsIMPDH ΔCBS.
Fig. 4: Mutations interfering with allosteric regulation of IMPDH activity perturb nucleotide metabolism of B. subtilis.
Fig. 5: Allosteric regulation of IMPDH activity is critical for tolerance of B. subtilis to heat shock conditions.

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Data availability

Structure factors and coordinates of X-ray crystallographic datasets have been deposited at the Protein Data Bank (www.rcsb.org) under the accession codes 7OJ1 and 7OJ2 for the Ap4A-bound BsIMPDH and BsIMPDH-ΔCBS, respectively. All other structural data employed in this manuscript (accession codes 3L2B, 3TSB, 4DQW, 4XTI, 4XWU, 5AHL, 5AHM, 5MCP, 6GJV, 6RPU) are publicly available in the Protein Data Bank. Source data for Figs. 1, 2, 4 and 5, and for Extended data Figs. 1, 2, 5, 6 and 8 are provided with this paper.

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Acknowledgements

This work was supported by the priority programme of the Deutsche Forschungsgemeinschaft (DFG) SPP1879 – ‘Nucleotide second messenger signalling in bacteria’ (to G.B.), the USA National Institute of Health R35 GM127088 and the Howard Hughes Medical Institute Faculty Scholars Award (to J.D.W.), and the National Science Foundation (NSF) grant award no. 1715710 (to D.A.-N.). We thank the European Synchrotron Radiation Facility (Grenoble, France) and the Electron Storage Ring BESSY II (Berlin, Germany) for the excellent beamline support. We acknowledge support from the ‘DFG-core facility for interactions, dynamics and macromolecular assembly structure’ at the Philipps-University Marburg (to G.B.). G.H. acknowledges support from the Free-floater programme of the Max Planck Society.

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Author notes

  1. These authors contributed equally: Pietro I. Giammarinaro, Megan K. M. Young.

Authors and Affiliations

  1. Department of Chemistry and Center for Synthetic Microbiology, Philipps University Marburg, Marburg, Germany

    Pietro I. Giammarinaro, Wieland Steinchen, Christopher-Nils Mais, Georg Hochberg, Anja Paulus & Gert Bange

  2. Department of Bacteriology, University of Wisconsin-Madison, Madison, WI, USA

    Megan K. M. Young, Jin Yang, David M. Stevenson, Daniel Amador-Noguez & Jue D. Wang

  3. Max Planck Institute for Terrestrial Microbiology, Marburg, Germany

    Georg Hochberg & Gert Bange

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  1. Pietro I. Giammarinaro
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Contributions

J.D.W. and G.B. conceptualized the project. G.H. and D.A.-N. developed the methodology. P.I.G., M.K.M.Y., W.S., C.-N.M., J.Y. and A.P. conducted the experimental investigations. J.D.W. and G.B. acquired funding. J.D.W. and G.B. supervised the project. P.I.G., M.Y., W.S., J.D.W. and G.B. wrote the original draft. All authors read and commented on the manuscript.

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Correspondence to Jue D. Wang or Gert Bange.

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Extended data

Extended Data Fig. 1 Binding of nucleotides and dinucleotides to BsIMPDH determined by isothermal titration calorimetry (ITC).

a-j. A 25 µM concentrated solution of purified BsIMPDH was titrated with a. AMP, b. ADP, c. ATP, d. Ap3A, e. Ap4A, f. Ap5A, g. Ap6A, h. Ap3G, i. Ap4G, or j. Ap5G. The differential power (DP, upper plot) for each injection of ligand was recorded and used to determine the dissociation constant Kd from a fitting of the calculated binding enthalpies (ΔH, lower plot).

Source data

Extended Data Fig. 2 Enzyme kinetic parameters obtained for BsIMPDH.

a-c. The maximal velocity (Vmax) and Michaelis-Menten constant (Km) of BsIMPDH activity were determined in the presence of various ligands. For Ap4A, the inhibitory constant (Ki) was fitted from the change in Vmax in the presence of different Ap4A concentrations. The mean ± SD of enzymological parameters was obtained with Graph Pad Prism from n = 2 replicates. For assay results displayed in a (related to Fig. 1e–h) and c (related to Extended Data Fig. 6a), the concentration of NAD+ was kept constant at 5 mM and the concentration of IMP variable between 25 and 1,000 µM. For the assay results displayed in b (related to Fig. 1e), the concentration of IMP was kept constant at 3 mM and the concentration of NAD+ variable between 25 and 5,000 µM. n.d., not determined.

