Allosteric regulation of inosine 5'-monophosphate dehydrogenase (IMPDH), an essential enzyme of purine metabolism, contributes to the homeostasis of adenine and guanine nucleotides. However, the precise molecular mechanism of IMPDH regulation in bacteria remains unclear. Using biochemical and cryo-EM approaches, we reveal the intricate molecular mechanism of the IMPDH allosteric regulation in mycobacteria. The enzyme is inhibited by both GTP and (p)ppGpp, which bind to the regulatory CBS domains and, via interactions with basic residues in hinge regions, lock the catalytic core domains in a compressed conformation. This results in occlusion of inosine monophosphate (IMP) substrate binding to the active site and, ultimately, inhibition of the enzyme. The GTP and (p)ppGpp allosteric effectors bind to their dedicated sites but stabilize the compressed octamer by a common mechanism. Inhibition is relieved by the competitive displacement of GTP or (p)ppGpp by ATP allowing IMP-induced enzyme expansion. The structural knowledge and mechanistic understanding presented here open up new possibilities for the development of allosteric inhibitors with antibacterial potential.

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Prague Czech Republic

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Hedstrom, L. IMP Dehydrogenase: Structure, Mechanism, and Inhibition. Chem. Rev.109 7, 2903–2928 (2009). 10.1021/cr900021w PubMed DOI PMC

Buey, R. M., Fernández-Justel, D., Jiménez, A. & Revuelta, J. L. The Gateway to Guanine Nucleotides: Allosteric Regulation of IMP Dehydrogenases. Protein Sci.31, e4399 (2022). 10.1002/pro.4399 PubMed DOI PMC

Burrell, A. L. et al. IMPDH1 Retinal Variants Control Filament Architecture to Tune Allosteric Regulation. Nat. Struct. Mol. Biol.29, 47–58 (2022). 10.1038/s41594-021-00706-2 PubMed DOI PMC

Hedstrom, L., Liechti, G., Goldberg, J. B. & Gollapalli, D. R. The Antibiotic Potential of Prokaryotic IMP Dehydrogenase Inhibitors. Curr. Med Chem.18, 1909–1918 (2011). 10.2174/092986711795590129 PubMed DOI PMC

Singh, V. et al. The Inosine Monophosphate Dehydrogenase, GuaB2, Is a Vulnerable New Bactericidal Drug Target for Tuberculosis. ACS Infect. Dis.3, 5–17 (2017). 10.1021/acsinfecdis.6b00102 PubMed DOI PMC

Chacko, S. et al. Expanding Benzoxazole-Based Inosine 5′-Monophosphate Dehydrogenase (IMPDH) Inhibitor Structure–Activity As Potential Antituberculosis Agents. J. Med. Chem.61, 4739–4756 (2018). 10.1021/acs.jmedchem.7b01839 PubMed DOI PMC

Park, Y. et al. Essential but Not Vulnerable: Indazole Sulfonamides Targeting Inosine Monophosphate Dehydrogenase as Potential Leads against Mycobacterium Tuberculosis. ACS Infect. Dis.3, 18–33 (2017). 10.1021/acsinfecdis.6b00103 PubMed DOI PMC

Alexandre, T., Rayna, B. & Munier-Lehmann, H. Two Classes of Bacterial IMPDHs According to Their Quaternary Structures and Catalytic Properties. PLoS One10, e0116578 (2015). 10.1371/journal.pone.0116578 PubMed DOI PMC

Ereño-Orbea, J., Oyenarte, I. & Martínez-Cruz, L. A. CBS Domains: Ligand Binding Sites and Conformational Variability. Arch. Biochem. Biophys.540, 70–81 (2013). 10.1016/j.abb.2013.10.008 PubMed DOI

Buey, R. M. et al. Guanine Nucleotide Binding to the Bateman Domain Mediates the Allosteric Inhibition of Eukaryotic IMP Dehydrogenases. Nat. Commun.6, 8923 (2015). 10.1038/ncomms9923 PubMed DOI PMC

Fernández-Justel, D. et al. Diversity of Mechanisms to Control Bacterial GTP Homeostasis by the Mutually Exclusive Binding of Adenine and Guanine Nucleotides to IMP Dehydrogenase. Protein Sci.31, e4314 (2022). 10.1002/pro.4314 PubMed DOI PMC

