GK PUA Domain Modulates Cables, Cooperativity, and Proline Feedback

Abstract

In E. coli, the committed step of proline biosynthesis is initiated by γ-glutamate kinase (GK); however, the regulatory logic governing this enzyme remains poorly understood. E. coli GK (EcGK) possesses an enigmatic C-terminal PUA domain—a motif historically linked to RNA binding but uncharacterized in this context. Here, we demonstrate that EcGK forms metabolic filaments and further assembles into four-stranded metabolic cables. Cryo-EM structures of EcGK filaments reveal two assembly interfaces: one analogous to the eukaryotic P5CS “hook” motif and a novel interface mediated by the PUA domain. Filament formation hinders substrate binding, whereas higher-order cable assembly introduces additional cooperativity in substrate binding. Moreover, we resolve the first proline-bound EcGK structure, revealing the function of the PUA domain and the E135-D148 loop in proline feedback inhibition. Thus, the PUA domain plays dual roles in EcGK regulation: it drives filament/cable assembly to reshape substrate binding kinetics, and also facilitates the E135–D148 loop to enable high sensitivity to proline feedback inhibition. In E. coli, the PUA domain thus modulates proline biosynthesis within physiological ranges of substrates and products.

Keywords

allosteric cooperativity / cytoophidium / metabolic cable / metabolic filament / proline biosynthesis / PUA domain / γ-glutamate kinase (GK)

References

[1] H. Garcia-Seisdedos, C. Empereur-Mot, N. Elad, and E. D. Levy, “Proteins Evolve on the Edge of Supramolecular Self-Assembly,” Nature 548 (2017): 244–247.

[2] K. L. Hvorecny and J. M. Kollman, “Greater Than the Sum of Parts: Mechanisms of Metabolic Regulation by Enzyme Filaments,” Current Opinion in Structural Biology 79 (2023): 102530.

[3] C. J. Guo and J. L. Liu, “Cytoophidia and Filaments: You Must Unlearn What You Have Learned,” Biochemical Society Transactions 51 (2023): 1245–1256.

[4] E. M. Lynch, J. M. Kollman, and B. A. Webb, “Filament Formation by Metabolic Enzymes—A New Twist on Regulation,” Current Opinion in Cell Biology 66 (2020): 28–33.

[5] J.-L. Liu, “The Cytoophidium and Its Kind: Filamentation and Compartmentation of Metabolic Enzymes,” Annual Review of Cell and Developmental Biology 32 (2016): 349–372.

[6] E. M. Lynch and J. M. Kollman, “Coupled Structural Transitions Enable Highly Cooperative Regulation of Human CTPS2 filaments,” Nature Structural & Molecular Biology 27 (2020): 42–48.

[7] C. J. Smith, A. H. Deutch, and K. E. Rushlow, “Purification and Characteristics of a Gamma-Glutamyl Kinase Involved in Escherichia coli Proline Biosynthesis,” Journal of Bacteriology 157 (1984): 545–551.

[8] L. T. Smith, “Characterization of a Gamma-Glutamyl Kinase From Escherichia coli That Confers Proline Overproduction and Osmotic Tolerance,” Journal of Bacteriology 164 (1985): 1088–1093.

[9] A. P. Seddon, K. Y. Zhao, and A. Meister, “Activation of Glutamate by γ-Glutamate Kinase: Formation of γ-cis-Cycloglutamyl Phosphate, an Analog of γ-Glutamyl Phosphate,” Journal of Biological Chemistry 264 (1989): 11326–11335.

[10] I. Pérez-Arellano, V. Rubio, and J. Cervera, “Dissection of Escherichia coli Glutamate 5-Kinase: Functional Impact of the Deletion of the PUA Domain,” FEBS Letters 579 (2005): 6903–6908.

[11] P. E. Hernandez, J. A. Ordoñez, and B. Sanz Perez, “Use of the Mud(Ap, lac) Bacteriophage to Study the Regulation of L-proline Biosynthetic Genes in Escherichia coli K-12,” Current Microbiology 9 (1983): 31–35.

