Activity modulation in anaerobic ribonucleotide reductases: nucleotide binding to the ATP-cone allosterically mediates substrate binding to the active site

Reviewer #1 (Public Review):

The goal of this study is to understand the allosteric mechanism of overall activity regulation in an anaerobic ribonucleotide reductase (RNR) that contains an ATP-cone domain. Through cryo-EM structural analysis of various nucleotide-bound states of the RNR, the mechanism of dATP inhibition is found to involve order-disorder transitions in the active site. These effects appear to prevent binding of substrate and a radical transfer needed to initiate the reaction.

Strengths of the manuscript include the comprehensive nature of the work - including both numerous structures of different forms of the RNR and detailed characterization of enzyme activity to establish the parameters of dATP inhibition. The manuscript has been improved in a revision by performing additional experiments to help corroborate certain aspects of the study. But these new experiments do not address all of the open questions about the structural basis for mechanism. Additionally, some questions about the strength of biochemical data and fit of binding or kinetic curves to data that were raised by other referees still remain. Some experimental observations are not consistent with the proposed model. For example, why does dATP enhance Gly radical formation when the proposed mechanism of dATP inhibition involves disorder in the Gly radical domain?

The work is impactful because it reports initial observations about a potentially new mode of allosteric inhibition in this enzyme class. It also sets the stage for future work to understand the molecular basis for this phenomenon in more detail.

Reviewer #3 (Public Review):

The manuscript by Bimai et al describes a structural and functional characterization of an anaerobic ribonucleotide reductase (RNR) enzyme from the human microbe, P. copri. More specifically, the authors aimed to characterize the mechanism by how (d)ATP modulates nucleotide reduction in this anaerobic RNR, using a combination of enzyme kinetics, binding thermodynamics, and cryo-EM structural determination, complemented by hydrogen-deuterium exchange (HDX). One of the principal findings of this paper is the ordering of a NxN 'flap' in the presence of ATP that promotes RNR catalysis and the disordering (or increased protein dynamics) of both this flap and the glycyl radical domain (GRD) when the inhibitory effector, dATP, binds. The latter is correlated with a loss of substrate binding, which is the likely mechanism for dATP inhibition. It is important to note that the GRD is remote (>30 Ang) from the binding site of the dATP molecule, suggesting long-range communication of the structural (dis)ordering. The authors also present evidence for a shift in oligomerization in the presence of dATP. The work does provide evidence for new insights/views into the subtle differences of nucleotide modulation (allostery) of RNR, in a class III system, through long-range interactions.

The strengths of the work are the impressive, in-depth structural analysis of the various regulated forms of PcRNR by (d)ATP using cryo-EM. The authors present seven different models in total, with striking differences in oligomerization and (dis)ordering of select structural features, including the GRD that is integral to catalysis. The authors present several, complementary biochemical experiments (ITC, MST, EPR, kinetics) aimed at resolving the binding and regulatory mechanism of the enzyme by various nucleotides. The authors present a good breadth of the literature in which the focus of allosteric regulation of RNRs has been on the aerobic orthologues.

The addition of hydrogen-deuterium exchange mass spectrometry (HDX-MS) complements the results originating from cryo-EM data. Most notably, is the observation of the enhanced exchange (albeit quite subtle) of the GRD domain in the presence of dATP that matches the loss of structural information in this region in the cryo-EM data. The most pronounced and compelling HDX results are seen in the form of dATP-induced protection of peptides immediately adjacent to the b-hairpin at the s-site, where dATP is expected to bind based on cryo-EM. It is clear that the presence of dATP increases the rigidity of this region.

Weaknesses: The discussion of the change in peptide mobility in the N-terminal region is complicated by the presence of bimodal mass spectral features and this may prevent detailed interpretation of the data, especially for select peptide region that shows opposite trends upon nucleotide association. Further, the HDX data in the NxN flap is unchanged upon nucleotide binding (ATP, dATP, or CTP), despite changes observed in the cryo-EM data.

