Tankyrase 1 (TNKS1) and tankyrase 2 (TNKS2) are two homologous proteins that are gaining increasing importance due to their implication in multiple pathways and diseases such as cancer. TNKS1/2 interact with a large variety of substrates through the ankyrin (ANK) domain, which recognizes a sequence present in all the substrates of tankyrase, called Tankyrase Binding Motif (TBM). One of the main functions of tankyrases is the regulation of protein stability through the process of PARylation-dependent ubiquitination (PARdU). Nonetheless, there are other functions less studied that are also essential in order to understand the role of tankyrases in many pathways. In this review, we concentrate in different tankyrase substrates and we analyze in depth the biological consequences derived of their interaction with TNKS1/2. We also examine the concept of both canonical and non-canonical TBMs and finally, we focus on the information about the role of TNKS1/2 in different tumor context, along with the benefits and limitations of the current TNKS inhibitors targeting the catalytic PARP domain and the novel strategies to develop inhibitors against the ankyrin domain. Available data indicates the need for further deepening in the knowledge of tankyrases to elucidate and improve the current view of the role of these PARP family members and get inhibitors with a better therapeutic and safety profile. TNKS is located on chromosome 8, contains 1327 amino acids and its primary structure can be classified into four different domains: The C-terminal catalytic PARP domain that mediates the PAR addition to its substrates, an sterile alpha module (SAM) responsible for the homo- and hetero-oligomer formation, the ankyrin (ANK) domain divided in 5 clusters (ARC 1–5) which serve as the substrate binding site, and the His, Pro and Ser (HPS) rich domain with unknown function at the N-terminus [TNKS2 is situated on chromosome 10, contains 1166 amino acids and presents a similar organization to TNKS. Both catalytic domains are highly conserved, with 94% homology; the SAM domain has 74% similarity and the ANK domain shares 83% identity. However, TNKS2 present a different N-terminal region, lacking the HPS domain [BRCA1/2 mutations [Tankyrase 1 (TNKS1) and tankyrase 2 (TNKS2) are two homologous proteins that belong to the Poly (ADP-ribose) polymerases (PARPs) family , a group of 17 members that catalyze the addition of ADP-ribose molecules into a large and variety amount of proteins, causing a reversible posttranslational modification (PTM) commonly named ADP-ribosylation . TNKS isterminus , 5. The terminus . TNKS2 iTNKS1 has been the best studied of the two isoforms due to its greater abundance. Nevertheless, since their discover, tankyrases have been found to be involved in several functions  such as One of the best characterized functions of tankyrases is the regulation of protein stability via proteasomal degradation. PARylation by tankyrase is tightly linked to ubiquitination by an E3 ubiquitin ligase called RNF146 (RING finger protein 146) or Iduna. Tankyrases recognize their binding partners and PARylate them through their ANK domain and PARP domain, respectively. Then, RNF146 recognizes PARylated proteins by its WWE domain, which specifically recognizes the iso-ADP-ribose moiety present only in PAR chains Fig. a.Fig. 2The telomeric repeat-binding factor 1 or TRF1 is a member of the six-subunit shelterin complex responsible for the protection and replication of telomeres. This factor contains a TBM indispensable for the recognition by tankyrase , 32 (TabRING finger protein 146 (RNF146) or Iduna is a RING E3 ubiquitin ligase responsible for the protein ubiquitination. This posttranslational modification is a tight regulated process and is implicated in a large variety of cellular functions carried out by a complex of proteins. It is necessary an E1 ubiquitin activating enzyme, an E2 ubiquitin conjugating enzyme and an E3 ubiquitin ligase, which confers the substrate specificity , 47. Theβ-TrCP in order to target it for proteasomal degradation [Axis inhibition 1 and 2 (Axin1/2) are considered negative regulators of the canonical Wnt pathway , 51. β-cradation . Axin waradation , 41, 47.radation , 50.Phosphatidylinositol -trisphosphate phosphatase or PTEN is an enzyme that acts as a negative regulator of the PI3K-Akt signaling pathway . PTEN meDespite our knowledge about tankyrases is increasing, some aspects remain unclear. One of them is how regulatory domains control TNKS1/2 catalytic activity. The ANK domain of TNKS1/2 is responsible for the interactions with a large variety of functional and structural binding partners , 32. Mos, and its activation is promoted by the formation of a complex with the proteins STRAD and MO25 [Liver kinase B1 or LKB1 is a serine/threonine kinase implicated in cellular energy homeostasis and is also the major upstream activator of AMPK . LKB1 reand MO25 . The binand MO25  and MO25and MO25 . Phosphoand MO25 . Recentland MO25 .GDP-Mannose-4, 6-Dehydratase or GMD is an enzyme necessary for de novo synthesis of fucose, a monosaccharide present in several glycoproteins and glycolipids and also the major donor in all fucosylation reactions , 72. GDPThe role of tankyrases in the cellular homeostasis is not only defined by the catalytic ability to PARylate a great number of proteins. TNKS1/2 can form multimeric structures with other TNKS1/2 via the SAM domain, acting as scaffolding proteins which promote the interaction between different substrates of tankyrases through the binding of their multiple targets to the ankyrin domain. The first evidence of this new role was the observation that tankyrases also participates in Wnt/β-catenin pathway independently of the PARP activity , 76, 77.TNKS/2 genes [TNKS and TNKS2 mutations  and melanoma, colorectal and prostate  , adenosine subsite (AS)  or dual binders (DB) if they recognize both nicotinamide and adenosine subsites (TNKS656)  is related to multiple diseases, including obesity , diabete/2 genes , 91. Forons Fig. a and c, sms Fig. a and c. nes Fig. b and d. in colon , 93 and in colon , 94, butin colon , breast in colon , ovarianin colon  and livein colon . The firin colon . Alteratin colon –103. TheAPC or AXIN or of the CTNNB1 oncogene. These genetic alterations cause the accumulation of active β-catenin protein within the cell nucleus and the subsequent activation of its target genes that promote cell proliferation and malignancy [Colorectal cancer is the third most common cancer and the second leading cause of cancer death worldwide  . The Wnlignancy , 118. Nolignancy , 102. ToG007-LK binds specifically to the adenosine subsite of the TNKS catalytic domain , 120. ThThis inhibitor was found to recognize the adenosine subsite of TNKS and stabilize and reduce Axin and active nuclear β-catenin levels, respectively. Besides, K-756 was reported to induce cell growth inhibition in colon COLO 320DM and SW403 lines, whose proliferation is dependent on the Wnt/β-catenin signaling due to a short truncating mutation in APC component. The efficacy of K-756 was shown in an oral-administered DLD-1/TCF-Luc colon cancer xenograft mice model, proving the antiproliferative activity of K-756 .RK-582 binds to the nicotinamide subsite of tankyrases with a 200-fold selectivity against other PARP members and presents a remarkable level of efficacy at low doses. This new inhibitor blockades the Wnt/β-catenin pathway by elevating Axin2 and reducing active nuclear β-catenin levels while exhibits a potent tumor growth inhibitory effect in the mouse COLO-320DM xenograft model with oral or intraperitoneal administration , 115.The binding mode of LZZ-02 is similar to XAV939, since both recognize the nicotinamide pocket of TNKS. However, LZZ-02 forms more additional interactions with the residues near the border of the nicotinamide subsite in TNKS, which could improve the binding position and also enhance the inhibitory activity. LZZ-02 blockades the proliferation of APC mutated SW480 and DLD1 cells through the inhibition of the Wnt/β-catenin signaling. In addition, this inhibitor showed a robust antitumor effect in a DLD1-derived colon tumor xenograft model, pointing out the promising therapeutic application .Despite tankyrase inhibitors have shown multiple benefits both in vitro and in vivo models; there are still remaining issues that have to be elucidated. Firstly, the status of the APC mutation, which has been considered as a potential biomarker for sensitivity to TNKS inhibitors in colon cancer . Most coLung cancer is the most diagnosed cancer and one of the foremost causes of cancer-associated mortality worldwide . Non-smaIWR-1 has the ability to inhibit TNKS binding the adenosine binding pocket with a specificity of 100-fold than XAV939 for TNKS enzymes over other PARPs , 125. ThAZ1366 occupies the nicotinamide binding site of the PARP catalytic domain and works stabilizing Axin1 and decreasing the mRNA levels of different β-catenin targets. The most common subtype of NSCLC is associated with activating mutations in the EGFR gene. AZ1366 has shown a synergistic effect suppressing NSCLC proliferation in combination with EGFR inhibitors. When analyzing Wnt-responsive tumors in mice, an improvement in tumor control and survival using this combined inhibition was found compared to the single use of EGFR inhibitors .This inhibitor is based on the optimization of XAV939, although it is consider a dual binder (DB) inhibitor since it binds both nicotinamide and adenoside subsites . The effThe role of tankyrases in the development of colon and lung cancer and the use of TNKS inhibitors are better known. Nevertheless, there is a growing knowledge concerning the use of TNKS inhibitors in other types of cancer Table . The TNKAll the current TNKS inhibitors are targeting the catalytic PARP domain. Tankyrase catalytic inhibition triggers the accumulation of many substrates and also TNKS1/2 . RecentlIn view of the current scientific literature, there is still a long way to go to study in a more precise and systematic manner the implications of TNKS1/2 in the development of cancer. The role of tankyrases in the optimization of the Wnt/β-catenin pathway, key in the development of colorectal cancer and with implications in numerous types of cancer, is clear. Therefore, it is necessary to consolidate clinical studies and global data analysis to improve our current view of the role of TNKS1/2 in the origin of the alterations that lead to tumor development and the use of new therapeutic agents based on inhibition by tankyrases. In addition, it will be necessary in the future to deepen the knowledge of the TNKS interactoma in normal and tumor cells to define and identify new TNKS substrates. Future investigations will be also needed to elucidate the new functions of tankyrases that will allow to desing pharmacological inhibitors disrupting the TBM-ARC interaction independtly of the poly (ADP-ribosylation) activity.