Source data

Extended Data Fig. 3 Coordination of Ap4A by the BsIMPDH CBS domains.

a. The CBS domains of the B. subtilis IMPDH (red, this study) were aligned with the CBS domains of P. aeruginosa IMPDH (left; pink, PDB-ID: 4DQW25), A. gossypii IMPDH (middle; light blue, PDB-ID: 6RPU26), and C. perfringens pyrophosphatase (right; salmon, PDB-ID: 3L2B63, revealing on overall similar topology. b. The unbiased Fobs-Fcalc difference electron density map (top) is coloured in green and red for positive and negative electron density, respectively, and contoured at 3.0 σ. The Ap4A ligand was not present during the refinement. The 2Fobs-Fcalc electron density map after final refinement (bottom), including the Ap4A ligand, is coloured in blue and contoured at 1.5 σ. c. Coordination of Ap4A in chains A and B of the crystal structure of Ap4A-bound BsIMPDH. Atoms are displayed as spheres and coloured as follows: carbon, black; oxygen, red; nitrogen, blue; phosphor, purple. Purple and orange solid lines illustrate ligand or non-ligand covalent bonds, and green dashed lines represent hydrogen bonds. Red semicircles denote hydrophobic interactions. The image was generated with LigPlot+64. d. Coordination of ligands by the CBS domains of B. subtilis IMPDH with Ap4A (left), P. aeruginosa IMPDH with 2 ATP (middle, PDB-ID: 4DQW25), and A. gossypii IMPDH with Ap5G (right, PDB-ID: 6RPU26). Ligands and amino acid residues lining the ligand-binding sites are shown as sticks and coloured by element (carbon, green/yellow/cyan; oxygen, red; nitrogen, blue; phosphor, orange). Green and purple spheres represent magnesium and manganese ions, respectively. Dashed lines denote hydrogen bonding interactions between the main chain carbonyl/amide groups and the adenine and guanine nucleobases. A. gossypii IMPDH does not require a divalent metal ion to coordinate the GDP ligand, whereas metal ion coordination in the prokaryotic B. subtilis and P. aeruginosa IMPDHs is achieved by E184 and E180 residues, respectively40.

Extended Data Fig. 4 Conformational flexibility of CBS domains in crystal structures of selected IMPDH proteins.

a-f. The crystal structures of IMPDH proteins from a. B. anthracis in apo-state (PDB-ID: 3TSB65), b. B. subtilis bound to Ap4A (this study), c. P. aeruginosa in apo-state (PDB-ID: 6GJV66), d. P. aeruginosa bound to ATP (PDB-ID: 4DQW25), e. A. gossypii bound to Ap5G and GDP (PDB-ID: 6RPU26), and f. A. gossypii bound to ATP (PDB-ID: 5MCP39) are shown as ribbon and colored to their B factors from 20 (blue) to 150 Å2 (red). For octameric biological assemblies, the bottom tetrameric ring is shown in grey surface. CBS domains are indicated by black dashed rectangles, and teal spheres denote the nucleotide ligands coordinates within the CBS domains.

Extended Data Fig. 5 Influence of Ap4A and ATP on conformational flexibility of BsIMPDH CBS domains by hydrogen/deuterium exchange mass spectrometry.

a. Difference in hydrogen/deuterium exchange (HDX) of representative peptides in the presence of Ap4A (top) or ATP (bottom), expressed as the difference in HDX of ligand-bound BsIMPDH versus its apo-state. b. Location of selected peptides exhibiting bimodality in HDX (EX1 kinetics) displayed on the adjacent CBS domains of two monomers of Ap4A-bound BsIMPDH. c-d. Mass spectra of two selected BsIMPDH peptides exhibiting EX1 kinetics for hydrogen/deuterium exchange, that is, c. the peptides containing residues 113–136, and d. residues 172–200 of BsIMPDH. The occurrence of a fast-exchanging population in the apo-state (red) is indicative for unfolding of secondary structures, which is restricted in samples of Ap4A-bound (blue) and ATP-bound (green) BsIMPDH.