Author Response

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

We thank the Reviewing Editor and two additional reviewers for the insightful input they gave us on the first version of our manuscript on allosteric activity regulation of the anaerobic ribonucleotide reductase from Prevotella copri. We have revised the manuscript in the light of the reviewers' comments. In particular, we have added additional experiments using hydrogen-deuterium exchange mass spectrometry (HDX-MS) to probe the accessibility and mobility of different parts of the protein structure in the apo-state and in the presence of dATP/CTP and ATP/CTP. The results strongly confirm the binding of nucleotides to the activity and specificity sites, as seen biochemically and structurally. In the question of mobility of the glycyl radical domain the HDX-MS experiments suggest an increased mobility in the presence of dATP, though the results are not as clear-cut as for the nucleotide binding. The HDX-MS analyses are complicated by the fact that they reflect all species in solution, which are evidently multiple for all states of PcNrdD. Finally, we have rephrased key parts of the results and discussion, and modified the title, to avoid any implication that we believe the glycyl radical domain becomes extensively disordered, rather that it becomes more mobile to the extent that it cannot be seen in the cryo-EM structures.

eLife assessment

This study advances our understanding of the allosteric regulation of anaerobic ribonucleotide reductases (RNRs) by nucleotides, providing valuable new structural insight into class III RNRs containing ATP cones. The cryo-EM structural characterization of the system is solid, but other aspects of the manuscript, which are incomplete, could be improved by including additional functional characterization and more evidence for the proposed mechanism of inhibition by dATP. The work will be of interest to biochemists and structural biologists working on ribonucleotide reductases and other allosterically regulated enzymes.

Public Reviews:

Reviewer #1 (Public Review):

The goal of this study is to understand the allosteric mechanism of overall activity regulation in an anaerobic ribonucleotide reductase (RNR) that contains an ATP-cone domain. Through cryo-EM structural analysis of various nucleotide-bound states of the RNR, the mechanism of dATP inhibition is found to involve order-disorder transitions in the active site. These effects appear to prevent substrate binding and a radical transfer needed to initiate the reaction.

Strengths of the manuscript include the comprehensive nature of the work - including numerous structures of different forms of the RNR and detailed characterization of enzyme activity to establish the parameters of dATP inhibition. The manuscript could be improved, however, by performing additional experiments to establish that the mechanism of inhibition can be observed in other contexts and it is not an artifact of the structural approach. Additionally, some of the presentations of biochemical data could be improved to comply with standard best practices.

The work is impactful because it reports initial observations about a potentially new mode of allosteric inhibition in this enzyme class. It also sets the stage for future work to understand the molecular basis for this phenomenon in more detail.

We thank the editor and reviewers for their positive evaluation of the potential impact of our work. We completely agree that hypotheses based on structural data require orthogonal experimental verification. However, the number and consistency of the cryo-EM structures speak in favour of the data being representative of conditions in solution. We feel that in particular cryo-EM data should be relatively free of artefacts, e.g. biased or incorrect relative domain orientations, compared to crystallography, where crystal packing effects can affect these parameters. As we write in response to Reviewer #2, it has been difficult to propose a direct structural mechanism for transmission of the allosteric signal from the a-site in the ATP-cone to the active site and GRD given that the ATP-cones and linker are disordered in the dATP-bound dimers and only partly ordered in the dATP-bound tetramers. Further verification experiments will be performed in future but are outside the scope of the present article.

We will improve the presentation of the biochemical data in a revised version.

General comments:

(1) It would be ideal to perform an additional experiment of some type to confirm the orderdisorder phenomena observed in the cryo-EM structures to rule out the possibility that it is an artifact of the structure determination approach. Circular dichroism might be a possibility?

Circular dichroism reports only on the approximate relative proportions of helix, sheet and loop structure in a protein, thus we believe that it would not be a sensitive enough tool to distinguish between ordered and disordered states. We are considering what alternative methods might be appropriate.

(2) Does the disordering phenomenon of one subunit in the ATP-bound structures have any significance - could it be related to half-of-sites activity? Does this RNR exhibit half-of-sites activity?

