Filamentation modulates allosteric regulation of PRPS
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Evaluation Summary:
The authors investigated the structure of phosphoribosyl pyrophosphate (PRPP) synthase (PRPPS) from Escherichia coli, a highly conserved enzyme from bacteria to mammals that catalyzes the synthesis of a key common compound for several metabolic pathways. Combining structural data with mutagenesis and activity assays, they demonstrate that the enzyme is regulated differently by allosteric effectors when assembled into one filament form or the other. The strength of the manuscript is the high-quality cryo-EM data, which allows the reconstruction of two different filament forms bound to different ligands.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Phosphoribosyl pyrophosphate (PRPP) is a key intermediate in the biosynthesis of purine and pyrimidine nucleotides, histidine, tryptophan, and cofactors NAD and NADP. Abnormal regulation of PRPP synthase (PRPS) is associated with human disorders, including Arts syndrome, retinal dystrophy, and gouty arthritis. Recent studies have demonstrated that PRPS can form filamentous cytoophidia in eukaryotes. Here, we show that PRPS forms cytoophidia in prokaryotes both in vitro and in vivo. Moreover, we solve two distinct filament structures of E. coli PRPS at near-atomic resolution using Cryo-EM. The formation of the two types of filaments is controlled by the binding of different ligands. One filament type is resistant to allosteric inhibition. The structural comparison reveals conformational changes of a regulatory flexible loop, which may regulate the binding of the allosteric inhibitor and the substrate ATP. A noncanonical allosteric AMP/ADP binding site is identified to stabilize the conformation of the regulatory flexible loop. Our findings not only explore a new mechanism of PRPS regulation with structural basis, but also propose an additional layer of cell metabolism through PRPS filamentation.
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Author Response
Reviewer #2 (Public Review):
Huan-Huan et al. investigated the structure of phosphoribosyl pyrophosphate (PRPP) synthase (PRPPS) from Escherichia coli, a highly conserved enzyme from bacteria to mammals that catalyzes the synthesis of a key common compound for several metabolic pathways. Although the structure of this enzyme was known, the mechanism of regulation by ADP and AMP remained uncharacterized. Previously, the group of JE. Wilhelm found that PRPP synthetase from different eukaryotes assembles into long filamentous structures (named cytophidia). The present study shows that PRPP synthetase filaments also form in bacteria, both in vitro and in vivo. Then, they determined the structure of two different forms of PRPPS filaments at atomic detail using cryo-electron microscopy. Combining structural data with …
Author Response
Reviewer #2 (Public Review):
Huan-Huan et al. investigated the structure of phosphoribosyl pyrophosphate (PRPP) synthase (PRPPS) from Escherichia coli, a highly conserved enzyme from bacteria to mammals that catalyzes the synthesis of a key common compound for several metabolic pathways. Although the structure of this enzyme was known, the mechanism of regulation by ADP and AMP remained uncharacterized. Previously, the group of JE. Wilhelm found that PRPP synthetase from different eukaryotes assembles into long filamentous structures (named cytophidia). The present study shows that PRPP synthetase filaments also form in bacteria, both in vitro and in vivo. Then, they determined the structure of two different forms of PRPPS filaments at atomic detail using cryo-electron microscopy. Combining structural data with mutagenesis and activity assays, they demonstrate that the enzyme is regulated differently by allosteric effectors when assembled into one filament form or the other.
The strength of the manuscript is the high-quality cryo-EM data, which allows the reconstruction of two different filament forms bound to different ligands, the identification of a new regulatory site, and the description of the movements of the regulatory loop at the active site, which either blocks the active site (in filament type B) or hampers the binding and inhibition of ADP to the allosteric site (in filament type A).
Based on the structural information, the authors designed point mutants that favor the formation of one filament type or the other. Using these mutants, the authors dissect the different responses of the two filament forms to the nucleotides that bind and regulate the reaction rate.
The authors conclude that filament formation is not needed for PRPPS activity, but that the formation of these filaments is an additional layer to fine-tune its activity.
Overall, the data are of high quality and the conclusions are of interest to understanding the significance of the organization of proteins into supramolecular membrane-less compartments.
