Structural basis of dynamic P5CS filaments
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Evaluation Summary:
This study describes the structures of filamentous forms of the enzyme P5CS from Drosophila, an enzyme important in the synthetic pathway for proline and ornithine. Three CryoEM experiments by the authors have resulted in structures of several apo and substrate-bound conformational states of the enzyme. The structures suggest that filamentation by P5CS may serve the purpose to facilitate the two-step enzymatic reaction by limiting the free diffusion of the reaction intermediate, the product of the first catalytic step and the substrate of the second, thereby increasing the reaction rate of the rate-limiting step (the second step) of the enzymatic reaction.
(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
The bifunctional enzyme Δ 1 -pyrroline-5-carboxylate synthase (P5CS) is vital to the synthesis of proline and ornithine, playing an essential role in human health and agriculture. Pathogenic mutations in the P5CS gene (ALDH18A1) lead to neurocutaneous syndrome and skin relaxation connective tissue disease in humans, and P5CS deficiency seriously damages the ability to resist adversity in plants. We have recently found that P5CS forms cytoophidia in vivo and filaments in vitro. However, it is difficult to appreciate the function of P5CS filamentation without precise structures. Using cryo-electron microscopy, here we solve the structures of Drosophila full-length P5CS in three states at resolution from 3.1 to 4.3 Å. We observe distinct ligand-binding states and conformational changes for the GK and GPR domains, respectively. Divergent helical filaments are assembled by P5CS tetramers and stabilized by multiple interfaces. Point mutations disturbing those interfaces prevent P5CS filamentation and greatly reduce the enzymatic activity. Our findings reveal that filamentation is crucial for the coordination between the GK and GPR domains, providing a structural basis for the catalytic function of P5CS filaments.
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Author Response
Reviewer #2 (Public Review):
P5CS is part of the proline and ornithine synthesis pathway, and catalyzes the reaction of L-glutamate to glutamate-gamma-semialdehyde in an ATP and NADPH dependent manner. Mutations in this enzyme lead to human disease and issues in agriculturally important plants. The authors present structures from three CryoEM analyses at moderate resolution (3.1-4.2 Agstrom) of P5CS showing filamentous structures in the presence of L-glutamate, L-glutamate and ATPgammaS, and L-glutamate, ATP, and NADPH in an effort to understand the enzyme mechanism and role of enzyme filamentation. Filamentation of enzymes is an important and newly appreciated mechanism of enzyme regulation, and this work provides important new information on how filamentation may enhance the enzymatic catalysis by P5CS. Large …
Author Response
Reviewer #2 (Public Review):
P5CS is part of the proline and ornithine synthesis pathway, and catalyzes the reaction of L-glutamate to glutamate-gamma-semialdehyde in an ATP and NADPH dependent manner. Mutations in this enzyme lead to human disease and issues in agriculturally important plants. The authors present structures from three CryoEM analyses at moderate resolution (3.1-4.2 Agstrom) of P5CS showing filamentous structures in the presence of L-glutamate, L-glutamate and ATPgammaS, and L-glutamate, ATP, and NADPH in an effort to understand the enzyme mechanism and role of enzyme filamentation. Filamentation of enzymes is an important and newly appreciated mechanism of enzyme regulation, and this work provides important new information on how filamentation may enhance the enzymatic catalysis by P5CS. Large conformational changes are seen in the enzyme between the different structures, representing different stages of the enzymatic reaction. The enzyme forms tetramers which then assemble into left-handed helical filaments with 68 degrees a rise of 60 Angstrom (roughly the height of a tetramer) between adjacent tetramers. The authors suggest, base on the structure of P5CS with L-glutamine and a structure with G5P and ADP (the product of the first reaction between ATP and L-glutamine) that conformational changes upon ATP binding lead to a shift of reactants L-glutamate and ATP towards each other, creating an active state for the reaction of the first enzymatic step. While an interesting suggestion, it should be noted that the structure with ATP is not known, and this suggestion is conjecture based on a structure with no ATP and with ADP. It is possible that the structure with ATP is yet distinct. Binding of NADPH further induces a conformational change bringing the NADPH towards residue C598 (a residue apparently important for enzyme function, though a figure showing NADPH and C598 together is not given, and no details on what function C598 perform is discussed). The authors show that the filament accommodates all conformations, and suggest that the filament is dynamic, performing multiple rounds