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.The bifunctional enzyme Δ Kinetic analysis suggest that bacterial GK and GPR form a complex , here we solve the structures of full-length P5CS in multiple filamentous states. We reconstruct The P5CS molecule contains two domains, GK and GPR, catalyzing the first and second steps in the biosynthesis of proline from glutamate. The GK domain catalyzes glutamate phosphorylation, and the GPR domain catalyzes the NADPH-dependent reduction of γ-glutamyl phosphate (G5P) to glutamate-γ-semialdehyde (GSA). The end product P5C, formed by a spontaneous cyclization reaction of GSA , will beDrosophila melanogaster full-length P5CS proteins. First, we analyzed the APO and substrate-bound states of P5CS by negative staining (Drosophila P5CS in the APO state is hard to form filaments at low concentrations (<0.05 μM). The addition of glutamate to the P5CS samples induces micron-scale filaments , (2) glutamate and ATPγS (P5CSGlu/ATPγS), and (3) glutamate, ATP, and NADPH (P5CSMix) were imaged in cryo-EM for single-particle analysis (SPA). Long and flexible filaments of P5CS were observed under all the three conditions. After 3D classification and 3D reconstruction, the electron density maps of the P5CSGlu, P5CSGlu/ATPγS, and P5CSMix filaments reached resolutions of 4.0 Å, 4.2 Å, and 3.6 Å, respectively  the glutamate-binding domain (GBD) and (2) the ATP-binding domain (ABD) at the GK domain; (3) the NADPH-binding domain (NBD), (4) the catalytic domain (CD), and (5) the oligomerization domain (OD) at the GPR domain . In the Mix filament to display the details  and all substrates were in excess during our sample preparation. Thus, no ligand was modeled in the GK domain structure of the P5CSGlu/ATPγS filament.We obtained a structure of the GK domain with the binding of glutamate in the P5CSfilament  and a sefilament . In the ensities . The GK filament . We specMix filament, and glutamate at the binding site is converted into the intermediate G5P. At ABD of the P5CSMix filament, the phosphate donor ATP becomes an ADP, associating with an Mg2+  in the P5CSMix filament  at region I of the P5CSfilament . This hefilament  and 3.Glu and P5CSMix filaments  of GBD shifting greatly by approximately 3 Å away . The funilaments . The GK + to catalyze the conversion of various aldehydes into their corresponding carboxylic acids. Many studies on ALDHs have shown that a conserved residue cysteine acts as the active site of nucleophile, forming thiohemiacetal intermediate with substrate  superfamily. ALDH family uses NAD(P)ubstrate . CuriousGlu filament, no ligand binds to the GPR domain -released states shows that the overall structures of the GPR domain are similar . The str2-β5-α2) architecture, which is known as the Rossmann-like fold for dinucleotide binding  binding, the residue R525 interacts with the adenine moiety. This interaction transforms the ike fold . Meanwhiike fold  and slidike fold . We hypoike fold . This trike fold .In our models, unliganded, G5P-binding and NADP(H)-released states represent as open conformation, and the NADP(H)-binding state represents as closed conformation. We propose that the P5CS filament accommodates the GPR domain in both open and closed conformations. Therefore, recurring transformations between these two conformations are essential for the catalytic cycle at the GPR domain.Glu, P5CSGlu/ATPγS, and P5CSMix filaments, neighboring GPR dimers interact with each other and form a helical structure. The interaction formed between F642-P644 of the contact loops in adjacent GPR domains, which appears as a CH/Pi interaction , nucleoside is important for the conformational change of the AAK domain, and the structures are similar when bound by ADP or AMPPNP  catalyzes NADPH-dependent reductive dephosphorylation of β-aspartyl phosphate to aspartate-β-semialdehyde . The GPRormation . Thus, w GSA/P5C . G5P canAs mentioned in the ‘Results’ section, we observed that mutated residues R124A and F642A do not directly participate in the active sites, while they are crucial for filamentation. This suggests that the P5CS filamentation couples the reaction catalyzed between the GK domain and GPR domain through transferring unstable intermediate G5P . ConsideDrosophila dynamic P5CS filament. In this proposed model, spontaneous filamentation occurs at the APO state, and elongation of P5CS filament is associated with the binding of glutamate. Upon the binding of glutamate, the binding pocket at the GK domain is bound by ATP; subsequently, conformational changes facilitate the formation of a hook structure and phosphorylation of glutamate, which produces G5P. When products of the GK domain dissociate from the active site, G5P would be trapped by the channel and chamber within the filament and further captured by the GPR domain. Next, NADPH binds to the GPR domain, triggering the conformational change into closed conformation, which brings the NADPH towards the catalytic residue C598 and facilitates the reaction. After this reaction, NADP+ and GSA will be released, and the GPR domain returns to its open conformation  is one of the causes of hyperammonemia, neurocutaneous syndromes, and motor neuron syndrome  cells overnight at 16°C after induction with 0.1 mM IPTG at OD600 range of 0.6–0.8. The remainder of purification was performed at 4°C. The harvested cells were sonicated under ice and purified by Ni-NTA agarose beads (QIAGEN) in lysis buffer . After in-column washing with lysis buffer, the proteins were eluted with elution buffer , peak fractions were treated with SUMO protease for 1 hr at 8°C. The P5CS proteins were further purified through HiLoad 16/600 Superdex 200pg gel-filtration chromatography  in column buffer (25 mM HEPES pH 7.5 and 100 mM KCl), peak fractions were collected, concentrated, and stored at –80°C before use.The full-length l-glutamate (Sigma), with added 20  mM MgCl2, 10  mM ATP (Takara), and 0.5  mM NADPH (Roche) used to initiate the reaction (The full-length wild-type or mutant P5CS (100 nM protein) activity was determined in the reaction buffer containing 25  mM HEPES pH 7.5, and 10 mM reaction , then th4, 100 mM l-glutamate, 10 mM ATP, and 0.5 mM NADPH. The prepared protein samples were applied to glow-discharged carbon-coated EM grids , and stained with 1% uranyl acetate. Negative-stain EM grids were photographed on a Tecnai Spirit G21 microscope (FEI).Wild-type or mutation P5CS proteins were mixed with different substrate conditions. In brief, the final concentration was as follows: 25 mM HEPES pH 7.5, 100 mM KCl, 10 mM MgSO4, and incubated with 20 mM l-glutamate for the P5CSGlu filament preparation. The P5CSGlu/ATPγS filament was added with an additional 0.5 mM ATPγS (Abcam) compared to the P5CSGlu filament. For the P5CSMix filament, P5CS proteins (2 μM) were incubated with 100 mM KCl, 10 mM MgSO4, 20 mM l-glutamate, 2 mM ATP, and 0.5 mM NADPH. All the samples were incubated for 1 hr on ice before vitrification. The P5CS filament samples were placed on H2/O2 glow-discharged holey carbon grids  or amorphous alloy film (CryoMatrix M024-Au300-R12/13). Then, the grids were immediately blotted for 3.0 s and plunge-frozen in liquid ethane cooled by liquid nitrogen using Vitrobot (Thermo Fisher) at 4°C with 100% humidity. Images were collected on Titan Krios G3 (FEI) equipped with a K3 Summit direct electron detector (Gatan), operating in counting super-resolution mode at 300 kV with a total dose of 72 e−/Å2, subdivided into 50 frames in 4 s exposure using SerialEM  and 2H5G (human GPR domain) as a reference, respectively. Manual adjustment and building the missing regions were done in Coot  feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: i) Decision letter after peer review:eLife. Your article has been reviewed by 4 peer reviewers, one of wom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by José Faraldo-Gómez as the Senior Editor. All reviewers have opted to remain anonymous.Thank you for submitting your article "Structural basis of dynamic P5CS filaments" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.1) The major criticism of all three reviewers was that the paper needs major rewriting both to improve the presentation and to improve its clarity.2) Significant questions were raised about the need for more mechanistic insight. This may require some additional experiments such as those suggested, and would also require a better discussion in the paper about mechanisms. Further, some claims may need to be tempered in the absence of a structure with bound ATP.Reviewer #1 (Recommendations for the authors):The writing would greatly benefit from a professional editor.The paper uses "spiral" in many places when they really mean "helical". A spiral is defined as: winding in a continuous and gradually widening (or tightening) curve, either around a central point on a flat plane or about an axis so as to form a cone. In contrast, a helix has a fixed radius.Reviewer #2 (Recommendations for the authors):1. Most importantly, how does filamentation facilitate the reaction? Is it merely by local concentration effects, a.k.a. "channeling" between active sites via the filament interior? Or is it via concerted or coupled conformational changes within the filament? Is it possible that particular interactions within the filament may be required to stabilize particular conformations of the enzyme required for its activity? If so, then the filament also performs this function in facilitating the enzyme reaction. One could address this by investigating whether or not each active site is fully functional in the absence of filamentation. This could be accomplished using the non-filament forming mutant enzymes, and testing for the presence of the product of the first step separately from that of the second, rather than for only the product of both steps, as done in Figure 5. If the reaction intermediate is too unstable, an active site mutation in the second step active site could be utilized to stall the second reaction step and enable G5P to be measured.2. None of the structures contain ATP. One contains ADP. Speculation on what ATP does to the structure should be tempered by this fact. Structures of ATP dependent enzymes generally show distinct structures when bound to ATP vs. ADP, although this may or may not be relevant here. Still, the qualification should be made and the language more careful to reflect this fact.3. Movement of NADPH towards a C598 is mentioned, and C598 is mentioned to be important for enzyme activity. What does C598 do? Where is it located? A figure showing the relationship between C598 and NADPH in the two conformations would be useful, as well as a description of why the close approach of these moieties is important. C598 is shown in Figure 4B, though not discussed in the text pertaining to that figure, and it is close to G5P. Could it merely be involved in G5P binding?4. Figure 4B, inset – the close proximity of D715 to G5P would appear to not be a favorable interaction. Are these mediated by any cations, or is the pKa of D715 known to be raised perhaps to be involved in the reaction mechanism?5. The document needs professional language editing throughout to make the text easier to read, although the issues did not impede this reviewer from understanding the main points of the manuscript.6. Legends of Figures 2 and 3 should explain what the dashed lines represent. From the text, these appear to be disordered residues, but that is not clear in viewing the figure alone.7. Figure 2: the structure with Glu/ATPgammaS is not shown, yet the text (page 9 line 156) implies it should be. Is it not shown because its conformation is the same as that with G5P-Mg-ADP? Also, the text refers to P5CS^Mix, but the figure shows "G5P-Mg-ADP bound". I gather that these are the same, but this should be made clear in one or both of the places they are discussed (i.e. the text and the figure). The figure legend mentions P5CS^Mix as having G5P-Mg-ADP bound, but the figure itself could also indicate which structure is which using the nomenclature of the text (i.e. P5CS^Glu/ATPgammaS and P5CS^Mix).8. Page 9, line 165 "L-glutamate is bound in a vertical way". This is not obvious in the figure. Also, it is difficult to see the L-glutamate with the color scheme chosen .9. Page 9, line 170, "the loop shifts away from the top of the binding pocket ". Which loop? Can