Structure of the Calvin-Benson-Bassham sedoheptulose-1,7-bisphosphatase from the model microalga Chlamydomonas reinhardtii

Reviewer #1 (Public Review):

In this study, Le Moigne and coworkers shed light on the structural details of the Sedoheptulose-1,7-Bisphosphatase (SBPase) from the green algae Chlamydomonas reinhardtii. The SBPase is part of the Calvin cycle and catalyzes the dephosphorylation of sedoheptulose-1,7-bisphosphate (SBP), which is a crucial step in the regeneration of ribulose-1,5-bisphosphate (RuBP), the substrate for Rubisco. The authors determine the crystal structure of the CrSBPase in an untreated, oxidized state. Based on this structure, potential active site residues and sites of post-translational modifications are identified. Furthermore, the authors determine the CrSBPase structure in a reduced state revealing the disruption of a disulfide bond in close proximity to the dimer interface. The authors then use molecular dynamics (MD) to gain insights into the redox-controlled dynamics of the CrSBPase and investigate the oligomerization of the protein using small-angle X-ray scattering (SAXS) and size-exclusion chromatography. Despite the difference in oligomerization, disruption of this disulfide bond did not impact the activity of CrSBPase, suggesting additional thiol-dependent regulatory mechanisms modulating the activity of the CrSBPase.

The authors provide interesting new findings on a redox-mechanism that modulates the oligomeric behavior of the SBPase. Comparisons of the Chlamydomonas structure to the previously determined SBPase structure from the moss Physcomitrium patens confirm a high structural similarity between the two proteins suggesting that this mechanism might be evolutionary conserved. Future research will have to address this question experimentally, also considering potential cooperativity between the subunits to confirm the link between oligomerization and SBPase activity.

Reviewer #2 (Public Review):

The central theme of the manuscript is to report on the structure of SBPase - an enzyme central to the photosynthetic Calvin-Benson-Bassham cycle. The authors claim that the structure is the first of its kind from a chlorophyte Chlamydomonas reinhardtii, a model unicellular green microalga. The authors use a number of methods like protein expression, purification, enzymatic assays, SAXS, molecular dynamics simulations and x-ray crystallography to resolve a 3.09 A crystal structure of the oxidized and partially reduced state. The results are supported by the claims made in the manuscript. While the structure is the first from a chlorophyte, it is not unique. Several structures of SBPase are available and a comparison has been made between the structure reported here and others that have been previously published.

Author response:

The following is the authors’ response to the original reviews.

eLife assessment

The manuscript reports useful findings by resolving the crystal structure of Sedoheptulose-1,7-Bisphosphatase (SBPase) from the green algae Chlamydomonas reinhardtii, which is involved in the Calvin cycle. The data presented are solid based on validated methodologies, which help in understanding the structure and function of this enzyme.

We thank the editors for this positive assessment.

Public Reviews:

Reviewer #1 (Public Review):

In this study, Le Moigne and coworkers shed light on the structural details of the Sedoheptulose-1,7-Bisphosphatase (SBPase) from the green algae Chlamydomonas reinhardtii. The SBPase is part of the Calvin cycle and catalyzes the dephosphorylation of sedoheptulose-1,7-bisphosphate (SBP), which is a crucial step in the regeneration of ribulose-1,5-bisphosphate (RuBP), the substrate for Rubisco. The authors determine the crystal structure of the CrSBPase in an oxidized state. Based on this structure, potential active site residues and sites of post-translational modifications are identified. Furthermore, the authors determine the CrSBPase structure in a reduced state revealing the disruption of a disulfide bond in close proximity to the dimer interface. The authors then use molecular dynamics (MD) to gain insights into the redox-controlled dynamics of the CrSBPase and investigate the oligomerization of the protein using small-angle X-ray scattering (SAXS) and size-exclusion chromatography. Despite the difference in oligomerization, disruption of this disulfide bond did not impact the activity of CrSBPase, suggesting additional thiol-dependent regulatory mechanisms modulating the activity of the CrSBPase.

We thank reviewer 1 for his/her careful reading of our manuscript.

