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Human PRPS1 filaments stabilize allosteric sites to regulate activity

Nature Structural & Molecular Biology volume 30, pages 391–402 (2023)Cite this article

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Abstract

The universally conserved enzyme phosphoribosyl pyrophosphate synthetase (PRPS) assembles filaments in evolutionarily diverse organisms. PRPS is a key regulator of nucleotide metabolism, and mutations in the human enzyme PRPS1 lead to a spectrum of diseases. Here we determine structures of human PRPS1 filaments in active and inhibited states, with fixed assembly contacts accommodating both conformations. The conserved assembly interface stabilizes the binding site for the essential activator phosphate, increasing activity in the filament. Some disease mutations alter assembly, supporting the link between filament stability and activity. Structures of active PRPS1 filaments turning over substrate also reveal coupling of catalysis in one active site with product release in an adjacent site. PRPS1 filaments therefore provide an additional layer of allosteric control, conserved throughout evolution, with likely impact on metabolic homeostasis. Stabilization of allosteric binding sites by polymerization adds to the growing diversity of assembly-based enzyme regulatory mechanisms.

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Fig. 1: Biochemical and structural overview of PRPP synthetase.
Fig. 2: Presence of phosphate or ADP dictate filament structure of PRPS1.
Fig. 3: The allosteric interface coordinates catalysis across protomers.
Fig. 4: Mutation of filament interface residues decreases catalysis.
Fig. 5: Filament formation stabilizes the C terminus and allosteric site.
Fig. 6: Mutations near the N and C termini alter filament formation, which correlates with catalysis.
Fig. 7: PRPS1 filaments stabilize the allosteric site, reinforcing the inhibited and active conformations and facilitating a catalytic reload mechanism.

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Data availability

The cryo-EM maps generated for this manuscript are available from the EMDB (https://www.ebi.ac.uk/emdb/) at the accession codes listed in Tables 1–3 of the manuscript (EMDB IDs: EMD-27279, EMD-27280, EMD-27281, EMD-27282, EMD-27283, EMD-27284, EMD-27285, EMD-27286, EMD-27287, EMD-27288, EMD-27289, EMD-27290, EMD-27291, EMD-27292, EMD-27293, EMD-27294, EMD-27295). The protein models generated for this manuscript are available from the RCSB PDB (https://www.rcsb.org/) at the accession codes listed in Tables 1–3 of the manuscript (PDB IDs: 8DBC, 8DBD, 8DBE, 8DBF, 8DBG, 8DBH, 8DBI, 8DBJ, 8DBK, 8DBL, 8DBM, 8DBN, 8DBO). Protein sequences identified by the NCBI online portal for BLAST (v2.13.0; https://blast.ncbi.nlm.nih.gov/Blast.cgi) were queried on the ‘non-redundant protein sequences (nr)’ database. Source data are provided with this paper.

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Acknowledgements

We thank the Arnold and Mabel Beckman Cryo-EM Center at the University of Washington for electron microscope use. We also thank members of the Kollman group for valuable feedback provided during cryo-EM data collection and processing. This work was supported by the US National Institutes of Health (grants nos. R01GM118396 and S10OD023476 to J.M.K. and 1F32AI145111 to K.L.H.)

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Authors and Affiliations

  1. Department of Biochemistry, University of Washington, Seattle, WA, USA

    Kelli L. Hvorecny, Kenzee Hargett, Joel D. Quispe & Justin M. Kollman

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  1. Kelli L. Hvorecny
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  2. Kenzee Hargett
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Contributions

K.L.H. performed experiments. K.H. optimized protein purification. J.D.Q. arranged, guided and provided support for EM data collection. K.L.H. and J.M.K. designed experiments, performed data analysis and interpretation, and wrote the manuscript.

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Correspondence to Justin M. Kollman.

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Nature Structural & Molecular Biology thanks Ambroise Desfosses, Arjen Jakobi and Menico Rizzi for their contribution to the peer review of this work. Primary Handling Editors: Florian Ullrich, Carolina Perdigoto and Katarzyna Ciazynska, in collaboration with the Nature Structural & Molecular Biology team.

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Extended data

Extended Data Fig. 1 Filament formation in PRPS1.

a. Section of negative stain EM of purified PRPS protein in a HEPES/Salt buffer used for purification. b. Panel of negative stain EM sections of PRPS1 in phosphate buffer in the presence of the indicated ligands. c. Elution profile from a size exclusion column (Superose 6) of PRPS1 in 50 mM phosphate buffer, pH 7.6. d. Elution profile from a size exclusion column (Superose 6) of PRPS1 in 100 mM KCl, 50 mM HEPES, pH 7.6 in the presence (black) or absence (grey) of 50 mM potassium phosphate, pH 7.6. PRPS1: monomer, 35 kDa; dimer, 70 kDa; hexamer, 210 kDa; filament, ≧ 420 kDa. e. Motion-corrected and summed cryo electron micrographs, Gaussian blurred and contrast adjusted for visualization, from four datasets presented in this manuscript, representing the cameras and tilts used in data collection. Microscopes, cameras, and stage tilts are listed in Data Table 1.