without depolymerization. This is an exciting possibility, but it should be noted that the authors do not have direct evidence that a depolymerization intermediate step is required (structures are of the final states, not the intermediate). The authors find in several of their new structures that an interface is formed by residues F642-P644 (which are distant from the active sites) in GPR domains of adjacent P5CS tetramers in the filament. They show that this interaction is responsible for the filamentation as a point mutation in the segment disrupts both filamentation and enzyme activity (which also shows the importance of filamentation to enzyme activity). They also show that a contact between adjacent GK domains forms a "hook" structure in some conformational states of the enzymes, which they suggest is formed upon ATP binding (though their structures show only ADP binding, not ATP). They find that mutations in this site do not disrupt filamentation in the apo and L-glutamate bound states, but found that addition of ATP results in depolymerization, and addition of NADPH induces the formation of filaments but that are much shorter than those of the wild type enzyme. The mutation in the hook region also strongly reduces enzyme activity. They conclude that ATP therefore initiates the reaction in the GK domain, and triggers the hook structure to stabilize the conformation necessary for the next step of the reaction. The authors speculate that the filament couples the reactions catalyzed at the two domains by a channeling effect - the intermediate of the two step reaction and product of the first step, G5P, is produced in an active site 60 Angstroms away from the active site of the second catalytic step. Both active sites face the interior of the filament, and therefore the filament may create a microenvironment to allow limited diffusion of G5P so that it may more efficiently diffuse from one active site to the other. In addition to showing new details of the enzymatic mechanism of P5CS, this work also contributes to our understanding of how filaments can facilitate enzymatic reactions (possibly via a caging effect). Finally, the authors do not discuss their structure in comparison to the known structure of human P5CS, which is an important omission.
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. Specifically, we have made the following revisions:
- We temper our claims in the absence of an ATP-bound structure.
- The function of C598 has now been discussed.
- We temper our claims in the absence of direct evidence of the requirement of a depolymerization intermediate step.
- We have added text and figures to compare the structure of P5CS in Drosophila and that in human.
Reviewer #3 (Public Review):
Jiale Zhong et al. investigated the structure of pyrroline-5 carboxylate synthase (P5CS) from Drosophila, a bifunctional enzyme composed of fused glutamate kinase (GK) and glutamyl phosphate reductase (GPR) domains. The crystal structure of human P5CS GPR domain was available in the Protein Data Bank and the structure of prokaryotic GKs had been previously reported, but there was no structure available for the full-length P5CS. Previously, the authors had shown that P5CS assembles into long filamentous structures both in vivo and in vitro. Now, they reported the detailed structural analysis of the full-length P5CS, showing that the protein folds into tetramers that assemble into a spiral filament.
The strength of the manuscript is the high-quality cryo-EM data, which allow the reconstruction of the protein filament in three different ligand-bound states at various resolutions: i) with glutamate in the GK domain and GPR free of ligands (4 Å); ii) with the product glutamate 5-phosphate in the GPR domain (it is unclear what is the content of GK in this structure) (4.2 Å); and iii) with glutamate 5-phosphate, ADP and Mg2+ in the GK and the GPR domain either free or bound to NADPH (3.6 Å). The study shows the structures of both enzymatic domains and provides some details of ligand binding and associated conformational changes.
Importantly, the structure reveals the contacts between P5CS tetramers along the filament axis. Based on this information, the authors designed point mutants that disrupt these contacts along the filament and showed that they also reduced severely the enzymatic activity. Thus, the authors conclude that filament formation is essential for P5CS activity. Given the distance between the GK and GPR active sites, they speculate that the filament grooves create a half-open chamber that accumulates the product of the GK reaction (glutamyl phosphate) and favors its diffusion to the GPR domains on the outer part of the filament. Overall, the data are of high-quality and the conclusions are of high interest to understand how the organization of proteins into supramolecular membraneless compartments regulate their activity.
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 proposed a mechanistic effect for one pathogenic variant that would affect the interaction of the tetramers along the filament.