The authors provide interesting new findings on a redox-mechanism that modulates the oligomeric behavior of the SBPase, however without investigating this potential mechanism in more detail. The conclusions of this manuscript are mostly supported by the data, but they should be more carefully evaluated in respect to what is known from other systems as e.g. the moss Physcomitrella patens. This is especially of interest, as SBPase was previously reported to be dimeric, whereas for FBPase a dimer/tetramer equilibrium has been observed.

We thank reviewer 1 for his/her comments on the novel or confirmatory character of our structure-function analysis onCrSBPase. We address the questions of oligomeric states later in this response.

(1) Given that PpSBPase has been already characterized in detail, the authors should provide a more rigorous comparison to the existing data on SBPases. This includes a more conclusive structural comparison but also the enzymatic assays should be compared to the findings from P. patens. Do the authors observe differences between the moss and the chlorophyte systems, maybe even in regard to the oligomerization of the SBPase?

Indeed, a previous study conducted by one of the authors of the current manuscript (Stéphane D. Lemaire) and collaborators determined the structure and regulatory properties of SBPase from the moss Physcomitrella patens (Gütle et al. 2018 https://doi.org/10.1073/pnas.1606241113). We added a clearer reference to this earlier work. The differences that we observed regarding the oligomeric states of SBPase from Chlamydomonas reinhardtii principally stem from our analytical method in vitro through size-exclusion chromatography, in comparison with crystal packing analysis in the reference study. We detailed PpSBPase/CrSBPase oligoimeric state comparison in the paragraph 'Oligomeric states of CrSBPase'. Besides, the asymmetric unit of our CrSBPase crystal structure is also a homodimer, similarly to PpSBPase, and we suggest that PpSBPase is also likely to adopt several oligomeric states in vitro. If this were confirmed by experiments, SBPase in several organisms would behave analogously to FBPase regarding the dimer/tetramer equilibrium.

In paragraph 'Crystal structure of CrSBPase' we added a comparison by alignment of our CrSBPase crystal structure to the previously reported _Pp_SBPase crystal structure, stating that with RMSD=0.478 Å the proteins are essentially identical.

In paragraph 'CrSBPase enzymatic activity' we compared the value we obtained for enzyme specific activity to those previously published on other SBPase from Chlamydomonas or the land plant Spinacia oleracea, highlighting the similarity of results in three different systems and teams (Seuter et al. 2002 https://doi.org/10.1023/A:1019297521424 and Tamoi et al. 2005 DOI: 10.1271/bbb.69.848).

(2) The authors should include the control experiments (untreated SBPase) and the assays performed with mutant versions of the SBPase, which are currently only mentioned in the text or not shown at all.

We add supplementary figure 14 in order to illustrate that since SBPase C115S or C120S mutants are still activated by reducing agent, the disulfide bridge between cysteines 115 and 120 is not the single control over SBPase activity but rather a control over the oligomeric exchange of the enzyme indirectly contributing to redox activation of the active site.

(3) The representation of the structure in figures (especially Figures 1 and 3) should be adjusted to match the author's statements. In Figure 1, the angle from which the structure is displayed changes over the entire figure making it difficult to follow especially as a non-structural biologist. Furthermore, important aspects of the structure mentioned in the text are not labeled and should be highlighted, by e.g. a close-up. Same holds true for Figure 3 that currently mostly shows redundant information.

We thank reviewer 1 for his/her advise on how to improve Figure 1. We drew new images for the complete figure, hopefully providing more consistent and clearer visual support to our text. For simplicity, protein is now always represented centered around its active site in the same orientation. We represent co-crystallized water in all projections as a guide to the eye.

Figure 3 and supplementary figure 3 were switched in order to better represent the experimental evidence provided by the resolution of SBPase structure under reducing conditions, i.e., the increase in local disorder around C115-C120 pair of cysteines in the 113-130 stretch forming a redox-conditionally dynamic loop and β-hairpin motif.

(4) The authors state that mutation of C115 and C120 to serine destabilize the dimer formation, while more tetramer and monomer is formed. As the tetramer is essentially a dimer of dimers, the authors should elaborate how this might work mechanistically. In my opinion, dimer formation is a prerequisite for tetramer formation and the two mutations rather stabilize the tetramer instead of destabilizing the dimer.