Source data

Extended Data Fig. 2 Data processing and statistics for cryo-EM datasets.

Left. Overview of the processing scheme for the datasets presented in this manuscript. Right. Fourier shell correlation curves calculated in Relion (black) and in Phenix (grey). One set of curves per dataset is shown. For full dataset statistics and information, see Data Table 1.

Source data

Extended Data Fig. 3 Volumes and models of filament interface residues.

a. Surface representation of filament interface in phosphate- or ADP-bound structures; orange patches indicate residues involved in the interface. b. Model and map of the primary interface residues of filament structures presented in this manuscript (the interface from the ADP bound filament can be found in Fig. 2c). Right panel shows the overlay of the interfaces when aligned by the bottom protomer. ADP-bound structure colored in orange and blue; all others in grey. c. Schematic of primary interactions across the filament interface; rectangular dashed lines indicate pi-stacking interactions and rounded dashed lines indicate hydrogen bonds. d. The C-terminal portion of a protein sequence alignment comparing PRPS across kingdoms. Identical residues are highlighted in orange. e. Alignment of the phosphate and ADP-bound structures on the allosteric domain (left) show minimal differences at the protomer level. f. Comparison of phosphate- (dark grey) and ADP-bound (orange/blue) structures to human crystal structures of wild type PRPS1 (light grey, PDB ID 2H06, 2HCR, 3EFH, and 3S5J). Structures have been aligned on the allosteric domain of protomer a. g. Differences in the filaments arise from the orientations of the protomers relative to each other in the hexamers, with rotations of neighboring protomers relative to a as indicated. h. Overlay of the phosphate- (dark grey) and ADP-bound (orange/blue) filament interfaces with the E. coli PRPS filament interfaces (light grey, PDB ID 7XMU, 7XMV) i. Comparison of phosphate- (dark grey) and ADP-bound (orange/blue) structures to E. coli PRPS filament structures (light grey, PDB ID 7XMV). Structures have been aligned on the allosteric domain of protomer a, and phosphates from the phosphate-bound human and E. coli structures have been omitted for clarity.

Extended Data Fig. 4 Substrate- and product-bound filaments.

a. Volume of PRPS1 filaments bound to phosphate/ATP (left), phosphate/ATP/R5P (middle), or phosphate/PRPP (right); protomers colored in blue or orange. b, top. Volume of one hexamer from a filament of PRPS1 bound to PRPP. Protomers are orange and blue, with the active site in yellow. b, bottom. Zoom in of active site indicated in (top), including the catalytic loop (dark blue), ATP (yellow), phosphate, magnesium, and coordinated waters. c–e. Volume showing the catalytic loop (dark blue or dark orange) and the ligands in the active site (yellow) for each of the filament structures presented in this work. f. Overlay of active sites shown in Main Text Fig. 3b–d and also including PRPS1 + ADP (light grey). g. Volume (top) describing location of slices (bottom) showing catalytic domains in two maps with well-resolved catalytic loops. h. Overlay of PRPS1 + ATP/R5P closed catalytic loop and key residues from the three PRPS structures from the PDB that also contain a closed catalytic loop (3MBI from Thermoplasma volcanium; 5T3O and 7PN0 from Thermus thermophilus). PRPS1 with ATP/R5P in blue, PDB models in grey. i. Overlay of PRPS1 with ATP/R5P (blue/orange) and 5T3O and 7PN0 from Thermus thermophilus (greys), showing the neighboring open and closed catalytic loops.

Extended Data Fig. 5 Example classification scheme and directional FSCs.

a. Classification scheme for PRPS1 + ATP/R5P after symmetry expansion. Particles were classified into ten classes, without alignment using a protomer mask. a subset of the resulting volumes was locally refined using a hexamer mask and exported to Phenix for density modification. Two volumes were then used for model building. b. Directional FSC for volumes derived from tilted datasets and volumes and models from the active sites from protomer a of each map.

Source data

Extended Data Fig. 6 Ligand volumes from substrate- and product-bound filaments.