A similar filamentous organization is expected for this enzyme in other higher eukaryotes, including humans. Defects in the human enzyme are the cause of rare congenital diseases. Based on the current data, the authors speculate that the mechanistic effect for certain pathogenic variants could be affecting the formation of the filaments.
This manuscript reveals that this enzyme is more complicated than initially expected. The newly proposed regulatory mechanism is not easy to understand, since ADP can inhibit or enhance the reaction depending on whether it binds to one regulatory site or to the other, but also by competing with ATP in the catalytic site. Some parts of the text and figures are not sufficiently clear and difficult to follow. The authors could make an effort to improve clarity and correct grammatical issues.
We would like to thank the reviewer for the informative comments on our manuscript. We have endeavored to address them as fully as possible in our revision. The manuscript has been rewritten to improve the clarity and the presentation.
Reviewer #3 (Public Review):
This work aims to investigate the role of self-assembly in the regulation of enzyme activity of E. coli PRPS (EcPRPS). EcPRPS is an important enzyme in the biosynthesis of nucleic acids and some amino acids, forms micron-sized self-assemblies in cells (cytoophidia), is allosterically inhibited by ADP, and is activated by inorganic phosphate, Pi. The authors set out to investigate the structure and function of the filamentous form of the enzyme responsible for cytoophidia formation.
The authors present two new, high-resolution cryo-EM structures of filamentous forms of EcPRPS, each assembling via unique stacking of EcPRPS hexamers. One form, type A, was formed in the presence of ATP and Mg2+, and the cryo-EM map was interpreted as containing one ADP and one R5P (ribose 5-phosphate) in the active site (as well as two Mg2+). The substrates of EcPRPS are ATP and R5P and the ADP occupies the expected ATP binding site, though neither ADP nor R5P was supplied in the experimental solution. Surprisingly, a second ADP molecule is also identified, bound near the active site at a location named site 2. In addition, one Pi is bound in the canonical allosteric site, site 1. A second type of filament (type B) was formed in the presence of Pi and contains two Pi, one in site 1 and one in the R5P binding site of the active site.
Analysis of these two structures revealed a significant change in the positioning of a segment of the enzyme, named the Regulatory Flexible (RF) loop. In the type A filament structure, with the active site occupied by ADP and R5P, the RF loop interacts with the ADP in site 2. In the type B structure, with an empty site 2, the RF loop sits in a different position and occupies the ATP binding site of the active site. The suggestion made by these observations is that the binding of ADP in site 2 stabilizes the RF loop such that ATP may bind the active site, and without ADP in site 2, the loop will block ATP from binding the active site. This suggests that ADP is not merely an allosteric inhibitor, but could also act as an activator.
As for allosteric site 1, which is occupied only by Pi in both structures, the authors suggest it binds ADP as seen in structures of homologous PRPS enzymes, and that this binding is the cause of allosteric inhibition by ADP. Structural comparisons suggest that the binding of ADP to site 1 is controlled by the position of the RF loop, which in turn can be controlled by interactions with ADP at site 2. The authors suggest that the binding of ADP to site 1 and to site 2 is mutually exclusive via this mechanism of RF positioning.
In addition to the structural analysis, a strength of the paper is the use of point mutations to investigate the effects of eliminating one or both types of filaments on enzyme activity, cell growth, and cytoophodia formation. When either one or the other type of filament form is knocked out, cytoophidia still forms, but when both types are knocked out using a double-mutant, no cytoophidia form. This suggests that both filament types form in cells and are responsible for cytoophidia formation. Effects on cellular growth were nominal, and largest only with the double-mutant, which grew much more slowly than the wild-type enzyme. At longer time points only, each single point-mutant showed faster growth than the wild-type enzyme. These results are interpreted by the authors to mean that both types of filaments form in cells and have functional consequences.
In activity assays, the double-mutant, which does not form either type of filament, showed a very high level of sensitivity to allosteric inhibition by ADP, suggesting that filament formation mitigates this to some degree (and that filament formation is not necessary for allosteric inhibition by ADP). The absence of type B and presence of type A filaments leads to lower sensitivity to allosteric inhibition by ADP: lower than wild type EcPRPS and lower than when neither filament forms. Hence the presence of the type A and absence of type B filament leads to greater enzymatic activity and less allosteric inhibition by ADP than no filamentation or when both types are present. Absence of the type A filament appears similar to the double-mutant (which does not have filament) in that it is very sensitive to ADP inhibition. The conclusion is that the type A filament mitigates allosteric inhibition by ADP, while the type B filament is allosterically inhibited by ADP (similar to the non-filamenting enzyme).