Half-of-sites activity has not been biochemically proven in any ribonucleotide reductase in spite of the fact that it was first suggested in 1987 (PMID: 3298261). However, strong structural indication was recently published in the form of the holo-complex of the class Ia ribonucleotide reductase from Escherichia coli, which is highly asymmetrical and in which productive contacts forming an intact proton-coupled electron transfer pathway are only formed between one of two pairs of monomers (PMID: 32217749). We have not been able to prove half-of-sites activity for PcNrdD due to low overall radical content, but the structural results are indeed consistent with such an activity.

(3) Does the disordering of the GRD with dATP bound have any long-term impact on the stability of the Gly radical? I realize that the authors tested the ability to form the Gly radical in the presence of dATP in Fig. 4 of the manuscript. But it looks like they only analyzed the samples after 20 min of incubation. Were longer time points analyzed?

Radical content was measured after 5 min and 20 min incubation; 5 min incubations (not included in the manuscript) consistently gave higher radical content compared to 20 min incubation. Longer time points were not analysed, as we assumed that the radical content would be even lower after 20 min.

(4) Did the authors establish whether the effect of dATP inhibition on substrate binding is reversible? If dATP is removed, can substrates rebind?

This is an interesting question. We measured KDs for dATP in the micromolar range and are hence confident that dATP binding is reversible. Our measurements do not, however, directly prove that inhibition of the enzyme is reversible. Nevertheless, it is worth noting that the protein as purified was precipitated and analysed by the UV-visible spectrum. The aspurified PcNrdD contained 30% nucleotide contamination. The as-purified sample was then analysed by HPLC and we identified a major peak, corresponding to dATP/dADP. Therefore, purification conditions had to be optimised to remove the nucleotides. This is evidence that PcNrdD that has “seen” dATP can subsequently bind substrates in the presence of ATP. We will describe the purification more clearly in a revision.

(5) In some figures (Fig. 6e, for example), the cryo-EM density map for the nucleotide component of the model is not continuous over the entire molecule. Can the authors comment on the significance of this phenomenon? Were the ligands validated in any way to ensure that the assignments were made correctly?

Indeed we sometimes saw discontinuous density for the nucleotides, both in the active site and in the specificity site. However, the break was almost always near the C5’ carbon atom, which is common to all nucleotides. While we cannot readily explain this phenomenon, the nucleotides refined well with full occupancy, giving B-factors similar to those of the surrounding protein atoms. The identity of the nucleotide could always be inferred from a) the size of the base (purine or pyrimidine); b) the known nucleotide combinations added to the protein before grid preparation; c) prior knowledge on the combinations of effector and substrate that have been found valid for all RNRs since the first studies of allosteric specificity regulation.

Reviewer #2 (Public Review):

This manuscript describes the functional and structural characterization of an anaerobic (Class III) ribonucleotide reductase (RNR) with an ATP cone domain from Prevotella copri (PcNrdD). Most significantly, the cryo-EM structural characterization revealed the presence of a flap domain that connects the ATP cone domain and the active site and provides structural insights about how nucleotides and deoxynucleotides bind to this enzyme. The authors also demonstrated the catalytic functions and the oligomeric states. However, many of the biochemical characterizations are incomplete, and it is difficult to make mechanistic conclusions from the reported structures. The reported nucleotide-binding constants may not be accurate because of the design of the assays, which complicates the interpretation of the effects of ATP and dATP on PcNrdD oligomeric states. Importantly, statistical information was missing in most of the biochemical data. Also, while the authors concluded that the dATP binding makes the GRD flexible based on the absence of cryo-EM density for GRD in the dATP-bound PcNrdD, no other supports were provided. There was also a concern about the relevance of the proposed GRD flexibility and the stability of Gly radical. Overall, the manuscript provides structural insights about Class III RNR with ATP cone domain and how it binds ATP and dATP allosteric effectors. However, ambiguity remains about the molecular mechanism by which the dATP binding to the ATP cone domain inhibits the Class III RNR activity.

Strengths:

(1) The manuscript reports the first near-atomic resolution of the structures of Class III RNR with ATP domain in complex with ATP and dATP. These structures revealed the NxN flap domain proposed to form an interaction network between the substrate, the linker to the ATP cone domain, the GRD, and loop 2 important for substrate specificity. The structures also provided insights into how ATP and dATP bind to the ATP cone domain of Class III RNR. Also, the structures suggested that the ATP cone domain is directly involved in the tetramer formation by forming an interaction with the core domain in the presence of dATP. These observations serve as an important basis for future study on the mechanism of Allosteric regulation of Class III RNR.