Panels show the volume and the ligands for (a) PRPS1 + ATP, (b) PRPS1 + ATP/R5P with open loop, (c) PRPS1 + ATP/R5P with closed loop, (d) PRPS1 + AMP/PRPP with closed loop, (e) PRPS1 + PRPP.

Extended Data Fig. 7 Mutation of filament interface residues.

a. Panel of negative stain EM sections of PRPS1 engineered mutations in phosphate buffer in the presence of the indicated ligands. b. Chromatography curves from a Superose 6 of PRPS1 and three engineered, filament-interface mutations. c. Assay performed in buffer containing: 50 mM Potassium HEPES pH 7.6, 6 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, 0.1 mg/mL bovine serum albumin. Left: Activity assay of the three engineered mutations with or without 50 mM potassium phosphate, pH 7.6 (N = 4 technical replicates). Right: Ratio of 50 mM phosphate: 0 mM phosphate activities (V) from the panel to the left. d. Substrate kinetics of the three engineered mutations at protein concentrations with detectable catalytic activity. Assay performed in buffer containing: 50 mM Potassium Phosphate pH 7.6, 6 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, 0.1 mg/mL bovine serum albumin. Triplicate readings of one well for a single replicate (N = 1 technical replicate) are shown as open circles. e. Kinetic parameters for the wild type protein and the three engineered mutations.

Source data

Extended Data Fig. 8 Control assays for catalysis experiments.

a. PRPS1 catalysis over time at the lowest ribose-5-phosphate concentration used (100 μM ATP, 1.5 μM ribose-5-phosphate), plotted before conversion to μM AMP. Individual data points are shown as open circles (N = 3). b. PRPS1 kinetic analysis varying ATP concentration and holding ribose-5-phosphate at 100 μM. Individual data points are shown as open circles. Solid circles and error bars represent mean ± standard deviation (N = 3). Calculated kinetic parameters in inset.

Source data

Extended Data Fig. 9 Volumes for C-termini of PRPS1-E307A mutations.

a–d. Panels detailing PRPS1-E307A maps and models, with protomers in blue/orange and C-termini highlighted in red. Row 1: Dataset, symmetry, and number of particles included in the map. Row 2: View of one face of the ResMap filtered volumes from PRPS1-E307A datasets. Row 3: Insert showing volume of C-termini of protomer a from Relion’s implementation of ResMap (grey box) or Phenix’s Density Modification (black/white box). Rows 4 & 5: View of both faces of the density modified volumes from PRPS1-E307A datasets.

Extended Data Fig. 10 Filament formation in PRPS1 disease mutants.

a. Kinetic parameters for the wild type protein and the four disease mutations as determined from the data shown in Main Text Fig. 6. b. Chromatography curves from a Superose 6 of PRPS1 and four disease mutations. c. Panel of negative stain EM sections of PRPS1 disease mutations in phosphate buffer in the presence of the indicated ligands.

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Supplementary Video 1

Morph between phosphate-bound PRPS1 filament and ADP-bound PRPS1 filament. Cartoon of backbone atoms, with the protomers colored in orange and blue, and a phosphate positioned in the allosteric site for reference. Filaments have been aligned at the central interface. The video progresses as follows: phosphate-bound to ADP-bound to phosphate-bound to ADP-bound.

Supplementary Video 2

Morph between the open and closed conformations of PRPS1 incubated with ATP and ribose-5-phosphate. Top and side views of cartoon of backbone atoms of hexamer, with the protomers colored in orange and blue and the catalytic loops in the a and b protomers in dark orange and dark blue, respectively. Active site ligands are shown in yellow. Hexamers have been aligned on protomers c–f. The video progresses from protomer b in the open-loop conformation to protomer b in the closed-loop conformation, back to protomer b in the open-loop conformation to protomer b in the closed-loop conformation.

Supplementary Table 1

Primers used in this manuscript.

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Source Data Fig. 4

Activity assay data points.

Source Data Fig. 6

Activity assay data points.

Source Data Extended Data Fig. 1

Size exclusion chromatography curve data.

Source Data Extended Data Fig. 2

Fourier shell correlation curve data points.

Source Data Extended Data Fig. 5

Three-dimensional Fourier shell correlation curve data points and histogram data.

Source Data Extended Data Fig. 7

Activity assay data points and size exclusion chromatography curve data.

Source Data Extended Data Fig. 8

Activity assay data points.

Source Data Extended Data Fig. 10

Size exclusion chromatography curve data.

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Hvorecny, K.L., Hargett, K., Quispe, J.D. et al. Human PRPS1 filaments stabilize allosteric sites to regulate activity. Nat Struct Mol Biol 30, 391–402 (2023). https://doi.org/10.1038/s41594-023-00921-z

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