it be identified in the figure?10. Page 9, line 171, M213 is mentioned, but not shown in the figure . What is the significance of M213? Why mention it?11. Page 11, line 207, "G5P-binging", is this "G5P-binding"?12. Page 12, line 221 and Figure 4F, why is it a "Rossmann-like fold" and not a "Rossman fold"?13. Page 12, line 228, beta9-beta10 is mentioned in Figure 4F and Figure 4-Video 1, but not identified in the figure or video. Labeling the CD would also be helpful to connect to the text.14. page 15, line 285. "First, the intermediate G5P is instability and the distance between the two reaction centers is about 60 Å". Does this mean G5P is unstable? How unstable? What is its lifetime under normal enzymatic conditions? A source for this information should also be cited.15. Figure 5, S2 – it would be helpful to show roughly where the filament axis is in panel B – there is some indication in A, but the map is truncated making it not as obvious to the reader without additional guesswork. This would help to see how the filament may sequester to some degree G5P. Also, a view from the top may be helpful to identify spaces within the filament and where pores are located.16. Page 16, line 307, "When the products of the GK domain dissociate from the pocket, the G5P is trapped within the filament and further captured by the GPR domain. Next, NADPH binds to the GPR domain, the conformational change brings the NADPH towards the catalytic residue C598, becoming the close conformation and facilitating the reaction". Is there evidence that NADPH does not bind until G5P is released? "close conformation" does this mean "closed conformation" or is it referring to the close proximity of C598? Again, the reader does not know the significance of C598 in how it participates in the reaction and this would be helpful to know here.17. Page 16, line 319. Please add references to the statement in the first sentence of the paragraph regarding mutations and diseases.18. Page 19, Enzyme assays. Please provide a reference for the method.19. This reviewer is not a cryo-electron microscopist, so cannot comment on the quality of the data or its processing. However, is there a means to assess map/model agreement? For example, in crystallography one has the Rfree  to determine the quality of the model. In cryoEM, perhaps a method such as cross-correlation and/or number of atoms within the envelope would be useful to assess how well the model and map agree?20. Page 22, line 446, say what 4Q1T and 2h5g are . It should be stated how these were used – was a homology model made with the P5CS sequence? How much of the model required changing?21. Figure 3 and 4 – a color purple is indicated, but the structures in question appear blue.22. Can the authors speculate on how ATP binding  results in the formation of the hook? If not, can this be mentioned?23. Page 12, Line 232, explain what ALDH family is and why this is relevant .24. Why is are these new structures not analyzed and compared to the known crystal structure of human P5CS?Reviewer #3 (Recommendations for the authors):The weakness of the paper is the presentation in general. The authors should make an effort to improve the clarity of their descriptions and review the text carefully to correct grammatical issues.This work reports high-quality cryo-EM reconstructions that reveal an impressive supramolecular filament of great beauty. My impression is that the description of the filament is correct, but that the structures offer a lot more of information that should allow to deepen in some crucial aspects such as the catalytic mechanisms, the communication of conformational changes or regulation between domains, which probably will be the subject of future publications.My major criticism is that the manuscript requires a carefully re-writing to improve clarity and correct many language issues.Some specific comments follow.Escherichia coli glutamate 5-kinase. J Mol Biol. 2007 Apr 13;367(5):1431-46. doi: 10.1016/j.jmb.2007.01.073.1. When describing the GK domain, the authors could reference the work describing the first G5K structure: Marco-Marín C, Gil-Ortiz F, Pérez-Arellano I, Cervera J, Fita I, Rubio V. A novel two-domain architecture within the amino acid kinase enzyme family revealed by the crystal structure of 2. The G5K fold is described as "a sandwich-like α3β8α4 structure". Perhaps the authors could mention that this is fold is characteristic of the amino acid kinase family (http://pfam.xfam.org/family/PF00696), as predicted in: Ramón-Maiques S, Marina A, Gil-Ortiz F, Fita I, Rubio V. Structure of acetylglutamate kinase, a key enzyme for arginine biosynthesis and a prototype for the amino acid kinase enzyme family, during catalysis. Structure. 2002 Mar;10(3):329-42. doi: 10.1016/s0969-2126(02)00721-9.3. When describing the structure of the GPR domain, the authors could mention that there is a crystal structure of the human P5CS GPR domain available in the PDB (entry 2H5G) without an accompanying publication.4. It is unclear what is the ligand content for each of the reported cryo-EM reconstructions. The filaments grown with glutamate and the non-hydrolyzable ATP analog (ATPgS) showed the product glutamate 5-phosphate in the GPR active site. But what is the content of the GK domain in this structure? The authors state that (Page 9. Line 160) "Three conformations of the GK domain with the different ligands were revealed clearly in our models". However, only two structures  are described and represented in the figures.The authors could consider adding images with the densities for the bound glutamate 5-phosphate, ADP and Mg 2+.Also, table 1 uses acronyms (GGL RGP NAP) for the bound ligands, but their meaning is not explained. The table also lists no ions bound in any structure, although the text mentions the presence of Mg5. (Page 10. Line 191-196) The authors refer to "ATP binding" and this is misleading because none of the structures has ATP bound.6. They refer to four different states for the GPR domain:  "… four different binding modes of GPR domain". But the differences between the apo state and the NADP(H)-released state are not clear since both show an empty active site.7. The work reports the structures of the GPR domain bound to glutamate 5-phosphate or to NADP. Perhaps the authors could combine these structures by superimposing the active sites and provide some additional details about the reaction mechanism. Perhaps a composed figure that shows the active site with the bound NADP and G5P could be illustrative. The description of the proposed catalytic mechanism  is not clear.8. The authors could discuss why in presence of glutamate and ATPgS, the GPR active site has glutamate 5-phosphate bound, whereas in the filament grown with all substrates , the GPR active site only shows NADP bound.9. There is certain inconsistency regarding the conditions for the formation of P5CS filament formation. It is not clear whether filament formation requires glutamate or not. In a previous paper (Zhang 2020), the authors stated that "Purified P5CS in apo state could hardly form filaments" and that "Removing glutamate from solution almost abolished P5CS filament formation". However, the current work describes that "P5CS proteins self-assemble into filaments without the requirement of ligands" but also that "addition of substrates could enhance the length of filaments" and that "In consistent (sic) with our previous study, L-glutamate (…) is critical in promoting the formation and stability of P5CS filament". Perhaps the authors should explain more clearly whether glutamate is needed or not, the required concentration, and provide any additional variables that influence the formation of the filaments in vitro.10.  "In the proposed model, spontaneous filamentation and elongation of P5CS is importantly associated with the binding of glutamate". To me, it is not clearly described in the manuscript how glutamate favors the formation or enhances the elongation of the P5CS filament. Perhaps the authors should reformulate this sentence or add a more detailed explanation.11. It is not explained why ATP triggers the depolymerization of the filaments formed by mutant R124A, since according to the results, ATP favors the formation of the hook structures that glue the protein tetramers along the filament. One would expect that the nucleotide enhances rather than destabilizes the mutated filaments.12. One important conclusion in the manuscript is that "The disruption of P5CS filament may result in uncoupled catalytic reactions of bifunctional P5CS and a reduced activity". The authors measured the complete two-step reaction in the WT and mutated proteins. The decreased activity could mean that in absence of filaments, the product of the GK reaction, is not properly channeled to the GPR domain. However, the decreased overall reaction activity could also be caused by a reduced G5K efficiency. Perhaps the authors could compare the G5K activity of the WT and mutated proteins. If the efficiency of the partial reaction is similar, this would strongly support that defects in filament formation are causing a defect in the channeling of the intermediate metabolite to the GPR domain.13. Some important references are missing. In addition to those indicated above (points 1 and 2), the authors should include references for RELION, COOT, Phenix, Chimera and for PDB entry 4Q1T cited in the methods.Reviewer #4 (Recommendations for the authors):A detailed list of issues and questions follows here.Figure 1 suppl 1 – scalebars should be included on all micrographs; panel 1B should include all 3 ligand states evaluated in the paper.Figure 1 suppl 2 – it is hard to know which regions of the filaments are covered by the masks. Please show the mask superimposed on the filament structure.Page 7 – the discussion of focused classification is a bit confusing. Does "multiple different conformational states" mean multiple conformations within each liganded state, or that each liganded state had a unique conformation. I think it's the later, but if the former then this should be explained and a supplemental figure showing the refinement classification strategy should be presented.E. coli enzyme, and the RMSD when the two structures are aligned . Similarly, there appears to be a structure of part of the human enzyme available, pdb id 2h5g, and it would be good to know how similar this structure is to the reported Drosophila structure.Page 8 – in comparison to existing crystal structures, it would be helpful to know the % identity to the Page 9 – disordered loop in region II – what is meant by "opening" and "closure" in terms of the disordered region. It is difficult to see in Figure 2 A/B, but it looks like the ordered region common to both structures is pretty much the same? If there are differences, this could perhaps be displayed differently.Page 9 – "with the binding of different ligands" – does this mean that the ligands induce different conformational states, or that the same three conformations were observed regardless of ligand state? – Also, what are the "three conformational states"? This paragraph describes two?Figure 2 A/B – very hard to assess the similarities and differences between the structures here. At a minimum the same regions should be shown for both structures. But from this it looks like the ordered part of the Glu-bound structure is the same as the "mix" structure, and the disordered loops have just been drawn in different positions. Maybe a superposition of the models would help clarify the differences? – Ah, there is a superposition in Figure 3A. Here it is clear, with the exception of one (or two?) residues if the Glu structure that point up  the structures are the same. Is there additional evidence  to support where the disordered loops have been drawn? Showing the quality of the em density in this region is important to judge the conclusions being drawn about the potential movement of this loop.Page 9, Figure 2 C/D – it is unclear where the "closure loop" is here, can this be highlighted?Page 9 – How was the conversion of substrate to phosphorylated intermediate (G5P) assessed? Is this based just on the fit to the density, or is there orthogonal evidence (mass spec or something)? It's hard to judge the quality of the fit into the partial map shown in Figure 2D – a supplemental figure with the region around the G5P/ADP