(2) The authors used a wide range of methodologies including activity assays, nucleotide binding assays, oligomeric state determination, and cryo-EM structural characterization, which were impressive and necessary to understand the complex allosteric regulation of RNR.

(3) The activity assays demonstrated the catalytic function of PcNrdD and its ability to be activated by ATP and low-concentration dATP and inhibited by high-concentration dATP.

(4) ITC and MST were used to show the ability of PcNrdD to bind NTP and dATP.

(5) GEMMA was used successfully to determine the oligomeric state of PcNrdD, which suggested that PcNrdD exists in dimeric and tetrameric forms, whose ratio is affected by ATP and/or dATP.

Weaknesses:

(1) Activity assays.

The activity assays were performed under conditions that may not represent the nucleotide reduction activity. The authors initiated the Gly radical formation and nucleotide reduction simultaneously. The authors also showed that the amount of Gly radical formation was different in the presence of ATP vs dATP. Therefore, it is possible that the observed Vmax is affected by the amount of Gly radical. In fact, some of the data fit poorly into the kinetic model. Also, the number of biological and technical replicates was not described, and no statistical information was provided for the curve fitting.

The highest turnover activity of PcNrdD measured in presence of ATP was 1.3 s-1 (470 nmol/min/mg), a kcat comparable to recently reported values for anaerobic and aerobic RNRs from Neisseria bacilliformis, Leeuwenhoekiella blandensis, Facklamia ignava, Thermus virus P74-23, and Aquifex aeolicus (PMID: 25157154, PMID: 29388911, PMID: 30166338, PMID: 34314684, PMID: 34941255). The general trend illustrated in Figure 1 is that ATP has an activating effect on enzyme activity, whereas high concentrations of dATP have an inactivating effect on activity, which cannot be explained by suboptimal assay conditions since our EPR results consistently show that more radical is formed in incubations with dATP compared to incubations with ATP. Curve fitting methods used are listed in Materials and Methods (as specified in the Figure 1 legend), and standard errors for all specified curve fitting results (from triplicate experiments) are shown in Figure 1.

(2) Binding assays.

The interpretation of the binding assays is complicated by the fact that dATP binds both a- and s-sites and ATP binds a- and active sites. dATP may also bind the active site as the product. It is unknown if ATP binds s-site in PcNrdD. Despite this complexity, the binding assays were performed under the condition that all the binding sites were available.

Therefore, it is not clear which event these assays are reporting.

Both ITC and MST experiments involving ATP and dATP binding to the a-site were performed in the presence of at least 1 mM GTP substrate (5 mM in MST) to fill the active site, and 1 mM dTTP effector to fill the s-site (specified in the legend to Figure 2). These conditions enable binding of ATP or dATP only to the a-site in the ATP-cone.

(3) Oligomeric states.

Due to the ambiguity in the kinetic parameters and the binding constants determined above, the effects of ATP and dATP on the oligomeric states are difficult to interpret. The concentrations of ATP used in these experiments (50 and 100 uM) were significantly lower than KL determined by the activity assays (780 uM), while it is close to the Kd values determined by ITC or MST (~25 uM). Since it is unclear what binding events ITC and MST are reporting, the data in Figure 3 does not provide support for the claimed effects of ATP binding. For the effects of dATP, the authors did not observe a significant difference in oligomeric states between 50 or 100 uM dATP alone vs 50 uM dATP and 100 uM CTP. The former condition has dATP ~ 2x higher than the Kd and KL (Figure 1b) and therefore could be considered as "inhibited". On the other hand, NrdD should be fully active under the latter condition. Therefore, these observations show no correlation between the oligomeric state and the catalytic activity.

The results in Figure 3 show that at in presence of 100 µM ATP plus 100 µM CTP the oligomeric equilibrium is 64% dimers plus 36% tetramers, and in presence of 50-100 µM dATP the oligomeric equilibrium is 32% dimers and 68% tetramers. We agree that there is no clear and strong correlation between oligomeric state and inhibition. We will also try to make it clearer in a revised version. Meanwhile, in order to add some clarity to our observations, SEC experiments at higher nucleotide concentrations will be done to strengthen our observations.