Rostirolla, D. C., Assunção, T. M., de; Bizarro, C. V., Basso, L. A. & Santos, D. S. Biochemical Characterization of Mycobacterium Tuberculosis IMP Dehydrogenase: Kinetic Mechanism, Metal Activation and Evidence of a Cooperative System. RSC Adv.4, 26271–26287 (2014).10.1039/C4RA02142H DOI

Usha, V. et al. Identification of Novel Diphenyl Urea Inhibitors of Mt-GuaB2 Active against Mycobacterium Tuberculosis. Microbiology157, 290–299 (2011). 10.1099/mic.0.042549-0 PubMed DOI

Makowska-Grzyska, M. et al. A Novel Cofactor-Binding Mode in Bacterial IMP Dehydrogenases Explains Inhibitor Selectivity. J. Biol. Chem.290, 5893–5911 (2015). 10.1074/jbc.M114.619767 PubMed DOI PMC

Labesse, G. et al. MgATP Regulates Allostery and Fiber Formation in IMPDHs. Structure21 6, 975–985 (2013). 10.1016/j.str.2013.03.011 PubMed DOI

Labesse, G., Alexandre, T., Gelin, M., Haouz, A. & Munier-Lehmann, H. Crystallographic Studies of Two Variants of Pseudomonas Aeruginosa IMPDH with Impaired Allosteric Regulation. Acta Crystallogr D. Biol. Crystallogr71, 1890–1899 (2015). 10.1107/S1399004715013115 PubMed DOI

Josephine, H. R., Ravichandran, K. R. & Hedstrom, L. The Cys319 Loop Modulates the Transition between Dehydrogenase and Hydrolase Conformations in Inosine 5′-Monophosphate Dehydrogenase. Biochemistry49, 10674–10681 (2010). 10.1021/bi101590c PubMed DOI PMC

Sintchak, M. D. et al. Structure and Mechanism of Inosine Monophosphate Dehydrogenase in Complex with the Immunosuppressant Mycophenolic Acid. Cell85, 921–930 (1996). 10.1016/S0092-8674(00)81275-1 PubMed DOI

Makowska-Grzyska, M. et al. Mycobacterium Tuberculosis IMPDH in Complexes with Substrates, Products and Antitubercular Compounds. PLoS One10, e0138976 (2015). 10.1371/journal.pone.0138976 PubMed DOI PMC

Zhang, R. et al. Characteristics and Crystal Structure of Bacterial Inosine-5‘-Monophosphate Dehydrogenase. Biochemistry38, 4691–4700 (1999). 10.1021/bi982858v PubMed DOI

Pimkin, M. & Markham, G. D. The CBS Subdomain of Inosine 5′-Monophosphate Dehydrogenase Regulates Purine Nucleotide Turnover. Mol. Microbiol.68, 342–359 (2008). 10.1111/j.1365-2958.2008.06153.x PubMed DOI PMC

Pimkin, M., Pimkina, J. & Markham, G. D. A Regulatory Role of the Bateman Domain of IMP Dehydrogenase in Adenylate. Nucleotide Biosynth. J. Biol. Chem.284, 7960–7969 (2009).10.1074/jbc.M808541200 PubMed DOI PMC

Giammarinaro, P. I. et al. Diadenosine Tetraphosphate Regulates Biosynthesis of GTP in Bacillus Subtilis. Nat Microbiol.7, 1442–1452 (2022). PubMed PMC

Makowska-Grzyska, M. et al. Bacillus Anthracis Inosine 5′-Monophosphate Dehydrogenase in Action: The First Bacterial Series of Structures of Phosphate Ion-, Substrate-, and Product-Bound Complexes. Biochemistry51, 6148–6163 (2012). PubMed PMC

Johnson, M. C. & Kollman, J. M. Cryo-EM Structures Demonstrate Human IMPDH2 Filament Assembly Tunes Allosteric Regulation. Elife9, e53243 (2020). 10.7554/eLife.53243 PubMed DOI PMC

Buey, R. M. et al. A Nucleotide-Controlled Conformational Switch Modulates the Activity of Eukaryotic IMP Dehydrogenases. Sci. Rep.7, 2648 (2017). 10.1038/s41598-017-02805-x PubMed DOI PMC

Knejzlík, Z. et al. The Mycobacterial guaB1 Gene Encodes a Guanosine 5′-Monophosphate Reductase with a Cystathionine-β-Synthase Domain. FEBS J.289, 5571–5598 (2022). 10.1111/febs.16448 PubMed DOI PMC