The work has a few weaknesses. First, R5P was not included in the solution used to prepare the type A filaments, yet is built into the cryoEM map. The map around the modeled R5P is not shown, making it difficult to assess this interpretation. Second, no filament structure with ADP in site 1 has been determined. Instead, the structure of a related PRPS with ADP in site 1 is shown, but the position of the RF loop in that structure does not occupy the ATP binding site as implied by the authors to be the function of this conformation (i.e. when ADP is bound in site 1).
The authors also claim that the type B filament enhances inhibition, but in fact, shows similar inhibition to the enzyme which cannot form filaments. However, when type A filaments are present, it appears that type B filaments are necessary to allow for some allosteric inhibition by ADP. Though not discussed, it may be that levels of the two types of filaments are altered to control overall enzyme activity. In addition, much of the discussion deals with interpretations about binding affinities of ligands to various sites, but all evidence used is indirect, as no binding affinities are measured directly.
Another weakness is in the investigation of site 2. The authors claim that ADP binding to site 2 enhances ATP binding in the active site, however, the mutation designed to disrupt ADP binding to site 2 results in reduced ATP in the absence of ADP, not in its presence.
Finally, a discussion of the role of Pi, and how the choice of the two filamentous forms is chosen is not addressed in the study. The authors show compelling evidence to support their coexistence in vitro and in cells, and differing activity, however, how and why one is formed over the other has yet to be uncovered.
We would like to thank the reviewer for the informative comments on our manuscript, and we have endeavored to address them as fully as possible in our revision. Specifically,
- We have added a supplementary figure showing the map around R5P.
- We did not propose when ADP bound to allosteric site 1, the RF loop occupy the ATP binding site.
- We temper our claims on type B filament in the absence of direct binding affinity measurement.
- We temper our claims on the role of ADP binding to site 2.
- We have added discussion on the role of Pi and how the choice of the two filamentous forms is chosen.
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Evaluation Summary:
The authors investigated the structure of phosphoribosyl pyrophosphate (PRPP) synthase (PRPPS) from Escherichia coli, a highly conserved enzyme from bacteria to mammals that catalyzes the synthesis of a key common compound for several metabolic pathways. Combining structural data with mutagenesis and activity assays, they demonstrate that the enzyme is regulated differently by allosteric effectors when assembled into one filament form or the other. The strength of the manuscript is the high-quality cryo-EM data, which allows the reconstruction of two different filament forms bound to different ligands.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to …
Evaluation Summary:
The authors investigated the structure of phosphoribosyl pyrophosphate (PRPP) synthase (PRPPS) from Escherichia coli, a highly conserved enzyme from bacteria to mammals that catalyzes the synthesis of a key common compound for several metabolic pathways. Combining structural data with mutagenesis and activity assays, they demonstrate that the enzyme is regulated differently by allosteric effectors when assembled into one filament form or the other. The strength of the manuscript is the high-quality cryo-EM data, which allows the reconstruction of two different filament forms bound to different ligands.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
This paper provides new insights into how polymerization into two different structures modulates the activity of the enzyme PRPS. The molecular mechanisms proposed are supported by the data, and likely to be of general interest.