showing the quality of the model in this region and demonstrating a better fit of G5P/ADP than Glu/ATP would be helpful. Or a comparison to the Glu/ATPgS structure might be convincing.Figure 3C – A supplementary figure showing the fit of the atomic model for the "hook" into density in Region 1 would be helpful in assessing the conformational change modeled here.Page 10, last paragraph – the residues contacting the ligands are all in nearly the same positions in the two structures. Conformational changes described above are distal to the binding site.Figure 5 supplement 1A – the quality of the negative stain images of F642A is insufficient to assess the protein quality. One cannot discern whether the protein is in a monomeric or tetrameric state, and the apo state micrograph would suggest that aggregation may be a problem for this mutant. Either better stain images or orthogonal data  are required to be certain that the protein is folded and stable upon introduction of the Phe to Ala mutation.Figure 5 supplement 1B – While it appears that ATP does limit polymerization of the R124A mutant, a more quantitative measurement would be helpful, especially in interpretation of the enzyme activity data in Figure 5D. I suggest light scattering or ultracentrifugation could be used to quantify the fraction of enzyme in polymers.Page 14, first paragraph – It is unclear why the R124A mutation would destabilize polymers. The P5CS(Glu) structure  shows that the polymers are stable in the absence of longitudinal "hook" interactions that are presumably disrupted by this mutation.Figure 5D – enzyme assays. The methods should indicate what concentration enzyme was used in this assay, and what concentration was imaged in negative stain. A major question is whether filaments are observed for the wildtype protein at whatever concentration was used for the enzyme assay. Presumably the answer is yes, but if this is not shown it would bring into question what causes the effect of the mutations on activity.These data should be quantified by calculating specific activity of the enzymes under these conditions, which would allow comparison of these data with published values for P5CS.This assay reads out the second step in a two-step reaction mechanism. If the function of filaments is to couple the two activities as asserted in the text (see comment below), then one would also expect that the point mutants would affect the rate of the first reaction. This should be tested using an assay to monitor ATP hydrolysis in the first step. If the rates of both reactions are reduced by the mutants this would be consistent with a coupling mechanism of the filament, but if only the second step is affected this would be consistent with the authors' hypothesis that the filament increases local intermediate concentration.Page 15 – The rationale behind "coupling" of catalytic reactions by filament assembly is not clear. As the enzyme appears to undergo a complete ligand binding and catalytic cycle in the context of the filament, it is not clear how the filament contacts are coupling activities. While I realize it is asking a lot to add another cryo-EM structure, it seems that the structure of free P5CS tetramers would be an important piece of data to have in interpreting how filaments might be increasing activity.Is there evidence to support an internal channel for G5P, as suggested here (at least I assume that is what is meant by "electrostatic channeling")? If there were a relevant channel stabilized by the filament, it should be seen in the wildtype enzyme filament structure.The proposal that filaments function to create a locally high concentration of intermediates is interesting, but should be tested. One way to do this would be to monitor NADPH production in the presence of G5P as a substrate – at high G5P concentrations one would expect the mutant protein to have the same enzymatic rate as the wildtype.At the bottom of page 15, in proposing the model for "filament catalysis" the statement that upon dissociation from the active site G5P is "trapped within the filament" is not well supported. The architecture of the filament is such that G5 would appear likely to be able to freely diffuse away from the filament.Figure 5, supplement 3 – It would be helpful to include subdomain delineation and the locations of regions I, II, and III with the sequence alignment.Drosophila.Page 16/17 – The potential link between filament assembly and human disease is intriguing. Is there evidence that the human enzyme forms filaments? It would be good to indicate how well-conserved the filament assembly interfaces are between human and Drosophila structure reported here.In a similar vein, the introduction mentions the role of P5CS in plants being of potential significance in agriculture. It would be good to indicate how well conserved filament assembly interfaces are in the plant sequences, and whether based on that one would anticipate that they assemble filaments similar to the  Reviewer #1 (Recommendations for the authors):The writing would greatly benefit from a professional editor.We have rewritten the paper to improve the clarity and the presentation.The paper uses "spiral" in many places when they really mean "helical". A spiral is defined as: winding in a continuous and gradually widening (or tightening) curve, either around a central point on a flat plane or about an axis so as to form a cone. In contrast, a helix has a fixed radius.Thanks for the explanation. We replaced “spiral” with “helical”.Reviewer #2 (Recommendations for the authors):1. Most importantly, how does filamentation facilitate the reaction? Is it merely by local concentration effects, a.k.a. "channeling" between active sites via the filament interior? Or is it via concerted or coupled conformational changes within the filament? Is it possible that particular interactions within the filament may be required to stabilize particular conformations of the enzyme required for its activity? If so, then the filament also performs this function in facilitating the enzyme reaction. One could address this by investigating whether or not each active site is fully functional in the absence of filamentation. This could be accomplished using the non-filament forming mutant enzymes, and testing for the presence of the product of the first step separately from that of the second, rather than for only the product of both steps, as done in Figure 5. If the reaction intermediate is too unstable, an active site mutation in the second step active site could be utilized to stall the second reaction step and enable G5P to be measured.By analyzing the arrangement of P5CS tetramers inside the filament, we believe that one role of filamentation is to facilitate the enzyme reaction. Having screened a large number of mutations, we were unable to obtain stable non-filament forming mutants.2. None of the structures contain ATP. One contains ADP. Speculation on what ATP does to the structure should be tempered by this fact. Structures of ATP dependent enzymes generally show distinct structures when bound to ATP vs. ADP, although this may or may not be relevant here. Still, the qualification should be made and the language more careful to reflect this fact.We temper our claims in the absence of an ATP-bound structure.3. Movement of NADPH towards a C598 is mentioned, and C598 is mentioned to be important for enzyme activity. What does C598 do? Where is it located? A figure showing the relationship between C598 and NADPH in the two conformations would be useful, as well as a description of why the close approach of these moieties is important. C598 is shown in Figure 4B, though not discussed in the text pertaining to that figure, and it is close to G5P. Could it merely be involved in G5P binding?We add text at lines 228-237 and a supplementary figure , highlighting the role and location of C598:+ to catalyze the conversion of various aldehydes into their corresponding carboxylic acids. Many studies on ALDHs have shown that a conserved residue cysteine acts as the active site of nucleophile, forming thiohemiacetal intermediate with substrate . Curiously, the NADPH-utilizing GPR domain of P5CS catalyzes the reverse reaction of ALDHs. ”“The GPR domain of P5CS belongs to aldehyde dehydrogenase (ALDH) superfamily. ALDHs family uses NAD(P)4. Figure 4B, inset – the close proximity of D715 to G5P would appear to not be a favorable interaction. Are these mediated by any cations, or is the pKa of D715 known to be raised perhaps to be involved in the reaction mechanism?Residue D715 is highly conserved and close to the active site, and it is not clear what role D715 plays in GPR domain reaction. It might be involved in the reaction mechanism.5. The document needs professional language editing throughout to make the text easier to read, although the issues did not impede this reviewer from understanding the main points of the manuscript.We have rewritten the paper to improve the clarity and the presentation.6. Legends of Figures 2 and 3 should explain what the dashed lines represent. From the text, these appear to be disordered residues, but that is not clear in viewing the figure alone.We have revised Figure legends of Figures 2 and 3:“The dashed lines represent disordered segments in this model.”“On the left panel, the dashed lines in the model represent the open-loop (blue-violet) and closed-loop (green) in region II.”7. Figure 2: the structure with Glu/ATPgammaS is not shown, yet the text (page 9 line 156) implies it should be. Is it not shown because its conformation is the same as that with G5P-Mg-ADP? Also, the text refers to P5CS^Mix, but the figure shows "G5P-Mg-ADP bound". I gather that these are the same, but this should be made clear in one or both of the places they are discussed (i.e. the text and the figure). The figure legend mentions P5CS^Mix as having G5P-Mg-ADP bound, but the figure itself could also indicate which structure is which using the nomenclature of the text (i.e. P5CS^Glu/ATPgammaS and P5CS^Mix).Glu/ATPγS filament:We add text at lines 162-169 and a supplementary figure  to depict the GK domain structure of the P5CSGlu filament  and a second structure of the GK domain with G5P-Mg-ADP in the P5CSMix filament . In the P5CSGlu/ATPγS filament, the ligands could not be determined due to incomplete densities . The GK domain structure of the P5CSGlu/ATPγS filament is virtually identical to that of the P5CSMix filament .”“We obtained a structure of the GK domain with the binding of glutamate in the P5CSThe ligand-bound states of the GK domain correspond to ligand-bound states of P5CS filament were indicated in Figure 2.8. Page 9, line 165 "L-glutamate is bound in a vertical way". This is not obvious in the figure. Also, it is difficult to see the L-glutamate with the color scheme chosen .We have added a supplementary figure  with a different angle to highlight the position of glutamate. We have selected an outline font for a clear display.9. Page 9, line 170, "the loop shifts away from the top of the binding pocket ". Which loop? Can it be identified in the figure?We have changed “the loop” into “a disordered segment” and highlighted it in the figure .10. Page 9, line 171, M213 is mentioned, but not shown in the figure . What is the significance of M213? Why mention it?We added text and a supplementary figure , highlighting the role and location of M213.11. Page 11, line 207, "G5P-binging", is this "G5P-binding"?Yes, it should be “G5P-binding”, we have revised it.12. Page 12, line 221 and Figure 4F, why is it a "Rossmann-like fold" and not a "Rossman fold"?The classical Rossmann fold contains six β-strands, whereas Rossmann-like folds contain only five β-strands, just like the GPR domain in figure 4F.13. Page 12, line 228, beta9-beta10 is mentioned in Figure 4F and Figure 4-Video 1, but not identified in the figure or video. Labeling the CD would also be helpful to connect to the text.We revised the description about NBD rotation at lines 272-275 and added a supplementary figure  to show the cylinder axis:“Meanwhile, the entire NBD rotates approximately 15° along the cylinder axis  and slides towards CD .”14. page 15, line 285. "First, the intermediate G5P is instability and the distance between the two reaction centers is about 60 Å". Does this mean G5P is unstable? How unstable? What is its lifetime under normal enzymatic conditions? A source for this information should also be cited.This means G5P is unstable; relevant references have been added.15. Figure 5, S2 – it would be helpful to show roughly where the filament axis is in panel B – there is some indication in A, but the map is truncated making it not as obvious to the reader without additional guesswork. This would help to see how the filament may sequester to some degree G5P. Also, a view from the top may be helpful to identify spaces within the filament and where pores are located.We have added a video  and a view from the top  for a better representation.16. Page 16, line 307, "When the products of the GK domain dissociate from the pocket, the G5P is trapped within the filament and further captured by the GPR domain. Next, NADPH binds to the GPR domain, the conformational change brings the NADPH towards the catalytic residue C598, becoming the close conformation and facilitating the reaction". Is there evidence that NADPH does not bind until G5P is released? "close conformation" does this mean "closed conformation" or is it referring to the close proximity of C598? Again, the reader does not know the significance of C598 in how it participates in the reaction and this would be helpful to know here.This means “closed conformation”. We have added supplementary figures , highlighting the role and location of C598. We have expanded the discussion of a catalytic model of the GPR domain at lines 359-365:Drosophila P5CS, our data suggest that the catalytic residue C598 of CD attacks the G5P to form the first tetrahedral thioacetal intermediate in the reaction, and then expulsion of phosphate collapses to form a stable thioacyl enzyme intermediate. A hydride is then transferred to this intermediate from NADPH, with subsequent collapse to release the product GSA.”