Imholz, N. C. E., Noga, M. J., van den Broek, N. J. F. & Bokinsky, G. Calibrating the Bacterial Growth Rate Speedometer: A Re-Evaluation of the Relationship Between Basal ppGpp, Growth, and RNA Synthesis in Escherichia Coli. Front. Microbiol.11, 574872 (2020). PubMed PMC

Steinchen, W., Zegarra, V. & Bange, G. (P)ppGpp: Magic Modulators of Bacterial Physiology and Metabolism. Front. Microbiol. 11, 2072 (2020). PubMed PMC

Fernández-Justel, D., Peláez, R., Revuelta, J. L. & Buey, R. M. The Bateman Domain of IMP Dehydrogenase Is a Binding Target for Dinucleoside Polyphosphates. J. Biol. Chem.294, 14768–14775 (2019). 10.1074/jbc.AC119.010055 PubMed DOI PMC

Despotović, D. et al. Diadenosine Tetraphosphate (Ap4A) – an E. Coli Alarmone or a Damage Metabolite? FEBS J.284, 2194–2215 (2017). 10.1111/febs.14113 PubMed DOI

Cox, J. A. G. et al. Novel Inhibitors of Mycobacterium Tuberculosis GuaB2 Identified by a Target Based High-Throughput Phenotypic Screen. Sci. Rep.6, 38986 (2016). 10.1038/srep38986 PubMed DOI PMC

Chen, L. et al. Triazole-Linked Inhibitors of Inosine Monophosphate Dehydrogenase from Human and Mycobacterium Tuberculosis. J. Med. Chem.53, 4768–4778 (2010). 10.1021/jm100424m PubMed DOI PMC

Trapero, A. et al. Fragment-Based Approach to Targeting Inosine-5′-Monophosphate Dehydrogenase (IMPDH) from Mycobacterium Tuberculosis. J. Med. Chem.61, 2806–2822 (2018). 10.1021/acs.jmedchem.7b01622 PubMed DOI PMC

Alexandre, T. et al. First-in-Class Allosteric Inhibitors of Bacterial IMPDHs. Eur. J. Medicinal Chem.167, 124–132 (2019).10.1016/j.ejmech.2019.01.064 PubMed DOI

Andersen, K. R., Leksa, N. C. & Schwartz, T. U. Optimized E. Coli Expression Strain LOBSTR Eliminates Common Contaminants from His-Tag Purification. Proteins81, 1857–1861 (2013). 10.1002/prot.24364 PubMed DOI PMC

Studier, F. W. Protein Production by Auto-Induction in High-Density Shaking Cultures. Protein Expr. Purif.41, 207–234 (2005). 10.1016/j.pep.2005.01.016 PubMed DOI

Zheng, S. Q. et al. MotionCor2: Anisotropic Correction of Beam-Induced Motion for Improved Cryo-Electron Microscopy. Nat. Methods14, 331–332 (2017). 10.1038/nmeth.4193 PubMed DOI PMC

Rohou, A. & Grigorieff, N. CTFFIND4: Fast and Accurate Defocus Estimation from Electron Micrographs. J. Struct. Biol.192, 216–221 (2015). 10.1016/j.jsb.2015.08.008 PubMed DOI PMC

Kimanius, D., Dong, L., Sharov, G., Nakane, T. & Scheres, S. H. W. New Tools for Automated Cryo-EM Single-Particle Analysis in RELION-4.0. Biochem J.478, 4169–4185 (2021). 10.1042/BCJ20210708 PubMed DOI PMC

Burnley, T., Palmer, C. M. & Winn, M. Recent Developments in the CCP-EM Software Suite. Acta Crystallogr D. Struct. Biol.73, 469–477 (2017). 10.1107/S2059798317007859 PubMed DOI PMC

Jakobi, A. J., Wilmanns, M. & Sachse, C. Model-Based Local Density Sharpening of Cryo-EM Maps. eLife6, e27131 (2017). 10.7554/eLife.27131 PubMed DOI PMC

Jumper, J. et al. Highly Accurate Protein Structure Prediction with AlphaFold. Nature596, 583–589 (2021). 10.1038/s41586-021-03819-2 PubMed DOI PMC

Pettersen, E. F. et al. UCSF ChimeraX: Structure Visualization for Researchers, Educators, and Developers. Protein Sci.30, 70–82 (2021). 10.1002/pro.3943 PubMed DOI PMC

Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and Development of Coot. Acta Cryst. D.66, 486–501 (2010). 10.1107/S0907444910007493 PubMed DOI PMC