The study falls short in addressing the catalytic mechanisms as well as the possible communication/regulation between protein domains within the tetramer and along the filament. Also, the study does not speculate on how the formation of the P5CS filament could depend on the interaction of the enzyme with CTP synthase, as was reported by the authors in a previous article.
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 discussion regarding the catalytic mechanisms and possible communication/regulation between protein domains within the tetramer and along the filament.
- The structural basis for the interaction between P5CS and CTPS is not the focus of this study. It will be an interesting topic for future studies.
Reviewer #4 (Public Review):
This paper reports the cryo-EM structures of Drosophila P5CS, an enzyme important in amino acid metabolism. This group had previously described P5CS filaments in Drosophila, and here show how the filaments are assembled. Overall, the paper describes structural changes that occur upon binding of substrates and reaction intermediates, making a strong case for a conformational cycle that involves some loop movements that will likely be of interest to researchers interested in the catalytic mechanism of P5CS. Importantly, the work shows that these movements occur in the context of the assembled filament. Point mutants that block filament assembly have reduce catalytic rates, suggesting that a role of the filament is to increase enzyme activity.
The cryo-EM reconstructions appear to be well executed, and the conclusions drawn are consistent with the reported resolutions of the structures. The structures clearly illustrate how filaments are assembled and that ligands induce conformational changes within the enzyme. My major concern with the paper is the limited mechanistic insight into: 1) the role of filaments in regulating P5CS activity, and 2) the role of conformational changes within the enzyme in driving the catalytic cycle. That is, there is no clear connection between the conformational changes observed on ligand binding and the catalytic mechanism, and no clear explanation for how filaments may increase enzyme activity.
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 made the following revisions:
- We have added discussion regarding the role of filaments in regulating P5CS activity.
- We have added discussion regarding the role of conformational changes within the enzyme in driving the catalytic cycle.
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Evaluation Summary:
This study describes the structures of filamentous forms of the enzyme P5CS from Drosophila, an enzyme important in the synthetic pathway for proline and ornithine. Three CryoEM experiments by the authors have resulted in structures of several apo and substrate-bound conformational states of the enzyme. The structures suggest that filamentation by P5CS may serve the purpose to facilitate the two-step enzymatic reaction by limiting the free diffusion of the reaction intermediate, the product of the first catalytic step and the substrate of the second, thereby increasing the reaction rate of the rate-limiting step (the second step) of the enzymatic reaction.
(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 …
Evaluation Summary:
This study describes the structures of filamentous forms of the enzyme P5CS from Drosophila, an enzyme important in the synthetic pathway for proline and ornithine. Three CryoEM experiments by the authors have resulted in structures of several apo and substrate-bound conformational states of the enzyme. The structures suggest that filamentation by P5CS may serve the purpose to facilitate the two-step enzymatic reaction by limiting the free diffusion of the reaction intermediate, the product of the first catalytic step and the substrate of the second, thereby increasing the reaction rate of the rate-limiting step (the second step) of the enzymatic reaction.
(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):
I have confined my remarks purely to the cryo-EM, image analysis and three-dimensional reconstruction, and leave the evaluation of the significance of the work to other reviewers. I had no concerns about the technical aspects of the work, and the resolution statements appear very reasonable. At this resolution there should be no ambiguities in the atomic models that have been generated.