“In the GPR domain of 17. Page 16, line 319. Please add references to the statement in the first sentence of the paragraph regarding mutations and diseases.Relevant references have been added.18. Page 19, Enzyme assays. Please provide a reference for the method.Relevant references have been added19. This reviewer is not a cryo-electron microscopist, so cannot comment on the quality of the data or its processing. However, is there a means to assess map/model agreement? For example, in crystallography one has the Rfree  to determine the quality of the model. In cryoEM, perhaps a method such as cross-correlation and/or number of atoms within the envelope would be useful to assess how well the model and map agree?To assess the agreement of map and model, the cross-correlation can be found in the PDB validation reports. As for the validation of the map, it is achieved by the gold standard FSC curve which is in Figure 1—figure supplement 2-4.20. Page 22, line 446, say what 4Q1T and 2h5g are . It should be stated how these were used – was a homology model made with the P5CS sequence? How much of the model required changing?Burkholderia thailandensis) and 2H5G (human GPR domain) were used as reference models to predict GK and GPR respectively. Then, we manually tuned each residue in Coot, using Phenix to refine and validate the final model.We use Swiss-model to build the initial model; 4Q1T  results in the formation of the hook? If not, can this be mentioned?We have expanded the discussion of nucleotides binding resulting in the formation of the hook structure at lines 335-346:Glu filament would transform upon the binding of ATP, thereby triggering the formation of hook structure and completing the catalytic reaction.”“We observed two ligand binding modes in the GK domain. Due to the lack of ATP-bound structure, it is difficult to determine whether ATP plays a decisive role in these conformational changes. According to a previous report on the N-Acetyl-L-glutamate kinase (NAGK), nucleoside is important for the conformational change of the AAK domain, and the structures are similar when bound by ADP or AMPPNP . Based on the similarity of sequences and structures between GK and NAGK , we propose that the conformation of the GK domain in the P5CS23. Page 12, Line 232, explain what ALDH family is and why this is relevant .Yes, P5CS is a member of the ALDH family. We have added text explaining the ALDH family.24. Why is are these new structures not analyzed and compared to the known crystal structure of human P5CS?Drosophila and that in human.We have added text and a supplementary figure  to compare the structure of P5CS in Reviewer #3 (Recommendations for the authors):The weakness of the paper is the presentation in general. The authors should make an effort to improve the clarity of their descriptions and review the text carefully to correct grammatical issues.This work reports high-quality cryo-EM reconstructions that reveal an impressive supramolecular filament of great beauty. My impression is that the description of the filament is correct, but that the structures offer a lot more of information that should allow to deepen in some crucial aspects such as the catalytic mechanisms, the communication of conformational changes or regulation between domains, which probably will be the subject of future publications.My major criticism is that the manuscript requires a carefully re-writing to improve clarity and correct many language issues.We have rewritten the paper to improve the clarity and the presentation.Some specific comments follow.Escherichia coli glutamate 5-kinase. J Mol Biol. 2007 Apr 13;367(5):1431-46. doi: 10.1016/j.jmb.2007.01.073.1. When describing the GK domain, the authors could reference the work describing the first G5K structure: Marco-Marín C, Gil-Ortiz F, Pérez-Arellano I, Cervera J, Fita I, Rubio V. A novel two-domain architecture within the amino acid kinase enzyme family revealed by the crystal structure of Relevant references have been added.2. The G5K fold is described as "a sandwich-like α3β8α4 structure". Perhaps the authors could mention that this is fold is characteristic of the amino acid kinase family (http://pfam.xfam.org/family/PF00696), as predicted in: Ramón-Maiques S, Marina A, Gil-Ortiz F, Fita I, Rubio V. Structure of acetylglutamate kinase, a key enzyme for arginine biosynthesis and a prototype for the amino acid kinase enzyme family, during catalysis. Structure. 2002 Mar;10(3):329-42. doi: 10.1016/s0969-2126(02)00721-9.We have added text at lines 152-158:Drosophila P5CS is conserved with the GK protein in E. coli. Alignments of sequences and structures indicate that their secondary structures are similar as both exhibit a sandwich-like α3β8α4 topological folding , which is a characteristic of the amino acid kinase (AAK) family .”“The GK domain of 3. When describing the structure of the GPR domain, the authors could mention that there is a crystal structure of the human P5CS GPR domain available in the PDB (entry 2H5G) without an accompanying publication.Drosophila and that in human.We have added text and a supplementary figure  to compare the structure of P5CS in 4. It is unclear what is the ligand content for each of the reported cryo-EM reconstructions. The filaments grown with glutamate and the non-hydrolyzable ATP analog (ATPgS) showed the product glutamate 5-phosphate in the GPR active site. But what is the content of the GK domain in this structure? The authors state that (Page 9. Line 160) "Three conformations of the GK domain with the different ligands were revealed clearly in our models". However, only two structures  are described and represented in the figures.The authors could consider adding images with the densities for the bound glutamate 5-phosphate, ADP and Mg 2+.Also, table 1 uses acronyms (GGL RGP NAP) for the bound ligands, but their meaning is not explained. The table also lists no ions bound in any structure, although the text mentions the presence of MgGlu/ATPγS filament:We add text at “lines 162-169” and a supplementary figure  to depict the GK domain structure of the P5CSGlu filament  and a second structure of the GK domain with G5P-Mg-ADP in the P5CSMix filament . In the P5CSGlu/ATPγS filament, the ligands could not be determined due to incomplete densities . The GK domain structure of the P5CSGlu/ATPγS filament is virtually identical to that of the P5CSMix filament .”“We obtained a structure of the GK domain with the binding of glutamate in the P5CSGlu filament.We added a supplementary figure  to show the cryo-EM densities for the bound G5P-Mg-ADP in the GK domain of P5CS2+ ion to the list.GGL, RGP, and NAP are component identifiers (3-letter code) which are consist of RCSB PDB, representing the bound ligands in our models. We also updated table 1 and added the Mg5. (Page 10. Line 191-196) The authors refer to "ATP binding" and this is misleading because none of the structures has ATP bound.We have removed this paragraph and expanded the discussion of ATP bound at lines 335-346:Glu filament would transform upon the binding of ATP, thereby triggering the formation of hook structure and completing the catalytic reaction.”“We observed two ligand binding modes in the GK domain. Due to the lack of ATP-bound structure, it is difficult to determine whether ATP plays a decisive role in these conformational changes. According to a previous report on the N-Acetyl-L-glutamate kinase (NAGK), nucleoside is important for the conformational change of the AAK domain, and the structures are similar when bound by ADP or AMPPNP . Based on the similarity of sequences and structures between GK and NAGK , we propose that the conformation of the GK domain in the P5CS6. They refer to four different states for the GPR domain:  "… four different binding modes of GPR domain". But the differences between the apo state and the NADP(H)-released state are not clear since both show an empty active site.We add text at lines 253-255 and a supplementary figure  to compare the NADP(H)-released state and the APO state.“The structure of NADP(H)-release state, which has no bound ligand, is identical to the unliganded state.”7. The work reports the structures of the GPR domain bound to glutamate 5-phosphate or to NADP. Perhaps the authors could combine these structures by superimposing the active sites and provide some additional details about the reaction mechanism. Perhaps a composed figure that shows the active site with the bound NADP and G5P could be illustrative. The description of the proposed catalytic mechanism  is not clear.We have added a figure , showing the active site with the bound NADP and G5P. The description of the proposed catalytic mechanism has been rewritten in Discussion at lines 359-365:Drosophila P5CS, our data suggest that the catalytic residue C598 of CD attacks the G5P to form the first tetrahedral thioacetal intermediate in the reaction, and then expulsion of phosphate collapses to form a stable thioacyl enzyme intermediate. A hydride is then transferred to this intermediate from NADPH, with subsequent collapse to release the product GSA.”“In GPR domain of 8. The authors could discuss why in presence of glutamate and ATPgS, the GPR active site has glutamate 5-phosphate bound, whereas in the filament grown with all substrates , the GPR active site only shows NADP bound.We have added text at lines 243-244 and supplementary figures  to discuss this result:“It might be a contamination of ATP, leading to the production of the substrate G5P.”“Figure 4—figure supplement 1. Representative cryo-electron microscopy (cryo-EM) densities for the active site of the γ-glutamyl phosphate reductase (GPR) domain.Glu/ATPγS filament. (B) Unmodeled densities in the active site of GPR domain at NADP(H)-binding state, which may be the reaction product: π or GSA/P5C.”