Croll, T. I. ISOLDE: A Physically Realistic Environment for Model Building into Low-Resolution Electron-Density Maps. Acta Cryst. D.74, 519–530 (2018).10.1107/S2059798318002425 PubMed DOI PMC

Afonine, P. V. et al. Real-Space Refinement in PHENIX for Cryo-EM and Crystallography. Acta Cryst. D.74, 531–544 (2018).10.1107/S2059798318006551 PubMed DOI PMC

Hoh, S. W., Burnley, T. & Cowtan, K. Current Approaches for Automated Model Building into Cryo-EM Maps Using Buccaneer with CCP-EM. Acta Cryst. D.76, 531–541 (2020).10.1107/S2059798320005513 PubMed DOI PMC

Stierand, K. & Rarey, M. Drawing the PDB: Protein−Ligand Complexes in Two Dimensions. ACS Med. Chem. Lett.1, 540–545 (2010). 10.1021/ml100164p PubMed DOI PMC

Roelofs, K. G., Wang, J., Sintim, H. O. & Lee, V. T. Differential Radial Capillary Action of Ligand Assay for High-Throughput Detection of Protein-Metabolite Interactions. Proc. Natl Acad. Sci.108, 15528–15533 (2011). 10.1073/pnas.1018949108 PubMed DOI PMC

Manalastas-Cantos, K. et al. ATSAS 3.0: Expanded Functionality and New Tools for Small-Angle Scattering Data Analysis. J. Appl Cryst.54, 343–355 (2021). 10.1107/S1600576720013412 PubMed DOI PMC

Svergun, D. I. Determination of the Regularization Parameter in Indirect-Transform Methods Using Perceptual Criteria. J. Appl Crystallogr25, 495–503 (1992).10.1107/S0021889892001663 DOI

Konarev, P. V., Volkov, V. V., Sokolova, A. V., Koch, M. H. J. & Svergun, D. I. PRIMUS: A Windows PC-Based System for Small-Angle Scattering Data Analysis. J. Appl Cryst.36, 1277–1282 (2003).10.1107/S0021889803012779 DOI

Franke, D. et al. ATSAS 2.8: A Comprehensive Data Analysis Suite for Small-Angle Scattering from Macromolecular Solutions. J. Appl Crystallogr50, 1212–1225 (2017). 10.1107/S1600576717007786 PubMed DOI PMC

Franke, D. & Svergun, D. I. DAMMIF, a Program for Rapid Ab-Initio Shape Determination in Small-Angle Scattering. J. Appl Crystallogr42, 342–346 (2009). 10.1107/S0021889809000338 PubMed DOI PMC

Svergun, D. I. Restoring Low Resolution Structure of Biological Macromolecules from Solution Scattering Using Simulated Annealing. Biophys. J.76, 2879–2886 (1999). 10.1016/S0006-3495(99)77443-6 PubMed DOI PMC

Sayers, E. A General Introduction to the E-Utilities. In Entrez Programming Utilities Help; National Center for Biotechnology Information (US), (2022).

Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic Local Alignment Search Tool. J. Mol. Biol.215, 403–410 (1990). 10.1016/S0022-2836(05)80360-2 PubMed DOI

Chapman, B. & Chang, J. Biopython: Python Tools for Computational Biology. SIGBIO Newsletter.20, 15–19 (2000).10.1145/360262.360268 DOI

Neuwald, A. F. & Poleksic, A. PSI-BLAST Searches Using Hidden Markov Models of Structural Repeats: Prediction of an Unusual Sliding DNA Clamp and of β-Propellers in UV-Damaged DNA-Binding Protein. Nucleic Acids Res.28, 3570–3580 (2000). 10.1093/nar/28.18.3570 PubMed DOI PMC

Camacho, C. et al. BLAST+: Architecture and Applications. BMC Bioinforma.10, 421 (2009).10.1186/1471-2105-10-421 PubMed DOI PMC

Rozewicki, J., Li, S., Amada, K. M., Standley, D. M. & Katoh, K. MAFFT-DASH: integrated protein sequence and structural alignment. Nucleic Acids Research47, W5–W10 (2019). PubMed PMC

Schneider, T. D. & Stephens, R. M. Sequence Logos: A New Way to Display Consensus Sequences. Nucleic Acids Res.18, 6097–6100 (1990). 10.1093/nar/18.20.6097 PubMed DOI PMC