Source data

Extended Data Fig. 6 Enzymatic activity and oligomeric state of BsIMPDH-WT and CBS domain variants.

a. Representative distributions of tetrameric and octameric BsIMPDH species for BsIMPDH-WT in the absence of IMP and NAD+ substrates and in dependence of Ap4A or ATP concentration. b-e. Representative distributions of tetrameric and octameric BsIMPDH species of b. BsIMPDH-WT, c. BsIMPDH-K202A, d. BsIMPDH-R141A/R144A, and e. BsIMPDH-ΔCBS, all in the presence of IMP and NAD+ substrates and in dependence of Ap4A or ATP concentration. f. Representative distributions of tetrameric and octameric BsIMPDH species for BsIMPDH-WT in the presence of IMP and NAD+ substrates and in dependence of the indicated adenosine nucleotides or ApnA and ApnG dinucleotides added. In a-f, the numbers in the diagrams reflect the molecular weight of the observed oligomeric species (mean and SD), the number of observed molecules for either species and their percentage of all observed molecules in the sample. g. Effect of 10 µM Ap4A or 2 mM ATP on the enzyme-kinetic behavior of BsIMPDH-WT and CBS domain variants K202A, R141A/R144A and ΔCBS for conversion of the IMP substrate into XMP. Individual data points of n = 2 replicates are shown, and parameters of the fits are given in Extended Data Fig. 2c. h. Correlation between observed fraction of IMPDH monomers in oligomeric state and the apparent Vmax of IMPDH activity. Linear regression was performed either with values for BsIMPDH WT only (red trace) or all depicted values including WT and variants (blue trace). Red and blue numbers denote values for the fraction and Vmax at the intersects with the x-axis and y-axis, respectively.

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Extended Data Fig. 7 Different conformations of catalytic flap and C-termini regions in crystal structures of IMPDH enzymes.

a-f. Cartoon representations of the crystal structures of a. Ap4A-bound full-length B. subtilis (Bs) IMPDH, b. B. subtilis (Bs) IMPDH-ΔCBS, c. P. aeruginosa (Pa) IMPDH-ΔCBS (PDB-ID: 5AHL31), d. P. aeruginosa (Pa) IMPDH-ΔCBS with substrate IMP in the active site (PDB-ID: 5AHM31), e. A. gossypii (Ag) IMPDH-ΔCBS (PDB-ID: 4XWU32), and f. A. gossypii (Ag) IMPDH-ΔCBS with substrate IMP in the active site (PDB-ID: 4XTI32). The catalytic cysteine (green), catalytic flap (red) and C-termini (orange) where colored and denoted where possible.

Extended Data Fig. 8 Quantification of rising intracellular Ap4A concentrations in B. subtilis after non-lethal heat shock.

In vivo quantification of Ap4A in wildtype (WT) B. subtilis 3610 and IMPDH mutant strains. Data represent mean ± SD of n = 3 biological replicates. Unpaired two-tailed t-tests were used to compare Ap4A levels for WT and IMPDH mutant strains of heat-shocked conditions (15 min, 30 min) versus the untreated control (0 min). Asterisks indicate p-values: * p ≤ 0.05; ns, not significant. Exact p-values are, WT: 0.1382 (15 vs. 0 min), 0.0943 (30 vs. 15 min), 0.0235 (30 vs. 0 min); K202A: 0.0191 (15 vs. 0 min), 0.5491 (30 vs. 15 min), 0.0223 (30 vs. 0 min); R141A/R144A: 0.4789 (15 vs. 0 min), 0.8778 (30 vs. 15 min), 0.3756 (30 vs. 0 min); ΔCBS: 0.0753 (15 vs. 0 min), 0.1146 (30 vs. 15 min), 0.0390 (30 vs. 0 min).

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Statistical source data – numerical raw data of experiment(s).

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Giammarinaro, P.I., Young, M.K.M., Steinchen, W. et al. Diadenosine tetraphosphate regulates biosynthesis of GTP in Bacillus subtilis. Nat Microbiol 7, 1442–1452 (2022). https://doi.org/10.1038/s41564-022-01193-x

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