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Reviewer #2 (Public Review):
P5CS is part of the proline and ornithine synthesis pathway, and catalyzes the reaction of L-glutamate to glutamate-gamma-semialdehyde in an ATP and NADPH dependent manner. Mutations in this enzyme lead to human disease and issues in agriculturally important plants. The authors present structures from three CryoEM analyses at moderate resolution (3.1-4.2 Agstrom) of P5CS showing filamentous structures in the presence of L-glutamate, L-glutamate and ATPgammaS, and L-glutamate, ATP, and NADPH in an effort to understand the enzyme mechanism and role of enzyme filamentation. Filamentation of enzymes is an important and newly appreciated mechanism of enzyme regulation, and this work provides important new information on how filamentation may enhance the enzymatic catalysis by P5CS. Large conformational changes …
Reviewer #2 (Public Review):
P5CS is part of the proline and ornithine synthesis pathway, and catalyzes the reaction of L-glutamate to glutamate-gamma-semialdehyde in an ATP and NADPH dependent manner. Mutations in this enzyme lead to human disease and issues in agriculturally important plants. The authors present structures from three CryoEM analyses at moderate resolution (3.1-4.2 Agstrom) of P5CS showing filamentous structures in the presence of L-glutamate, L-glutamate and ATPgammaS, and L-glutamate, ATP, and NADPH in an effort to understand the enzyme mechanism and role of enzyme filamentation. Filamentation of enzymes is an important and newly appreciated mechanism of enzyme regulation, and this work provides important new information on how filamentation may enhance the enzymatic catalysis by P5CS. Large conformational changes are seen in the enzyme between the different structures, representing different stages of the enzymatic reaction. The enzyme forms tetramers which then assemble into left-handed helical filaments with 68 degrees a rise of 60 Angstrom (roughly the height of a tetramer) between adjacent tetramers. The authors suggest, base on the structure of P5CS with L-glutamine and a structure with G5P and ADP (the product of the first reaction between ATP and L-glutamine) that conformational changes upon ATP binding lead to a shift of reactants L-glutamate and ATP towards each other, creating an active state for the reaction of the first enzymatic step. While an interesting suggestion, it should be noted that the structure with ATP is not known, and this suggestion is conjecture based on a structure with no ATP and with ADP. It is possible that the structure with ATP is yet distinct. Binding of NADPH further induces a conformational change bringing the NADPH towards residue C598 (a residue apparently important for enzyme function, though a figure showing NADPH and C598 together is not given, and no details on what function C598 perform is discussed). The authors show that the filament accommodates all conformations, and suggest that the filament is dynamic, performing multiple rounds without depolymerization. This is an exciting possibility, but it should be noted that the authors do not have direct evidence that a depolymerization intermediate step is required (structures are of the final states, not the intermediate). The authors find in several of their new structures that an interface is formed by residues F642-P644 (which are distant from the active sites) in GPR domains of adjacent P5CS tetramers in the filament. They show that this interaction is responsible for the filamentation as a point mutation in the segment disrupts both filamentation and enzyme activity (which also shows the importance of filamentation to enzyme activity). They also show that a contact between adjacent GK domains forms a "hook" structure in some conformational states of the enzymes, which they suggest is formed upon ATP binding (though their structures show only ADP binding, not ATP). They find that mutations in this site do not disrupt filamentation in the apo and L-glutamate bound states, but found that addition of ATP results in depolymerization, and addition of NADPH induces the formation of filaments but that are much shorter than those of the wild type enzyme. The mutation in the hook region also strongly reduces enzyme activity. They conclude that ATP therefore initiates the reaction in the GK domain, and triggers the hook structure to stabilize the conformation necessary for the next step of the reaction. The authors speculate that the filament couples the reactions catalyzed at the two domains by a channeling effect - the intermediate of the two step reaction and product of the first step, G5P, is produced in an active site 60 Angstroms away from the active site of the second catalytic step. Both active sites face the interior of the filament, and therefore the filament may create a microenvironment to allow limited diffusion of G5P so that it may more efficiently diffuse from one active site to the other. In addition to showing new details of the enzymatic mechanism of P5CS, this work also contributes to our understanding of how filaments can facilitate enzymatic reactions (possibly via a caging effect). Finally, the authors do not discuss their structure in comparison to the known structure of human P5CS, which is an important omission.
-
Reviewer #3 (Public Review):
Jiale Zhong et al. investigated the structure of pyrroline-5 carboxylate synthase (P5CS) from Drosophila, a bifunctional enzyme composed of fused glutamate kinase (GK) and glutamyl phosphate reductase (GPR) domains. The crystal structure of human P5CS GPR domain was available in the Protein Data Bank and the structure of prokaryotic GKs had been previously reported, but there was no structure available for the full-length P5CS. Previously, the authors had shown that P5CS assembles into long filamentous structures both in vivo and in vitro. Now, they reported the detailed structural analysis of the full-length P5CS, showing that the protein folds into tetramers that assemble into a spiral filament.