(4) Effects of dATP binding on GRD structure

One of the key conclusions of this manuscript is that dATP binding induces the dissociation of GRD from the active site. However, the structures did not provide an explanation for how the dATP binding affects the conformation of GRD or whether the dissociation of GRD is a direct consequence of dATP binding or it is due to the absence of nucleotide substrate. Also, Gly radical is unlikely to be stable when it is not protected from the bulk solvent. Therefore, it is unlikely that the GRD dissociates from the active site unless the inhibition by dATP is irreversible. Further evidence is needed to support the proposed mechanism of inhibition by dATP.

We admit that it has been difficult to propose a direct structural mechanism for transmission of the allosteric signal from the a-site in the ATP-cone to the active site and GRD given that the ATP-cones and linker are disordered in the dATP-bound dimers and that the linker can only be partly modelled in the dATP-bound tetramers. Most likely dATP binding causes a change in the dynamics of the linker region and NxN flap that directly affects substrate binding and simultaneously causes disorder of the GRD, given that all are part of a connected system (described as “nexus” in the manuscript). The structures determined in the presence of dATP and CTP show that CTP cannot bind in the absence of an ordered NxN flap.

In any case a major conclusion of the work is that dATP does not inhibit the anaerobic RNR by prevention of glycyl radical formation but by prevention of its subsequent transfer. We agree that further evidence is required to support the proposed mechanism, but given the extent of the data already presented in the manuscript, we feel that such studies should be the subject of a future publication.

(5) Functional support for the observed structures.

Evidence for connecting structural observations and mechanistic conclusions is largely missing. For example, the authors proposed that the interactions between the ATP cone domain and the core domain are responsible for tetramer formation. However, no biochemical evidence was provided to support this proposal. Similarly, the functional significance of the interaction through the NxN flap domain was not proved by mutagenesis experiments.

We did actually make mutants to verify the observed interactions, but several of them did not behave well in our hands, e.g. with regard to protein stability. Since we have no evidence that oligomerisation is coupled to inhibition, and since we did not observe any conservation between protein sequences in the interaction area, we chose not to pursue this point further. The main merit of the tetramer structures is that they allowed a high-resolution view of dATP binding to the ATP-cone and a comparison to previously-observed ATP-cones. Nevertheless, mutation experiments, also including the NxN flap, could be the subject of future work.

Reviewer #3 (Public Review):

The manuscript by Bimai et al describes a structural and functional characterization of an anaerobic ribonucleotide reductase (RNR) enzyme from the human microbe, P. copri. More specifically, the authors aimed to characterize the mechanism by how (d)ATP modulates nucleotide reduction in this anaerobic RNR, using a combination of enzyme kinetics, binding thermodynamics, and cryo-EM structural determination. One of the principal findings of this paper is the ordering of a NxN 'flap' in the presence of ATP that promotes RNR catalysis and the disordering of both this flap and the glycyl radical domain (GRD) when the inhibitory effector, dATP, binds. The latter is correlated with a loss of substrate binding, which is the likely mechanism for dATP inhibition. It is important to note that the GRD is remote (>30 Ang) from the binding site of the dATP molecule, suggesting long-range communication of the structural (dis)ordering. The authors also present evidence for a shift in oligomerization in the presence of dATP. The work does provide evidence for new insights/views into the subtle differences of nucleotide modulation (allostery) of RNR through long-range interactions.

The strengths of the work are the impressive, in-depth structural analysis of the various regulated forms of PcRNR by (d)ATP using cryo-EM. The authors present seven different models in total, with striking differences in oligomerization and (dis)ordering of select structural features, including the GRD that is integral to catalysis. The authors present several, complementary biochemical experiments (ITC, MST, EPR, kinetics) aimed at resolving the binding and regulatory mechanism of the enzyme by various nucleotides. The authors present a good breadth of the literature in which the focus of allosteric regulation of RNRs has been on the aerobic orthologues.