[12] R. A. Brady and L. N. Csonka, “Transcriptional Regulation of the proC Gene of Salmonella typhimurium,” Journal of Bacteriology 170 (1988): 2379–2382.

[13] K. Omori, et al., “Analysis of the Serratia marcescens proBA Operon and Feedback Control of Proline Biosynthesis,” Microbiology 137 (1991): 509–517.

[14] D. Limauro, A. Falciatore, A. L. Basso, G. Forlani, and M. De Felice, “Proline Biosynthesis in Streptococcus Thermophilus: Characterization of the proBA Operon and Its Products,” Microbiology 142 (1996): 3275–3282.

[15] T. Sander, N. Farke, C. Diehl, M. Kuntz, T. Glatter, and H. Link, “Allosteric Feedback Inhibition Enables Robust Amino Acid Biosynthesis in E. coli by Enforcing Enzyme Overabundance,” Cell Systems 8 (2019): 66–75.e8.

[16] I. Pérez-Arellano, V. Rubio, and J. Cervera, “Mapping Active Site Residues in Glutamate-5-Kinase. The Substrate Glutamate and the Feed-Back Inhibitor Proline Bind at Overlapping Sites,” FEBS Letters 580 (2006): 6247–6253.

[17] C. Marco-Marín, F. Gil-Ortiz, I. Pérez-Arellano, J. Cervera, I. Fita, and V. Rubio, “A Novel Two-Domain Architecture Within the Amino Acid Kinase Enzyme family Revealed by the Crystal Structure of Escherichia coli Glutamate 5-Kinase,” Journal of Molecular Biology 367 (2007): 1431–1446.

[18] L. N. Csonka and T. Leisinger, “Biosynthesis of Proline,” EcoSal Plus 2 (2007), https://doi.org/10.1128/ecosalplus.3.6.1.4.

[19] J. Liu, M. Liu, T. Shi, et al., “CRISPR-Assisted Rational Flux-Tuning and Arrayed CRISPRi Screening of an L-Proline Exporter for L-proline Hyperproduction,” Nature Communications 13 (2022): 891.

[20] M. Long, M. Xu, Z. Ma, et al., “Significantly Enhancing Production of Trans-4-Hydroxy-l-Proline by Integrated System Engineering in Escherichia coli,” Science Advances 6 (2020): eaba2383.

[21] I. Pérez-Arellano, F. Carmona-Álvarez, J. Gallego, and J. Cervera, “Molecular Mechanisms Modulating Glutamate Kinase Activity. Identification of the Proline Feedback Inhibitor Binding Site,” Journal of Molecular Biology 404 (2010): 890–901.

[22] L. Aravind and E. V. Koonin, “Novel Predicted RNA-Binding Domains Associated With the Translation Machinery,” Journal of Molecular Evolution 48 (1999): 291–302.

[23] I. Pérez-Arellano, J. Gallego, and J. Cervera, “The PUA Domain−a Structural and Functional Overview,” The FEBS Journal 274 (2007): 4972–4984.

[24] C. S. Cerrudo, P. D. Ghiringhelli, and D. E. Gomez, “Protein Universe Containing a PUA RNA -Binding Domain,” The FEBS Journal 281 (2014): 74–87.

[25] B. Zhang, Ö. Y. Tastan, X. Zhou, et al., “The Proline Synthesis Enzyme P5CS Forms Cytoophidia in Drosophila,” Journal of Genetics and Genomics 47 (2020): 131–143.

[26] J. Zhong, et al., “Structural Basis of Dynamic P5CS Filaments,” eLife 11 (2022): e76107.

[27] J. Abramson, J. Adler, J. Dunger, et al., “Accurate Structure Prediction of Biomolecular Interactions With AlphaFold 3,” Nature 630 (2024): 493–500.

[28] B. D. Bennett, E. H. Kimball, M. Gao, R. Osterhout, S. J. Van Dien, and J. D. Rabinowitz, “Absolute Metabolite Concentrations and Implied Enzyme Active Site Occupancy in Escherichia coli,” Nature Chemical Biology 5 (2009): 593–599.