Time-dependent dynamic character of SBPase oligomer exchange is not resolved by the current study because we essentially combined size-exclusion chromatography (SEC) and X-ray crystallography to define quaternary structures at equilibrium. Overall, homodimer is the dominant state of wild-type SBPase by abundance in the purified recombinant form and by forming the constitutive asymmetric unit in all crystal packings. Dimer is indeed present in the tetramer state, a dimer of dimers, as pertinently stated by reviewer 1.

This being recognized, we tried to explain the systematic co-elution of the principal dimeric form with an additional species of smaller size on SEC (supplementary figure 1, right-side shoulder of the peak), at the apparent mass of a monomer. When solving the crystal structures of SBPase we realized that the dimer interface is contributed by residues 113-130 forming a loop and β-hairpin motif. Notably, in this loop cysteine 115 (C115) maps at bonding distance of 3.9 Å of side chain of arginine 220 (R220) from dimer partner subunit. In loop 113-120, cysteine pair C115 and C120 are subject to redox switching between disulfide (closed) and dithiol (open) conformations, as shown in our structures 7B2O and 7ZUV, respectively. Given that the reduction of C115-C120 disulfide bridge correlates with a higher flexibility of this motif that contributes to dimer interface (figure S3), we hypothesized that reduction of SBPase would destabilize dimer state to the benefit of transitory monomer state, and indeed point mutagenesis of C115S or C120S caused a large modification of oligomer equilibrium in favour of the monomer (figure S1C).

Mechanistically, we suggest two scenarios for the tetramer formation: either monomers first interact as in the crystallographic dimer before pairing such dimers into tetramers (as proposed by reviewer 1), or monomers start tetramerization by favoring the alternative subunit interface (figure 5B, between cyan and magenta chains) before stabilizing the crystallographic homodimer interface. In this latter case, monomerization would be necessary to efficiently re-arrange SBPase dimers into tetramers.

In physiological conditions the re-arrangement switch would be controlled by C115-C120 reduction through ferredoxin-thioredoxin redox cascade. Structural studies in dynamic conditions like native mass spectroscopy/photometry would be necessary to solve this speculation unambiguously although at this stage of our investigation there seem little doubt to us that C115-C120 disulfide-dithiol exchange is essential to control a dimer/monomer balance in first instance.

Reviewer #2 (Public Review):

The central theme of the manuscript is to report on the structure of SBPase - an enzyme central to the photosynthetic Calvin-Benson-Bassham cycle. The authors claim that the structure is first of its kind from a chlorophyte Chlamydomonas reinhardtii, a model unicellular green microalga. The authors use a number of methods like protein expression, purification, enzymatic assays, SAXS, molecular dynamics simulations and xray crystallography to resolve a 3.09 A crystal structure of the oxidized and partially reduced state. The results are supported by the claims made in the manuscript. One of the main weakness of the work is the lack of wider discussion presented in the manuscript. While the structure is the first from a chlorophyte, it is not unique. Several structures of SBPase are available. As the manuscript currently reads, the wider context of SBPase structures available and comparisons between them is missing from the manuscript. Another important point is that the reported structure of crSBPase is 0.453A away from the alphafold model. Though fleetingly mentioned in the methods section, it should be discussed to place it in the wider context.

We thank reviewer 2 for his/her assessment of our manuscript. In response to his/her suggestion to better compare our SBPase structure from the model microalga Chlamydomonas reinhardtii to that of the ortholog from Physcomitrium patens previously reported by an author of this manuscript (Stéphane D. Lemaire) and collaborators (Gütle et al. 2018), we wish to point out that paragraph 3 of the introduction was dedicated to this reference along with a mention to related Thermosynechococcus elongatus dual function fructose-1,6-bisphosphatase sedoheptulose-1,7-bisphosphatase (F/SBPase). We nevertheless follow his/her suggestion to better detail comparison between chloroplastic SBPase structures in the first result section 'Crystal structure of CrSBPase', consistently with response 1 to reviewer 1 (see above).

Regarding the integration of AlphaFold (AF) computational models in a general discussion about SBPase molecular structure, we wish to point out that our initial 7B2O crystallographic model of CrSBPase was deposited in PDB on 2020-11-27 before AlphaFold2 was available for the scientific community (Jumper et al. publication date is 15 July 2021).