Given the resolution of some of the structures in the remote regions that appear to be of importance, the rigor of the work could have been improved by complementing this experimental studies with molecular dynamics (MD) simulations to reveal the dynamics of the GRD and loops/flaps at the active site.

We have discussed with expert colleagues the possibility of carrying out MD simulations on the different states in order to study the differential effects of ATP and dATP binding on the dynamics of the GRD. However, they felt that the chance of obtaining meaningful results was low, particularly since some structural elements are missing from the models for both forms, in particular the linker between the ATP-cone and the core.

The biochemical data supporting the loss of substrate binding with dATP association is compelling, but the binding studies of the (d)ATP regulatory molecules are not; the authors noted less-than-unity binding stoichiometries for the effectors.

Most of the methods used measure only binding strength, not the number of binding sites (N), whereas ITC also measures number of sites. N is dependent on the integrity of the protein, i.e. the number of protein molecules in a preparation that are involved in binding, and quite often gives lower values than the theoretical number of binding sites.

Also, the work would benefit from additional support for oligomerization changes using an additional biochemical/biophysical approach.

SEC (chromatography), GEMMA (mass spectrometry) and cryo-EM were used to study oligomerization. Since each method has restrictions on nucleotide concentrations as well as protein concentrations that can be used, the results are not directly comparable, but all three methods indicate nucleotide dependent oligomerization changes. The SEC results will be included in a revised version.

Overall, the authors have mostly achieved their overall aims of the manuscript. With focused modifications, including additional control experiments, the manuscript should be a welcomed addition to the RNR field

Recommendations for the authors: Reviewer #1 (Recommendations For The Authors):

(1) The last sentence of the abstract is not complete. The structures implicate a complex network of interactions in ... ? What do they implicate?

A couple of words seem to have been missed from the abstract. We have rewritten the end of the abstract to emphasise better that the dynamical transitions involve a linked network of interactions and not just the GRD.

(2) A reference is needed in the second sentence of the introduction.

We have added a reference as requested.

(3) Page 2, paragraph 2. The authors state "two beta subunits (NrdB) harboring a stable radical." This is not accurate. First of all, each beta subunit harbors its own cysteine oxidant.

And in several subclasses, that oxidant is not a stable radical but an oxidized metal cluster. Please revise to improve accuracy and also provide appropriate references.

We have revised the description and added a recent reference.

(4) Page 4, Fig. 1, panels C and D. The fit of the curve to the data is pretty poor. Is there an explanation? Could the data be improved in some way? In general, it is also best practice nowadays to show the individual data points in addition to the error bars in plots like the ones shown in Figure 1. Please modify the plots to include the individual data points in this figure - and probably also the subsequent figures showing binding data.

We have modified relevant panels in Figures 1, 2 and 5 as requested.

(5) Page 12, first paragraph. The authors state that one of the monomers in the ATP-CTP structure is well ordered and the other is less ordered. It would be ideal to show in a figure the basis for this conclusion using the cryo-EM maps. The "less ordered" monomer appears to be fully modeled.

Since the 2-fold axis of the dimer is vertical, the GRD of the left-hand monomer is hidden from view at the back of the molecule in Figure 6. For this monomer there was a small amount of density that allowed modelling of part of the glycyl radical loop (though not the tip containing the radical Gly itself) and the NxN flap, albeit with significantly higher mobility. We have illustrated this through an additional supplement for Figure 6 (figure supplement 2) in which the B-factors of the residues are shown both as a ribbon with radius proportional to the B-factor and through colouring. We hope that the four views in Figure 6 (figure supplement 2) together illustrate the relative mobility of different parts of the dimer.

It would also be ideal to show the basis for the conclusion that the entire GRD is disordered in the dATP-bound dimer structure.

Thank you for this suggestion. We have added a fifth supplement to Figure 8 in which we show the cryo-EM reconstruction for the dATP-bound dimer in two orientations, with the ATP-CTP-bound structure superimposed, which clearly shows that the entire GRD, the ATPcones, linker and NxN flap are all disordered in both monomers.

Reviewer #2 (Recommendations For The Authors):

(1) Units to describe enzyme activity.

• The unit for the specific activity in the main text (nmol/min•mg) is unusual. It is most likely a typo of nmol/min/mg or nmol/(min•mg).