[29] D. Radoš, “Homeostasis of the Biosynthetic E. coli Metabolome,” iScience 25 (2022): 104503.

[30] H. Yaginuma, S. Kawai, K. V. Tabata, et al., “Diversity in ATP Concentrations in a Single Bacterial Cell Population Revealed by Quantitative Single-Cell Imaging,” Scientific Reports 4 (2014): 6522.

[31] I. Pérez-Arellano, F. Gil-Ortiz, J. Cervera, and V. Rubio, “Glutamate-5-Kinase From Escherichia coli: Gene Cloning, Overexpression, Purification and Crystallization of the Recombinant Enzyme and Preliminary X-Ray Studies,” Acta Crystallographica Section D Biological Crystallography 60 (2004): 2091–2094.

[32] Y. Fichman, S. Y. Gerdes, H. Kovács, L. Szabados, A. Zilberstein, and L. N. Csonka, “Evolution of Proline Biosynthesis: Enzymology, Bioinformatics, Genetics, and Transcriptional Regulation,” Biological Reviews 90 (2015): 1065–1099.

[33] M. Ingerson-Mahar, A. Briegel, J. N. Werner, G. J. Jensen, and Z. Gitai, “The Metabolic Enzyme CTP Synthase Forms Cytoskeletal filaments,” Nature Cell Biology 12 (2010): 739–746.

[34] K. W. Ryu, T. S. Fung, D. C. Baker, et al., “Cellular ATP Demand Creates Metabolically Distinct Subpopulations of Mitochondria,” Nature 635 (2024): 746–754.

[35] P. Juda, J. Šmigová, L. Kováčik, E. Bártová, and I. Raška, “Ultrastructure of Cytoplasmic and Nuclear Inosine-5′-Monophosphate Dehydrogenase 2 “Rods and Rings” Inclusions,” Journal of Histochemistry & Cytochemistry 62 (2014): 739–750.

[36] Y. Fu, C.-J. Guo, Z.-J. Liu, and J.-L. Liu, “Architecture of CTPS Filament Networks Revealed by Cryo-Electron Tomography,” Experimental Cell Research 442 (2024): 114262.

[37] J. M. Hansen, A. Horowitz, E. M. Lynch, et al., “Cryo-EM Structures of CTP Synthase Filaments Reveal Mechanism of pH-sensitive Assembly During Budding Yeast Starvation,” eLife 10 (2021): e73368.

[38] C.-J. Guo, T. Zhang, Q. Leng, X. Zhou, J. Zhong, and J.-L. Liu, “Dynamic Arabidopsis P5CS Filament Facilitates Substrate Channelling,” Nature Plants 10 (2024): 880–889.

[39] K. L. Hvorecny, K. Hargett, J. D. Quispe, and J. M. Kollman, “Human PRPS1 Filaments Stabilize Allosteric Sites to Regulate Activity,” Nature Structural & Molecular Biology 30 (2023): 391–402.

[40] H.-H. Hu, G.-M. Lu, C.-C. Chang, et al., “Filamentation Modulates Allosteric Regulation of PRPS,” eLife 11 (2022): e79552.

[41] P. Pony, C. Rapisarda, L. Terradot, E. Marza, and R. Fronzes, “Filamentation of the Bacterial Bi-Functional Alcohol/Aldehyde Dehydrogenase AdhE Is Essential for Substrate Channeling and Enzymatic Regulation,” Nature Communications 11 (2020): 1426.

[42] C. Guo, Z. Wang, and J.-L. Liu, “Filamentation and Inhibition of Prokaryotic CTP Synthase With Ligands,” mLife 3 (2024): 240–250.

[43] M. C. Johnson and J. M. Kollman, “Cryo-EM Structures Demonstrate Human IMPDH2 Filament Assembly Tunes Allosteric Regulation,” eLife 9 (2020): e53243.

[44] D. N. Mastronarde, “Automated Electron Microscope Tomography Using Robust Prediction of Specimen Movements,” Journal of Structural Biology 152 (2005): 36–51.

Rights & permissions

2026 The Author(s). Aggregate published by SCUT, AIEI, and John Wiley & Sons Australia, Ltd.