AF2 entry AF-P46284-F1-model_v4 from AlphaFold Protein Structure Database aligns with our crystal structure 7B2O chain E with RMSD = 0.434 Å, showing excellent agreement between experiment and prediction at the level of protein main chain. It must still be pointed out that it is the AF2 model which is at 0.434 Å away from the experiment, and not the opposite. Exceptions of alignments are in local differences in several loops conformations and in the length of secondary structure elements. Many amino acid residues side chains adopt distinct orientations between the computational model and the experimental structure.

AF3 was recently communicated (Abramson et al. 2024) along with its online prediction server hosted at https://golgi.sandbox.google.com. CrSBPase model from AF3 align to our crystal structure 7B2O chain A with RMSD = 0.489 Å showing again their strong similarity and with a smaller discrepancy between AF2 and AF3 of RMSD = 0.216 Å. The only significant deviations between 7B2O and AF3 are in the orientation of several side chains and notably on the conformation of region 114-131 that contain the redox sensor motif.

We added the last two paragraphs to the revised version of the manuscript, after the results section presenting our crystallographic work.

Recommendations for the authors:

We made all recommended modifications as detail below.

Reviewer #1 (Recommendations For The Authors):

I have outlined a number of minor points below.

We addressed all minor points listed.

Line 220: The asymmetric unit only contains three dimers. The dimer of dimer or tetramer can only be reconstituted by displaying the symmetry mates.

We corrected our sentence for 'The asymmetric unit is composed of six polypeptide chains packing as three dimers'.

I also suggest that the authors separate the description of the asymmetric unit content from the modeled water molecules and rephrase e.g. „..and four water molecules could be modeled."

We rephrased as suggested.

I appreciate that the authors uploaded the structure in advance of this article, which allowed to evaluate the quality of the structure. Although this does not add valuable information, I have identified several unmodeled blobs, which possibly also account for waters.

Unmodeled blobs were tentatively assigned to water but had to be removed during later refinements. We used Coot Validate tools 'Unmodelled blobs' and 'Check/Delete water' to progress towards the current optimal refinement statistics. We admit that the resolution of the crystallographic dataset (3.09 Å) is limiting to reliably model mobile or less resolved elements like water molecules. Overall, we estimate that the functional elements of the structure are modeled to the best of our knowledge and with minimal subjectivity.

Line 222: Please write 309 instead of spelling the number.

We corrected for 309 instead of spelling the number.

Line 223: The structure representation in Figure 1A/B has to be improved. The authors might consider labeling the two domains & color them in two colors instead of the rainbow color coding. Furthermore, the 90{degree sign} rotation does not add much information. Here, turning the model in a different direction that allows to see the central b-sheet of domain 2 might be better suited. Furthermore, instead of describing b-strands first, followed by a-helices, I suggest describing which secondary structure elements form the two domains.

We improved Figure 1A as suggested while keeping Figure 2B with 90° rotation as rainbow color gradient in order to display with clarity the secondary structure content and connectivity. The orientation was tilted to better display the central β-sheet. This new version of Figure 1A/B should facilitate the text description of SBPase architecture that we amended as suggested.

Line 229: The information on A113-120 should be depicted in a closeup in Figure 1A.

We made a close-up view of sequence 113-120 as added figures 1C-D and modified the rest of the figure and legend accordingly.

Line 234: Please provide an r.m.s.d here.

We now provide r.m.s.d. for all structural alignments.

Line 242: Please introduce the domain labeling in Fig 1C to make it easier to track the exact region within SBP here. Is the residue numbering according to SBP or the human FBP?

Modified version of figure 1 now shows SBPase in the same orientation for panels A, E, F, G, H for simplicity. Domains labeling is indicated in panel A with NTD/CTD distinct colors as suggested. We explicited the position of W401 on all panels as a guide to the eye. We indicated in figure legend that residue numbering is according to Chlamydomonas SBPase Uniprot entry P46284.

Line 244: Is Figure 1D in the same orientation as C? I suggest making the surface transparent and showing the cartoon below, which will allow to easier see the solvent accessibility of the residues. Also, clearly label W401 (although it's the only water shown/modeled in this region).

We modified figure 1 to show all equivalent panels (ie. A-E-F-G-H) with the same orientation. In this new form we think that solvent accessibility and the relative position of significant residues is easier to interpret for the reader. W401 is consistently labeled throughout figure 1 panels.