(A) Cryo-EM map quality of G5P ligand in the active site of GPR domain in P5CS9. There is certain inconsistency regarding the conditions for the formation of P5CS filament formation. It is not clear whether filament formation requires glutamate or not. In a previous paper (Zhang 2020), the authors stated that "Purified P5CS in apo state could hardly form filaments" and that "Removing glutamate from solution almost abolished P5CS filament formation". However, the current work describes that "P5CS proteins self-assemble into filaments without the requirement of ligands" but also that "addition of substrates could enhance the length of filaments" and that "In consistent (sic) with our previous study, L-glutamate (…) is critical in promoting the formation and stability of P5CS filament". Perhaps the authors should explain more clearly whether glutamate is needed or not, the required concentration, and provide any additional variables that influence the formation of the filaments in vitro.We have revised the main text at lines 99-110 in response to the comment:Drosophila P5CS in the APO state is hard to form filaments at low concentrations (<0.05 μM). The addition of glutamate to the P5CS samples induces micron-scale filaments . Here, we observe that increasing P5CS concentration (>1 μM) also promotes the formation of filaments in the APO state. Our results show that the P5CS proteins can be self-assembled into filaments without ligands, and adding substrates increases the length of filaments at the same concentration of the P5CS proteins. Consistent with our previous study, glutamate (a substrate of P5CS) promotes the formation and maintenance of Drosophila P5CS filaments .”“In our previous study, we found that 10.  "In the proposed model, spontaneous filamentation and elongation of P5CS is importantly associated with the binding of glutamate". To me, it is not clearly described in the manuscript how glutamate favors the formation or enhances the elongation of the P5CS filament. Perhaps the authors should reformulate this sentence or add a more detailed explanation.Revised as the following:“In this proposed model, spontaneous filamentation occurs at APO state, and elongation of P5CS filament is associated with the binding of glutamate.”We have expanded the discussion at lines 346-349 in response to the comment:Glu filament, further research is needed to understand how the conformation of glutamate binding contributes to the extension of P5CS filaments.”“Although we solved the clear structure of the P5CS11. It is not explained why ATP triggers the depolymerization of the filaments formed by mutant R124A, since according to the results, ATP favors the formation of the hook structures that glue the protein tetramers along the filament. One would expect that the nucleotide enhances rather than destabilizes the mutated filaments.R124AP5CS filaments at lines 423-430:We have added text and expanded the discussion of short R124A mutant cannot stabilize the filament structure in the catalytic process and lose the ability to form the long filaments.”“We speculate that the swing of GPR in the catalytic reaction could destabilize the interaction between adjacent GPR domain dimers in the filament. Therefore, the extra interaction at the hook structure of the GK domain may be required for the stabilization of the filament. This proposed stabilization is consistent with negative stain data showing that the P5CS12. One important conclusion in the manuscript is that "The disruption of P5CS filament may result in uncoupled catalytic reactions of bifunctional P5CS and a reduced activity". The authors measured the complete two-step reaction in the WT and mutated proteins. The decreased activity could mean that in absence of filaments, the product of the GK reaction, is not properly channeled to the GPR domain. However, the decreased overall reaction activity could also be caused by a reduced G5K efficiency. Perhaps the authors could compare the G5K activity of the WT and mutated proteins. If the efficiency of the partial reaction is similar, this would strongly support that defects in filament formation are causing a defect in the channeling of the intermediate metabolite to the GPR domain.We have tempered our claims and added a Discussion section for the potential working model.13. Some important references are missing. In addition to those indicated above (points 1 and 2), the authors should include references for RELION, COOT, Phenix, Chimera and for PDB entry 4Q1T cited in the methods.Relevant references have been added.Reviewer #4 (Recommendations for the authors):In support of, and addition to, the broad comments included in the public review, a detailed list of issues and questions follows here.Figure 1 suppl 1 – scalebars should be included on all micrographs; panel 1B should include all 3 ligand states evaluated in the paper.We have added scale bars to all micrographs, and cryo-EM micrographs of three ligand states are provided .Figure 1 suppl 2 – it is hard to know which regions of the filaments are covered by the masks. Please show the mask superimposed on the filament structure.We have superimposed masks on corresponding filament structures in our flowchartPage 7 – the discussion of focused classification is a bit confusing. Does "multiple different conformational states" mean multiple conformations within each liganded state, or that each liganded state had a unique conformation. I think it's the later, but if the former then this should be explained and a supplemental figure showing the refinement classification strategy should be presented.It is the later. The word “different” has been removed at lines 121-124:“Using separate focused refinement strategy, we obtained multiple conformational states of the GK domain tetramer (3.1 to 3.5 Å) and the GPR domain dimer (3.6 to 4.3 Å).”E. coli enzyme, and the RMSD when the two structures are aligned . Similarly, there appears to be a structure of part of the human enzyme available, pdb id 2h5g, and it would be good to know how similar this structure is to the reported Drosophila structure.Page 8 – in comparison to existing crystal structures, it would be helpful to know the % identity to the E. coli GK and the GK domain of Drosophila P5CS:We have added text at lines 825-827 and a supplementary figure  to show the comparison of E. coli GK structure without PUA domain  and Drosophila GK domain structure of P5CS , with 30.56% sequence identity and RMSD value of 1.363 Å (198 atom pairs).”“(A) The comparison of Drosophila and that in human:We also add text at lines 464-449, 892-894, and a supplementary figure  to compare the structure of P5CS in Drosophila P5CS .”“In the protein structure database, there is only the GPR domain structure available for human P5CS (PDB: 2H5G). Its overall structure is similar to the GPR domain of Drosophila P5CS at NADP(H)-binding state (blue-violet), the GPR domain of human P5CS  has 56.74% sequence identity and RMSD value of 1.531 Å (398 atom pairs).”“Compared with the GPR domain of Page 9 – disordered loop in region II – what is meant by "opening" and "closure" in terms of the disordered region. It is difficult to see in Figure 2 A/B, but it looks like the ordered region common to both structures is pretty much the same? If there are differences, this could perhaps be displayed differently.We have rewritten the description of the disordered region and suggested that they referred to open-loop and closed-loop at lines 190-202, which were shown in Figure 3A and Figure 3—figure supplement 1:Glu filament, we speculate that the disordered segment in region II acts as a closed-loop, which traps glutamate in GBD . In the P5CSMix filament, the same segment shifts away from the top of the binding pocket and forms an open-loop, in which residue M213 interacts with G5P . We notice that closed-loop has a steric clash with G5P, preventing the binding of G5P under such a conformation . Our findings support the idea that region II at the GK domain engages in regulating the catalytic reaction.”“Meanwhile, based on the disorder densities in region II , we modeled the possible trend of the missing segment with a dashed line . In the P5CSPage 9 – "with the binding of different ligands" – does this mean that the ligands induce different conformational states, or that the same three conformations were observed regardless of ligand state? – Also, what are the "three conformational states"? This paragraph describes two?We have rewritten the main text at lines161-168, and added supplementary figures  to show three conformational states of the GK domain:Glu filament  and a second structure of the GK domain with G5P-Mg-ADP in the P5CSMix filament . In the P5CSGlu/ATPγS filament, the ligands could not be determined due to incomplete densities . The GK domain structure of the P5CSGlu/ATPγS filament is virtually identical to that of the P5CSMix filament .”“We obtained a structure of the GK domain with the binding of glutamate in the P5CSFigure 2 A/B – very hard to assess the similarities and differences between the structures here. At a minimum the same regions should be shown for both structures. But from this it looks like the ordered part of the Glu-bound structure is the same as the "mix" structure, and the disordered loops have just been drawn in different positions. Maybe a superposition of the models would help clarify the differences? – Ah, there is a superposition in Figure 3A. Here it is clear, with the exception of one (or two?) residues if the Glu structure that point up  the structures are the same. Is there additional evidence  to support where the disordered loops have been drawn? Showing the quality of the em density in this region is important to judge the conclusions being drawn about the potential movement of this loop.We add text and supplementary figures  to show the quality of the EM density in region II.Page 9, Figure 2 C/D – it is unclear where the "closure loop" is here, can this be highlighted?We highlighted the “open-loop” and “closed-loop” in the Figure 2C and D.Page 9 – How was the conversion of substrate to phosphorylated intermediate (G5P) assessed? Is this based just on the fit to the density, or is there orthogonal evidence (mass spec or something)? It's hard to judge the quality of the fit into the partial map shown in Figure 2D – a supplemental figure with the region around the G5P/ADP showing the quality of the model in this region and demonstrating a better fit of G5P/ADP than Glu/ATP would be helpful. Or a comparison to the Glu/ATPgS structure might be convincing.We have added a supplementary figure  to show that the binding modes of Glu/ATP and G5P/ADP ligands, indicating that the conformation of bound G5P/ADP is better than Glu/ATP.Figure 3C – A supplementary figure showing the fit of the atomic model for the "hook" into density in Region 1 would be helpful in assessing the conformational change modeled here.Glu filament shown as mesh.We have added a supplementary figure  to show the conformational change of hook structure in region I, and cartoon models have overlaid the cryo-EM density of P5CSAnd hook structure fitting into density was shown in Figure 1—figure supplement 4C.Page 10, last paragraph – the residues contacting the ligands are all in nearly the same positions in the two structures. Conformational changes described above are distal to the binding site.We have rewritten the description of GK domain structural changes at lines 221-224:“By comparing the structures of GK domain with various ligands, we demonstrate the conformational changes, which may be associated with phosphorylation of the substrate glutamate.”Figure 5 supplement 1A – the quality of the negative stain images of F642A is insufficient to assess the protein quality. One cannot discern whether the protein is in a monomeric or tetrameric state, and the apo state micrograph would suggest that aggregation may be a problem for this mutant. Either better stain images or orthogonal data  are required to be certain that the protein is folded and stable upon introduction of the Phe to Ala mutation.Drosophila P5CS cannot form a stable free tetramer, or the tetramer form only exists in the filament. We selected the high concentration protein, which will indeed affect the quality of staining. We found that aggregation is indeed a problem of F642A mutant. In our purification process, this mutant showed very low activity and was easy to inactivate, and the irregular spherical protein and no regular monomer or tetramer were observed by TEM.According to a large number of mutant screening and structural analysis, we speculate that Figure 5 supplement 1B – While it appears that ATP does limit polymerization of the R124A mutant, a more quantitative measurement would be helpful, especially in interpretation of the enzyme activity data in Figure 5D. I suggest light scattering or ultracentrifugation could be used to quantify the fraction of enzyme in polymers.R124A filaments at lines 316-321:We have rewritten the description of P5CSR124A mutant proteins formed long filaments in the APO state as well as in the presence glutamate . We observed that glutamate-bound P5CSR124A filaments disassembled at the initial phase of adding ATP. Being incubated with all substrates, P5CSR124A formed shorter filaments than P5CSWT .”