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Reviewer #2 (Public Review):
Huan-Huan et al. investigated the structure of phosphoribosyl pyrophosphate (PRPP) synthase (PRPPS) from Escherichia coli, a highly conserved enzyme from bacteria to mammals that catalyzes the synthesis of a key common compound for several metabolic pathways. Although the structure of this enzyme was known, the mechanism of regulation by ADP and AMP remained uncharacterized. Previously, the group of JE. Wilhelm found that PRPP synthetase from different eukaryotes assembles into long filamentous structures (named cytophidia). The present study shows that PRPP synthetase filaments also form in bacteria, both in vitro and in vivo. Then, they determined the structure of two different forms of PRPPS filaments at atomic detail using cryo-electron microscopy. Combining structural data with mutagenesis and activity …
Reviewer #2 (Public Review):
Huan-Huan et al. investigated the structure of phosphoribosyl pyrophosphate (PRPP) synthase (PRPPS) from Escherichia coli, a highly conserved enzyme from bacteria to mammals that catalyzes the synthesis of a key common compound for several metabolic pathways. Although the structure of this enzyme was known, the mechanism of regulation by ADP and AMP remained uncharacterized. Previously, the group of JE. Wilhelm found that PRPP synthetase from different eukaryotes assembles into long filamentous structures (named cytophidia). The present study shows that PRPP synthetase filaments also form in bacteria, both in vitro and in vivo. Then, they determined the structure of two different forms of PRPPS filaments at atomic detail using cryo-electron microscopy. Combining structural data with mutagenesis and activity assays, they demonstrate that the enzyme is regulated differently by allosteric effectors when assembled into one filament form or the other.
The strength of the manuscript is the high-quality cryo-EM data, which allows the reconstruction of two different filament forms bound to different ligands, the identification of a new regulatory site, and the description of the movements of the regulatory loop at the active site, which either blocks the active site (in filament type B) or hampers the binding and inhibition of ADP to the allosteric site (in filament type A).
Based on the structural information, the authors designed point mutants that favor the formation of one filament type or the other. Using these mutants, the authors dissect the different responses of the two filament forms to the nucleotides that bind and regulate the reaction rate.
The authors conclude that filament formation is not needed for PRPPS activity, but that the formation of these filaments is an additional layer to fine-tune its activity.
Overall, the data are of high quality and the conclusions are of interest to understanding the significance of the organization of proteins into supramolecular membrane-less compartments.
A similar filamentous organization is expected for this enzyme in other higher eukaryotes, including humans. Defects in the human enzyme are the cause of rare congenital diseases. Based on the current data, the authors speculate that the mechanistic effect for certain pathogenic variants could be affecting the formation of the filaments.
This manuscript reveals that this enzyme is more complicated than initially expected. The newly proposed regulatory mechanism is not easy to understand, since ADP can inhibit or enhance the reaction depending on whether it binds to one regulatory site or to the other, but also by competing with ATP in the catalytic site. Some parts of the text and figures are not sufficiently clear and difficult to follow. The authors could make an effort to improve clarity and correct grammatical issues.
-
Reviewer #3 (Public Review):
This work aims to investigate the role of self-assembly in the regulation of enzyme activity of E. coli PRPS (EcPRPS). EcPRPS is an important enzyme in the biosynthesis of nucleic acids and some amino acids, forms micron-sized self-assemblies in cells (cytoophidia), is allosterically inhibited by ADP, and is activated by inorganic phosphate, Pi. The authors set out to investigate the structure and function of the filamentous form of the enzyme responsible for cytoophidia formation.
The authors present two new, high-resolution cryo-EM structures of filamentous forms of EcPRPS, each assembling via unique stacking of EcPRPS hexamers. One form, type A, was formed in the presence of ATP and Mg2+, and the cryo-EM map was interpreted as containing one ADP and one R5P (ribose 5-phosphate) in the active site (as well …
Reviewer #3 (Public Review):
This work aims to investigate the role of self-assembly in the regulation of enzyme activity of E. coli PRPS (EcPRPS). EcPRPS is an important enzyme in the biosynthesis of nucleic acids and some amino acids, forms micron-sized self-assemblies in cells (cytoophidia), is allosterically inhibited by ADP, and is activated by inorganic phosphate, Pi. The authors set out to investigate the structure and function of the filamentous form of the enzyme responsible for cytoophidia formation.
The authors present two new, high-resolution cryo-EM structures of filamentous forms of EcPRPS, each assembling via unique stacking of EcPRPS hexamers. One form, type A, was formed in the presence of ATP and Mg2+, and the cryo-EM map was interpreted as containing one ADP and one R5P (ribose 5-phosphate) in the active site (as well as two Mg2+). The substrates of EcPRPS are ATP and R5P and the ADP occupies the expected ATP binding site, though neither ADP nor R5P was supplied in the experimental solution. Surprisingly, a second ADP molecule is also identified, bound near the active site at a location named site 2. In addition, one Pi is bound in the canonical allosteric site, site 1. A second type of filament (type B) was formed in the presence of Pi and contains two Pi, one in site 1 and one in the R5P binding site of the active site.