Line 263: Please provide a close-up of the C222 and C231 including measured distance. It's clearly not visible from this view. It might even be helpful to provide close-ups of all cysteine residues that are mentioned in the text.

In the modified version of figure 1 we estimate that C222 and C231 are more easily visible. We added a close-up view of C22-C231 environment in a new supplementary figure 2. Since we do not explore further the functional relevance of this redox pair we chose not include C222-C231 close-up view in main figure 1. We added legends and modified supplementary figures numbering accordingly.

Line 276: As already mentioned earlier, none of the panels in Figure 1 provide a close-up of this loop. This should be added.

This loop is now displayed as a close-up view in panels C and D of main figure 1.

Line 284: It is difficult to follow the relative positions of the potential modification sites if the model is always depicted from a different angle in Figure 1. The authors might want to change this across Figure 1 or show the rotation angle.

This problem was addressed in the revised figure 1, panels A-E-F-G-H are in the same orientation now. Panel B was kept at a rotation of 90° with corresponding annotation.

Line 290: Please label W401. Also stick to one nomenclature (W or H20).

We labeled W401 and kept nomenclature consistent throughout the manuscript.

For comparative reasons, a full kinetic measurement (determination of Km and kcat) of the SBPase would also be helpful here.

We resolved to avoid a full kinetic measurement of CrSBPase because we could neither identify a reliable chemical provider nor synthesize ourselves the physiological substrate sedoheptulose-1,7-bisphosphate (SBP) and only characterized the reaction with fructose-1,6-bisphosphate. However, in the revised form of the manuscript we added in main text paragraph 'CrSBPase enzymatic activity' the kinetic constants from the previous reference study conducted on spinach SBPase (Cadet and Meunier, Biochem. J. 1988) with KMSBP=0.05 mM and kcatSBP=81 sec-1 of fully active enzyme with SBP as a substrate. For comparison, the authors of this study report that activity of SBPase on FBP is in the same range but lower, with KMFBP=0.38 mM and kcatFBP=21 sec-1. We also added a comparison of specific activities of our CrSBPase and spinach SBPase in the main text, showing that our enzyme behaves as previously reported ortholog from land plant.

Line 303: How much MgSO4 was used for the experiment shown in Figure 2A?

10 mM of MgS04 was used for experiment shown in Figure 2A. We added this information in the figure legend. We also added in the legend that 10 mM DTT is present in the experiment of Figure 2B and that 10 mM of MgSO4 and 1 mM of DTT are present in the experiment of Figure 2C.

Line 321: In my opinion it is not necessary to show the regions of all molecules here. I was rather expecting a superposition of the two structures (oxidized and reduced) with a close-up of the respective disulfide in the two states.

We agree that the initial version of Figure 3 panels showing side-by-side all conformational variants of the redox motif appear redundant. We switched initial Figure 3 to supplementary data and replaced it with the crystallographic b-factor mapping of the redox motif, in the variable conditions resolved by the crystals. We would like to stress that all these conformations were experimentally determined through X-ray crystallography, whether of the crystal of pure inactive enzyme that proved to be oxidized on the redox motif, or of the equivalent crystals submitted to activating treatment by the chemical reductant TCEP. As an attempt to clarification we added visual boxes to better appreciate this reduction-induced conformational plasticity that we interpreted as a local conditional disorder.

Line 331: Could the authors provide movies of the MD simulation? Otherwise, interpretation of the MD simulation results might be difficult for non-experts.

We added two movies of 20-µsec MD simulations as supplementary data to help non-expert readers.

Line 343: It might be helpful to label the structure elements in Figure 4 accordingly (e.g. residues, etc.)

We added secondary structure labeling in Figure 4.

Line 381: Should be changed to Figure 5A.

We changed reference to figure 6 that is a renumbering of figure 5 with changes included from suggestions below. Figure 6 now includes chromatograms of recombinant SBPase in panel A and chromatogram and western blot analysis of Chlamydomonas extracts in panel B.

Line 383: See above, figure 5B. Which structure is shown in the figure? 7zuv or 7b2o? Maybe include both structures in the figure in a side-by-side view. The authors might also want to include the SEC chromatograms in the main figure. Especially the purification from Chlamydomonas is helpful to estimate whether post-translational modifications have an impact on the oligomerization. This should also be mentioned in the text.