“In contrast, the P5CSFor negative stain sample preparation, all substrates are excessive. The effect of ATP on the R124A mutant can only be captured in the initial phase of the GK domain reaction. With the extension of incubation time, a short filament will form eventually. Our data show that ATP can affect the R124A mutant, highlighting the importance of the hook structure.Page 14, first paragraph – It is unclear why the R124A mutation would destabilize polymers. The P5CS(Glu) structure  shows that the polymers are stable in the absence of longitudinal "hook" interactions that are presumably disrupted by this mutation.R124AP5CS filaments at lines 423-430:We have added text and expanded the discussion of short R124A mutant cannot stabilize the filament structure in the catalytic process and lose the ability to form the long filaments.”“We speculate that the swing of GPR in the catalytic reaction could destabilize the interaction between adjacent GPR domain dimers in the filament. Therefore, the extra interaction at the hook structure of the GK domain may be required for the stabilization of the filament. This proposed stabilization is consistent with negative stain data showing that the P5CSFigure 5D – enzyme assays. The methods should indicate what concentration enzyme was used in this assay, and what concentration was imaged in negative stain. A major question is whether filaments are observed for the wildtype protein at whatever concentration was used for the enzyme assay. Presumably the answer is yes, but if this is not shown it would bring into question what causes the effect of the mutations on activity.These data should be quantified by calculating specific activity of the enzymes under these conditions, which would allow comparison of these data with published values for P5CS.This assay reads out the second step in a two-step reaction mechanism. If the function of filaments is to couple the two activities as asserted in the text (see comment below), then one would also expect that the point mutants would affect the rate of the first reaction. This should be tested using an assay to monitor ATP hydrolysis in the first stepWe used 100 nM protein in enzyme assays and revised in our Methods, this protein concentration actually can induce the formation of filaments. And all the protein concentrations are marked on negative stain micrographs.Our aim for enzyme activity assays is to test whether the filament structure is necessary for its reaction. As for the mechanism, more studies are needed. From our results, we observed that the activity of the R124A mutant was greatly reduced. We speculate that it will not have much effect on the rate of the first reaction, because the residue is not close to the active site. If the rate of the first reaction decreases, the destruction of the hook structure may lead to the conformational change of the GK domain tetramer. We have expanded this discussion at lines 423-430:R124A mutant cannot stabilize the filament structure in the catalytic process and lose the ability to form the long filaments.”“We speculate that the swing of GPR in the catalytic reaction could destabilize the interaction between adjacent GPR domain dimers in the filament. Therefore, the extra interaction at the hook structure of the GK domain may be required for the stabilization of the filament. This proposed stabilization is consistent with negative stain data showing that the P5CSIf the rates of both reactions are reduced by the mutants this would be consistent with a coupling mechanism of the filament, but if only the second step is affected this would be consistent with the authors' hypothesis that the filament increases local intermediate concentration.Page 15 – The rationale behind "coupling" of catalytic reactions by filament assembly is not clear. As the enzyme appears to undergo a complete ligand binding and catalytic cycle in the context of the filament, it is not clear how the filament contacts are coupling activities. While I realize it is asking a lot to add another cryo-EM structure, it seems that the structure of free P5CS tetramers would be an important piece of data to have in interpreting how filaments might be increasing activity.Is there evidence to support an internal channel for G5P, as suggested here . Considering the distance between the GK and GPR domains is about 60 Å , we propose a model that P5CS filament may exhibit a scaffold architecture that stabilizes the relative position of the GK and GPR domains, the cooperation between which may produce electrostatic substrate channels that mediate the transfer of unstable intermediate G5P. In addition, P5CS filamentation may create a half-opened chamber with the active sites located at the inner part of the filament. Since the GK domain is catalytically faster than the GPR domain, the unstable intermediates G5P accumulate within the filament. This microenvironment may reduce the amount of G5P escaped into the solvent, thereby facilitating the rate-limiting reaction at the GPR domain.”In our results, P5CS protein showed high filament-forming ability. The filamentation of P5CS is like a highly ordered factory to facilitate the two-step reaction, which is also an important feature of P5CS filament. Whether dynamic P5CS filaments produce transient conformations to promote the transfer of intermediates, resulting in the formation of the channel or chamber to couple filament reactions, more structural studies of P5CS filaments are required to determine an underlying regulatory mechanism that transmits information between the GK and GPR domain in the tetramer and along the filament.We have updated this supplementary figure. Subdomain and these regions are labeled now.Drosophila.Page 16/17 – The potential link between filament assembly and human disease is intriguing. Is there evidence that the human enzyme forms filaments? It would be good to indicate how well-conserved the filament assembly interfaces are between human and Drosophila structure reported here.In a similar vein, the introduction mentions the role of P5CS in plants being of potential significance in agriculture. It would be good to indicate how well conserved filament assembly interfaces are in the plant sequences, and whether based on that one would anticipate that they assemble filaments similar to the We have added text at lines 445-452:Drosophila P5CS .Although it is still unknown whether human P5CS can form filament structure in vitro, it is reasonable to suspect that the filament-forming property is conserved between human and Drosophila P5CS based on their structural similarity.”“In the protein structure database, there is only the GPR domain structure available for human P5CS (PDB: 2H5G). Its overall structure is similar to the GPR domain of Arabidopsis. According to the P5CS structure of Arabidopsis predicted by AlphaFold2, its secondary structure on the interfaces are similar to that of Drosophila.In the species we compared, the sequence of the interfaces is relatively conservative in animals, and less conservative in


Quantitative data on in vivo vertebral disc deformations are critical for enhancing our understanding of spinal pathology and improving the design of surgical materials. This study investigated in vivo lumbar intervertebral disc deformations during axial rotations under different load-bearing conditions.Twelve healthy subjects  between the ages of 25 and 39 were recruited. Using a combination of a dual fluoroscopic imaging system (DFIS) and CT, the images of L3–5 segments scanned by CT were transformed into three-dimensional models, which matched the instantaneous images of the lumbar spine taken by a double fluorescent X-ray system during axial rotations to reproduce motions. Then, the kinematic data of the compression and shear deformations of the lumbar disc and the coupled bending of the vertebral body were obtained.Relative to the supine position, the average compression deformation caused by rotation is between + 10% and − 40%, and the shear deformation is between 17 and 50%. Under physiological weightbearing loads, different levels of lumbar discs exhibit similar deformation patterns, and the deformation patterns of left and right rotations are approximately symmetrical. The deformation patterns change significantly under a 10 kg load, with the exception of the L3–4 disc during the right rotation.The deformation of the lumbar disc was direction-specific and level-specific during axial rotations and was affected by extra weight. These data can provide new insights into the biomechanics of the lumbar spine and optimize the parameters of artificial lumbar spine devices. As the most common movement unit, axial rotation movement occurs frequently in daily activities. To obtain the movement characteristics of axial rotation, a large number of in vivo and in vitro studies have been carried out. Recently, cadaver studies have investigated external motion parameters by applying external axial rotational torque and compression loads –4. Many However, most of these experiments focused on the contribution of each vertebral body to rotational motion, rather than on the changes in intervertebral disc stress. Recent studies have investigated the deformation parameters of lumbar discs –11, withIn this article, the deformation characteristics of the lumbar disc during axial rotation were analyzed by DFIS technology, which has been widely used in various bone and joint studies. The DFIS builds the human motion model, which determines the relative position of the 3D vertebral body in the software using two orthogonal fluoroscopic images.A series of experiments were performed to validate the accuracy and repeatability of our equipment. Bai et al.  showed tSpecifically, we determined the compression and shear deformations in different areas of the lumbar discs and verified the influence of different loads on the deformation pattern during axial rotation. We assumed that in each region, the deformation of the disc during the axial rotation process was level-dependent and affected by the load.Twelve subjects  were recruited. Inclusion criteria included: normal spine development, age 18 ~ 40 years old, BMI 18.5 ~ 25 Kg/m2, Pfirrmann grade ≤ II (MRI), normal bone density. Exclusion criteria included: current or prior low-back pain, previous spinal surgery, anatomic abnormalities, pregnancy, or any spinal disorders. All subjects received a supine CT scan . Parallel digital images of the lumbar spine with a thickness of 0.625 mm and a resolution of 512 × 512 pixels were obtained. These CT images were input into Mimics software  for the construction of 3D models of the vertebrae from L3 to L5 , based on vertebral symmetry Fig. a. In the2  or a 10 kg load (carrying 5 kg sandbags front and back) was imaged using a dual fluoroscopic imaging system (DFIS). After unified instructional training, the subjects freely rotated their bodies from the standing position to the maximum position and maintained that position for a period of time, during which the researchers assisted in correcting the pelvis and buttocks. Custom-made lead clothing was used to protect the subjects’ thyroid and gonads Fig. a. The twUsing the established protocol, the vertebral model and paired fluoroscopy images were used to reproduce the motion of the vertebral body in Rhinoceros software  , 13, 14.The disc deformation was calculated using mesh vertices evenly distributed on the upper and lower endplates (approximately 3000 points per surface). The coordinate system of the upper endplate surface of the lower vertebra was used as a reference for calculating the displacement of each corresponding point on the lower endplate of the upper vertebra Fig. b. The no+, Tensile Deformation; -,Compressive Deformation. Tension (+) means that the disc height during rotation is greater than that during supine, and compression (−) means that the height during rotation is smaller than that of during supine.The overall compression deformation was measured in the reference coordinate system along the z-axis and plotted on a heat map Fig. . In addip < 0.05. Statistical analysis was performed with SPSS 18.0 software .A two-way