Analysis of these two structures revealed a significant change in the positioning of a segment of the enzyme, named the Regulatory Flexible (RF) loop. In the type A filament structure, with the active site occupied by ADP and R5P, the RF loop interacts with the ADP in site 2. In the type B structure, with an empty site 2, the RF loop sits in a different position and occupies the ATP binding site of the active site. The suggestion made by these observations is that the binding of ADP in site 2 stabilizes the RF loop such that ATP may bind the active site, and without ADP in site 2, the loop will block ATP from binding the active site. This suggests that ADP is not merely an allosteric inhibitor, but could also act as an activator.
As for allosteric site 1, which is occupied only by Pi in both structures, the authors suggest it binds ADP as seen in structures of homologous PRPS enzymes, and that this binding is the cause of allosteric inhibition by ADP. Structural comparisons suggest that the binding of ADP to site 1 is controlled by the position of the RF loop, which in turn can be controlled by interactions with ADP at site 2. The authors suggest that the binding of ADP to site 1 and to site 2 is mutually exclusive via this mechanism of RF positioning.
In addition to the structural analysis, a strength of the paper is the use of point mutations to investigate the effects of eliminating one or both types of filaments on enzyme activity, cell growth, and cytoophodia formation. When either one or the other type of filament form is knocked out, cytoophidia still forms, but when both types are knocked out using a double-mutant, no cytoophidia form. This suggests that both filament types form in cells and are responsible for cytoophidia formation. Effects on cellular growth were nominal, and largest only with the double-mutant, which grew much more slowly than the wild-type enzyme. At longer time points only, each single point-mutant showed faster growth than the wild-type enzyme. These results are interpreted by the authors to mean that both types of filaments form in cells and have functional consequences.
In activity assays, the double-mutant, which does not form either type of filament, showed a very high level of sensitivity to allosteric inhibition by ADP, suggesting that filament formation mitigates this to some degree (and that filament formation is not necessary for allosteric inhibition by ADP). The absence of type B and presence of type A filaments leads to lower sensitivity to allosteric inhibition by ADP: lower than wild type EcPRPS and lower than when neither filament forms. Hence the presence of the type A and absence of type B filament leads to greater enzymatic activity and less allosteric inhibition by ADP than no filamentation or when both types are present. Absence of the type A filament appears similar to the double-mutant (which does not have filament) in that it is very sensitive to ADP inhibition. The conclusion is that the type A filament mitigates allosteric inhibition by ADP, while the type B filament is allosterically inhibited by ADP (similar to the non-filamenting enzyme).
The work has a few weaknesses. First, R5P was not included in the solution used to prepare the type A filaments, yet is built into the cryoEM map. The map around the modeled R5P is not shown, making it difficult to assess this interpretation. Second, no filament structure with ADP in site 1 has been determined. Instead, the structure of a related PRPS with ADP in site 1 is shown, but the position of the RF loop in that structure does not occupy the ATP binding site as implied by the authors to be the function of this conformation (i.e. when ADP is bound in site 1).
The authors also claim that the type B filament enhances inhibition, but in fact, shows similar inhibition to the enzyme which cannot form filaments. However, when type A filaments are present, it appears that type B filaments are necessary to allow for some allosteric inhibition by ADP. Though not discussed, it may be that levels of the two types of filaments are altered to control overall enzyme activity. In addition, much of the discussion deals with interpretations about binding affinities of ligands to various sites, but all evidence used is indirect, as no binding affinities are measured directly.
Another weakness is in the investigation of site 2. The authors claim that ADP binding to site 2 enhances ATP binding in the active site, however, the mutation designed to disrupt ADP binding to site 2 results in reduced ATP in the absence of ADP, not in its presence.
Finally, a discussion of the role of Pi, and how the choice of the two filamentous forms is chosen is not addressed in the study. The authors show compelling evidence to support their coexistence in vitro and in cells, and differing activity, however, how and why one is formed over the other has yet to be uncovered.
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