7b2o and 7zuv are illustrated side-by-side in panels A and B of figure 5. This was indicated in the figure legend, we now added the information on the figure. As suggested above we included chromatograms initially presented as supplementary material in a new main figure 6, panel A for recombinant proteins and panel B for proteins extracted from Chlamydomonas. Initial figures 5D-E, showing surface conservation of the dimeric SBPase, is moved to supplementary figure 5.

Line 385: I don't find the cultivation of Chlamydomonas in the method section. It should be added.

We added a methods paragraph dedicated to « Cultivation of Chlamydomonas for native SBPase analysis ».

Line 390-392: This information is not really helpful. Concentrated purified proteins might precipitate after a week storage without physiologically relevant effects being the reason.

We agree that the observation of a precipitate building up in vitro after a week of storage bears no particular physiological implications. We rather intended to report that an aggregated form of purified protein can be turned to droplets under the redox conditions that activate the enzyme. We reformulated these lines for clarification.

Line 397: I would appreciate having the SEC-chromatograms of the mutants also in the main figure.

Size-exclusion chromatograms that were initially in supplementary figures are now shown in main text figure 6 panel A, with the profiles WT and mutants aligned.

Line 402: Where are these data shown? They should be included in Figure 5.

We added a figure to present these data, not shown in the initial version of the manuscript. We preferred to place it as supplementary material because C115S and C120S mutant catalytic activity is essentially the same as WT and do not reveal a direct mechanistic effect of C115-C120 reduction over the catalytic pocket.

Line 427: Did the authors look into a possible cooperativity of their SBPase?

We did not observe direct positive cooperativity that could be ascribed to allostery in our enzymatic assays. It was previously reported for spinach SBPase that SBP saturation functions were hyperbolic with no evidence of homotropic interactions in the enzyme oligomer (Cadet and Meunier Biochem J. 1988 253, 249-254). The authors of this kinetic study however present a clear sigmoid response of SBPase to Mg2+ concentration, suggestive of an activating cross-talk between active sites in the oligomer. We consider this hypothesis of interest and wish we could further investigate allosteric conformational changes when SBP physiological substrate would be available.

Line 428-434: I don't really understand how the proteome mapping fits in here. Do the authors speculate that SBPase is recruited by some of the identified enzymes or directly interacts with them or that rather the spatial distribution optimizes the reaction kinetics?

We indeed want to correlate our in vitro observations of CrSBPase conditions of activity to those recently published by the group of Dr. Martin Jonikas in a physiological, in vivo setup of Chlamydomonas reinhardtii (Wang, Patena et al. Cell 2023 186, 3499–3518). We have no experimental evidence demonstrating the first suggestion that SBPase is recruited or directly interacts with partner enzymes but we privilege the second suggestion that local spatial distribution in the chloroplast stroma optimizes enzyme reaction kinetic thanks to Calvin-Benson-Bassham enzymes proximity. We rephrased these lines to clarify our hypothesis and express its speculative character.

Reviewer #2 (Recommendations For The Authors):

To make the manuscript stronger, the authors are recommended to do the following:

We followed given recommendations.

(1) include a wider discussion on the other SBPase structures that are available. A detailed comparison should be made between the oxidized and reduced structures present in the PDB with the structures that are being reported in the manuscript.

Consistently with reviewer #1 suggestion, and as detailed in response to public review above, we followed the recommendation to better report previous structural studies of SBPase in the results section. We also added comparisons with computational models from AlphaFold2 and AlphaFold3.

(2) The authors mention co-operativity between the subunits. With excellent sampling from molecular dynamics simulations, the authors should demonstrate co-operativity between the subunits.

Our molecular dynamic (MD) simulations span 20 µsec of SBPase in the dimeric state, starting from the experimental structures determined by XRC. In the considered time window, the only significant events that we observed are the local reorganization of the LBH motif that is a prerequisite for dimer rearrangement. We infer that local disorder contributes a separation of the pair of subunits in order to later allow for the building of the active homotetramer, at longer time scales that are outside the capacities used in this work. Moreover, demonstrating cooperativity with MD simulations would require more than a single event to ensure that results are significant, and performing series of 20µs-MD of SBPase is also outside the available capacities.