analysis of variance was used to compare the differences in shear and tensile deformation, as well as the coupled bending of the lumbar spine during axial rotation motion at different load-bearing levels. The statistical significance was set at The overall compression deformation was shown on a heat map Fig. . Going fCompared with under weightbearing, the compression deformation mode of the disc changed significantly under the extra 10 kg load. Except for 1/20 of the L4–5 disc area during left rotation, the remaining discs produced similar tensile deformation in the anterior larger region . The changing trend of the L3–4 disc during right rotation under 10 kg load was comparable to that under weightbearing. However, in other cases, the compression deformation trend direction under 10 kg load was closer to the sagittal direction.Graphs Fig.  and 9-sqP = 0.02).The shear deformation of the lumbar intervertebral disc at the 9 positions was relatively constant Table . On averP = 0.04).The range of motion (ROM) of the lumbar vertebrae during axial rotation is equal to the degree difference between maximal left-right rotation of each subject Table . DynamicRotation motion is common in a variety of swing sports and daily activities. The acquisition of quantitative data is essential for correctly understanding spine pathology and improving the surgical treatment of spine diseases. In this study, a DFIS was used to investigate the in vivo deformation data of the lumbar disc during axial rotation and compare the effects of different weight loads. Other studies have used this method to measure disc deformation data in the standing position .Our data showed that, relative to the supine position, the average compression deformation caused by rotation motion was between + 10% and − 40% and the shear deformation was between 17 and 50%. Under physiological weightbearing, different levels of lumbar discs exhibited similar deformation patterns, and the left and right rotation deformation patterns were approximately symmetrical. Among them, the posterior side opposite the rotational motion experienced the largest compression deformation. Lower discs had a larger area of tensile deformation than upper discs on the anterior of the same side as the direction of rotational motion. It was also found that the deformation pattern changed significantly under a 10 kg load, with the exception of the L3–4 disc during the right rotation. The disc deformation changed from front to back, as opposed to the diagonal change under weightbearing. The shear deformation range was relatively constant, and no obvious regularity was found.A dynamic axial rotation of the body is complicated and needs a coordinated dynamic coupling of flexion-extension, left-right bending, and left-right axial rotations of the lumbar vertebrae to maintain the global dynamic balance of the body. While the facet joints bear the compressive load of the spine, the facet morphology restricts the movement of the vertebral body. Quantitative knowledge of lumbar kinematics during axial rotation has been investigated in various in vitro and in vivo studies. Li et al.  found thThese studies mainly focused on the ROM of rotational motion and rarely considered disc deformation. There have been many studies focusing on stress changes in the disc, mainly in the standing position and during flexion-extension motion. Wang et al.  measuredIn our study, under a 10 kg load, the coupled bending of the lumbar spine was significantly reduced, resulting in a change in the deformation pattern. The coupled lateral bending assisted in maintaining body balance during body axial rotation, but it may be affected by the extra weight. The combination of extra weight and axial rotation causes the lumbar spine to endure a bending moment during axial rotation, which may lead to an increase in muscle strength and changes in facet joint stress, thus changing the biomechanics of the lumbar spine. This discovery of the changes in disc stress during in vivo loading movement may be vital; it provides a new perspective on the model of three-pillar vertebral segment degeneration after long-term loading.The study has several limitations. Because of small sample size, our observation ability is limited, resulting in some differences that are not statistically significant and a large SD. In addition, due to the size limitations of the fluoroscope and the influence of the machine, we only checked the end-of-motion state of the L3–5 disc and did not analyze the instantaneous position. Despite these limitations, we measured the disc deformation pattern in conjunction with rotation and bending from a new perspective. Future research should include more segments and weightbearing loads.In summary, we found that the deformation of the lumbar disc exhibited direction specificity and level specificity during axial rotation, which was coupled with lateral bending movement to maintain body balance, which can be affected by weight bearing. These findings can provide new insights into the biomechanics of the human lumbar spine and references for finite element and degeneration studies.The deformation of the lumbar disc was direction-specific and level-specific during axial rotations and was affected by extra weight. These data can provide new insights into the biomechanics of the lumbar spine and optimize the parameters of artificial lumbar spine devices.


The histidine–aspartate (HD)-domain protein superfamily contains metalloproteins that share common structural features but catalyze vastly different reactions ranging from oxygenation to hydrolysis. This chemical diversion is afforded by (i) their ability to coordinate most biologically relevant transition metals in mono-, di-, and trinuclear configurations, (ii) sequence insertions or the addition of supernumerary ligands to their active sites, (iii) auxiliary substrate specificity residues vicinal to the catalytic site, (iv) additional protein domains that allosterically regulate their activities or have catalytic and sensory roles, and (v) their ability to work with protein partners. More than 500 structures of HD-domain proteins are available to date that lay out unique structural features which may be indicative of function. In this respect, we describe the three known classes of HD-domain proteins  and identify their apparent traits with the aim to portray differences in the molecular details responsible for their functional divergence and reconcile existing notions that will help assign functions to yet-to-be characterized proteins. The present review collects data that exemplify how nature tinkers with the HD-domain scaffold to afford different chemistries and provides insight into the factors that can selectively modulate catalysis. These six features appear to distinctly influence the range of chemistries these proteins perform. Despite variations in primary sequence, all HD-domain proteins share the HD residue dyad that coordinates transition metal ions such as Fe, Mn, Co, Mg, Cu, Zn, and Ni  as well The HD-domain superfamily fosters three different enzymatic classes: hydrolases, oxygenases, and lyases, with the hydrolases being the largest and best-characterized group. Hydrolases are further subdivided into (i) phosphatases, including dGTPases , RelA/Sp1.2.4A ribonucleotides and signaling molecules involved in bacterial stress responses ,10,16,424A . Phospha4A , which iIn most cases, phosphate hydrolysis is supported by non-redox metal ions, with the most active cofactors being Co or Mn for monophosphatases, Mn for diphosphatases, and Mg or Mn for triphosphatases . With a 1.2.1.cat/KM of 25.6 × 103 M−1s−1 . Th. Th22]. 1.3.PDEs can harbor both mononuclear and polynuclear cofactors. A common feature of all known polynuclear (di-or tri-nuclear) PDEs is an extra histidine residue in the active site such that their characteristic metal binding sequence is “H … HD … H … HH … D” . The rol1.3.1.Clustered regularly interspaced short palindromic repeats (CRISPR)-associated systems (Cas) are major players in prokaryotic adaptive immunity and RNA-based defense ,51. TypeThermobifida fusca Cas3 shows a diiron active site (Pyrococcus furiosus Cmr2 (dinuclear) and the Cas3′’ proteins (mononuclear in the published structures) exhibit an Mg-dependent PDE activity .,35.14,35HD-GYPs differ on the basis of their active metal cofactor and possible catalytic outcomes. While most commonly harbor a dimetal cofactor, some incorporate a trinuclear cofactor by involving a glutamate residue as an eighth ligand to the site  36]. Th. Th36]. Persephonella marina is the prototypical trinuclear HD-GYP and the first to be crystallographically characterized . Th. Th15]. enerated , and thu2 binding and subsequent turnover and most likely serves as a protective mechanism to prevent oxidative inactivation of the active site  is oriented toward the active site and binds to the Fe2 site, while the glutamate (E27 in PhnZ and E33 in TmpB) faces away from the active site ,15. Subsive site .Collectively, HD-domain oxygenases have catalytic and structural features that differ significantly not only from that of other nonheme–iron oxygenases, but also of HD-domain hydrolases. This divergence is useful as it can provide some descriptors to distinguish oxygenases from hydrolases within the HD-domain family. It is likely that these characteristics are conserved among all HD-domain oxygenases and may provide a critical first step into the characterization of other HD-domain proteins of unknown function.1.5.Myrothecium verrucaria and Saccharomyces (S.) cerevisiae, have developed cyanamide biodegradation pathways as a likely defense mechanism against cyanamide-producing plants. The cyanamide hydratase Ddi2, although currently not well characterized, catalyzes the conversion of cyanamide to urea  metal ions.To date, there is no established correlation between the chemical nature of the metallocofactor and the type of the reaction performed by the HD-domain enzymes. The employment of an Fe-based cofactor was initially thought to occur solely in oxygenases and be unfavorable for hydrolases due to the redox-active nature of Fe. However, more and more hydrolytic enzymes are found to incorporate catalytically relevant Fe-based cofactors, revoking this notion. Whereas some HD-domain hydrolases are selective for the types of divalent metal ions that can stimulate activity , others are rather promiscuous and can utilize most of the first-row transition metal ions . It is still puzzling why the metal ion selectivity is wide and why some of the proteins are highly selective, while others are highly unselective, especially when considering the high degree of conservation of the HD-domain binding motif. It could be that metal ion selection is linked to the environmental niches of the organisms in which the related HD-domain proteins are found or could reflect the apparent independence of a specific reaction on the exact chemical nature of the metal ion. Overall, there appears to be a loose relationship between the type of cofactor and the type of reaction catalyzed. All currently reported HD-GYP PDEs are dinuclear (or trinuclear), highlighting perhaps the importance of both metal ions in substrate binding and hydrolysis. All presently known oxygenases are also dinuclear. On the basis of their structure and mechanism, one of the metal ions is employed in molecular oxygen binding and the second one is involved in cosubstrate binding. However, cofactor selection on the basis of activity type breaks down for phosphatases, as both mono- and dinuclear active site configurations can act on similar (if not the same) substrates. There is an observed trend, in which bulkier substrates or compounds with multiple hydrolyzable bonds are most likely to be processed by multinuclear HD-domain enzymes. Presumably, in these cases the extended active site structure facilitates substrate binding and catalysis.The diversity of the HD-domain metalloproteins is continuing to expand as more members become biochemically characterized, allowing for the identification of specific traits that can act as molecular descriptors and predictors of function. Within this ever-growing family, these features may aid in the assignment of HD-domain proteins with unknown activities as well as new hydrolases, oxygenases, lyases, or proteins of new unidentified chemistries.