We have changes to nmol/min/mg in the text.

• The unit for the Vmax is unusual and should not be confused with the specific activity. By definition, Vmax is the velocity of a reaction at a defined enzyme concentration/amount. For example, if an assay of 10 mg enzyme yielded 470 nmol of product in 1 min, Vmax is 470 nmol/min, whereas the specific activity is 47 nmol/min/mg.

The velocity as calculated above is ca 1.3 s-1. We have added kcat values to accompany the specific activities given.

(2) Steady-state kinetic analysis.

• The steady-state kinetic analysis in Figure 1 needs to be repeated. While the nonlinear curve fitting for Figure 1a is reasonable, those in Figures 1b, 1c, and 1d were outside the error range. Consequently, the reported kinetic parameters are unlikely accurate. The authors should repeat the assays with different enzyme preparation to account for all the errors. If the fit curve is still outside the error range, the kinetic model is likely incorrect, and the authors need to investigate different kinetic models.

The replotted Figure 1 now includes two different experiments for 1b (four replicates in total).

• The authors should report the number of replicates and the statistical data for the curve fitting.

The figure legend has been updated with statistical data for all curve fits, and the number of replicates has been added.

• The authors should report Vmax, Ki, and KL for Figure 1d.

Results in Figures 1c and 1d are less straightforward than those in Figures 1a and 1b where the s-site is filled with dTTP, favouring binding of GTP to the active site. The curve fit in Figure 1c is disturbed at high concentrations of ATP, which plausibly competes with the CTP substrate and results in inhibition by formed dATP. The curve fit in Figure 1d is less certain since reduction of substrate is low due to intrinsic CTP reduction in absence of effector and partially overlapping activation and inhibition effects of dATP.

• The authors should consider presenting the data in a log scale because of the complex nature of the activation/inhibition at the lower concentrations of dATP.

Log scale plots are included as insets in Figures 1b and 1d.

• The basal level of CPT reduction in the absence of an effector nucleotide should be reported with an error.

The error value has been added in the figure legend for the basal level of CTP reduction in the absence of effector.

(3) Equations for the kinetic analysis.

-The equations should be numbered and referred to in the Figure 1 legend.

All equations are specified and numbered in Materials and Methods. The equation used for each curve fit in the panels in Figure 1 is specified in the figure legend.

-KL must be defined in the main text. I suppose this is Kd for ATP or dATP. The equation for KL determination is missing brackets for dNTP.

KL (the concentration of an allosteric effector that gives half maximal enzyme activity) is defined in Materials and Methods where the equation is described. KL is not the same as KD (the dissociation constant for a ligand and its receptor). Brackets have been added to equation 1.

• I believe dNTP in the first equation is incorrect because ATP was the ligand for Figures 1A and 1C.

[dNTP] in the first equation has been changed to [NTP/dNTP] to indicate that both ribonucleotides and deoxyribonucleotides can bind.

• The second equation can be expressed as dATP as I believe this is the only ligand that inhibits the enzyme.

We prefer to keep the more general [dNTP] in the equation.

• The equation used for the fitting in Figure 1d must be defined more clearly than "a combination of the two equations".

The equation used for the curve fit in Figure 1d has been specified as equation 3 in Materials and Methods.

(4) Design of the activity assays

It is not clear if the activity assays report the rate of glycyl radical formation or nucleotide reduction. The authors mixed NrdD and NrdG and initiated the reaction by adding formate (essential for nucleotide reduction) and dithionite (Gly radical formation). The Gly radical formation is slow (in min time scale). The authors reported that ATP/dATP affected the rate of Gly radical formation and in the presence of ATP, Gly radical formation was incomplete even after 20 min. Therefore, it is possible that within the timescale of the activity assays (5 min), the reactions could be partially limited by the Gly radical formation, which may be the reason for the poor curve fitting.