The strength of the manuscript is the high-quality cryo-EM data, which allow the reconstruction of the protein …
Reviewer #3 (Public Review):
Jiale Zhong et al. investigated the structure of pyrroline-5 carboxylate synthase (P5CS) from Drosophila, a bifunctional enzyme composed of fused glutamate kinase (GK) and glutamyl phosphate reductase (GPR) domains. The crystal structure of human P5CS GPR domain was available in the Protein Data Bank and the structure of prokaryotic GKs had been previously reported, but there was no structure available for the full-length P5CS. Previously, the authors had shown that P5CS assembles into long filamentous structures both in vivo and in vitro. Now, they reported the detailed structural analysis of the full-length P5CS, showing that the protein folds into tetramers that assemble into a spiral filament.
The strength of the manuscript is the high-quality cryo-EM data, which allow the reconstruction of the protein filament in three different ligand-bound states at various resolutions: i) with glutamate in the GK domain and GPR free of ligands (4 Å); ii) with the product glutamate 5-phosphate in the GPR domain (it is unclear what is the content of GK in this structure) (4.2 Å); and iii) with glutamate 5-phosphate, ADP and Mg2+ in the GK and the GPR domain either free or bound to NADPH (3.6 Å). The study shows the structures of both enzymatic domains and provides some details of ligand binding and associated conformational changes.
Importantly, the structure reveals the contacts between P5CS tetramers along the filament axis. Based on this information, the authors designed point mutants that disrupt these contacts along the filament and showed that they also reduced severely the enzymatic activity. Thus, the authors conclude that filament formation is essential for P5CS activity. Given the distance between the GK and GPR active sites, they speculate that the filament grooves create a half-open chamber that accumulates the product of the GK reaction (glutamyl phosphate) and favors its diffusion to the GPR domains on the outer part of the filament. Overall, the data are of high-quality and the conclusions are of high interest to understand how the organization of proteins into supramolecular membraneless compartments regulate their activity.
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 proposed a mechanistic effect for one pathogenic variant that would affect the interaction of the tetramers along the filament.
The study falls short in addressing the catalytic mechanisms as well as the possible communication/regulation between protein domains within the tetramer and along the filament. Also, the study does not speculate on how the formation of the P5CS filament could depend on the interaction of the enzyme with CTP synthase, as was reported by the authors in a previous article.
-
Reviewer #4 (Public Review):
This paper reports the cryo-EM structures of Drosophila P5CS, an enzyme important in amino acid metabolism. This group had previously described P5CS filaments in Drosophila, and here show how the filaments are assembled. Overall, the paper describes structural changes that occur upon binding of substrates and reaction intermediates, making a strong case for a conformational cycle that involves some loop movements that will likely be of interest to researchers interested in the catalytic mechanism of P5CS. Importantly, the work shows that these movements occur in the context of the assembled filament. Point mutants that block filament assembly have reduce catalytic rates, suggesting that a role of the filament is to increase enzyme activity.
The cryo-EM reconstructions appear to be well executed, and the …
Reviewer #4 (Public Review):
This paper reports the cryo-EM structures of Drosophila P5CS, an enzyme important in amino acid metabolism. This group had previously described P5CS filaments in Drosophila, and here show how the filaments are assembled. Overall, the paper describes structural changes that occur upon binding of substrates and reaction intermediates, making a strong case for a conformational cycle that involves some loop movements that will likely be of interest to researchers interested in the catalytic mechanism of P5CS. Importantly, the work shows that these movements occur in the context of the assembled filament. Point mutants that block filament assembly have reduce catalytic rates, suggesting that a role of the filament is to increase enzyme activity.
The cryo-EM reconstructions appear to be well executed, and the conclusions drawn are consistent with the reported resolutions of the structures. The structures clearly illustrate how filaments are assembled and that ligands induce conformational changes within the enzyme. My major concern with the paper is the limited mechanistic insight into: 1) the role of filaments in regulating P5CS activity, and 2) the role of conformational changes within the enzyme in driving the catalytic cycle. That is, there is no clear connection between the conformational changes observed on ligand binding and the catalytic mechanism, and no clear explanation for how filaments may increase enzyme activity.
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