Activity assays were performed with 5 min pre-incubation without dithionite and formate (no glycyl radical formation) and 10 min incubation after addition of dithionite and formate (glycyl radical formation plus substrate reduction). During earlier tests, NrdD and NrdG were first preincubated in the presence of dithionite (glycyl radical formation) and after addition of formate the substrate reduction was monitored during 20 min. These experiments resulted in lower enzyme activity, whereas higher activity was achieved only upon formate addition to the preincubation reaction. We suppose that the presence of dithionite, which is a strong reducing agent, affected NrdD stability and the reaction was stabilised by the presence of formate at an earlier stage of the reaction. For the EPR conditions used in the paper, 5 min incubation gave higher radical content compared to 20 min, and the reported activity assay gave highest activity after 10 min incubation; kcat of 1.3 s-1.

(5) Methods section for the activity assays.

• The concentration of dTTP, ATP, and dATP used in the assays must be described.

We thank the reviewer for pointing out this omission and we have now specified the concentrations used.

• Although the authors mentioned that they changed the concentration of dTTP, such data were not presented. Is this correct? Did the authors fix the dTTP concentration for the GTP reduction?

We apologise for the ambiguity and have specified that the dTTP concentration was fixed at 1 mM in the GTP experiments and that only the ATP or dATP concentrations were varied.

(6) Discrepancy between Ki/KL and Kd.

• There is a significant ambiguity remaining about the binding event that the ITC and MST results are reporting. Although dATP binds to both a- and s-sites and ATP binds to both active site and a-site, only a single binding event was observed in both cases. To distinguish the dATP binding to a- and s-sites and the active site, the authors should perform binding assays using mutant enzymes with only one of the binding sites available for dATP/ATP binding.

MST and ITC were performed in presence of substrate (1 mM GTP) and s-site effector (1 mM dTTP in ITC experiments, and 5 mM dTTP in MST experiments), thus dATP is blocked from binding to the s-site and ATP from binding to the active site.

• There are significant differences between Kd determined by MST or ITC and Ki/KL determined by the activity assays. Kd measurements were performed in the absence of the substrate nucleotides, while the assays required substrates. There may be complications from the presence of NrdG and the Gly radical formation. The authors must clearly describe all these complications and the discrepancy between Kd and Ki/KL.

MST, ITC and enzyme assays were all performed in the presence of substrate, and enzyme assays also contained NrdG, which was not present in the MST and ITC analyses. While KD is a thermodynamic constant representing the affinity of ligand to its binding site - in our case an effector nucleotide to the ATP-cone, KL is a kinetic constant (the allosteric effector concentration that gives half maximal activity) representing the relationship between the effector concentration and the reaction speed and is affected by the enzyme turnover number (kcat). The relationship between KD, KL and Ki is further complicated by conformational and possibly oligomeric state changes of NrdD upon binding of allosteric effectors, which occurs on a slower time scale than the rapid exchange of nucleotides in allosteric sites.

• The results of ATP/dATP copurification experiments shown in Figure 2 - figure supplement 1 show the preference of dATP binding over ATP. However, the results do not necessarily support the competition between ATP and dATP for binding to the ATP cone domain. It is still possible that dATP binding to the s-site diminishes the binding of ATP to the a-site.

Our aim was to exclude the possibility that ATP and dATP can bind to the ATP-cone at the same time and not to study competition between the two. Nevertheless, to eliminate the possibility that dATP binding to the s-site could affect nucleotide binding to the a-site, in two out of three conditions described in the supplementary figure, the experiments were performed in the presence of dTTP to prevent binding of dATP to the s-site.

(7) Oligomeric states.

• The authors must present the GEMMA results without ATP or dATP. Otherwise, the effects of ATP and dATP on the oligomeric state are not clear.

We cannot report GEMMA results without ATP or dATP because apo-PcNrdD was unstable in the GEMMA buffer and clogged the capillaries. Instead, SEC analysis was performed on apo-PcNrdD in a more suitable buffer and showed a homogeneous peak corresponding to a dimer (included as Figure 3 - figure supplement 1).

• Figure 3 does not support the induction of a2 upon ATP binding. The concentrations of ATP used in these experiments (50 and 100 uM) were significantly lower than KL determined by the activity assays (780 uM), while it is close to the Kd values determined by ITC or MST (~25 uM). Since it is unclear what binding events ITC and MST are reporting, the data in Figure 3 does not provide support for the claimed effects of ATP binding.

MST and ITC were performed in the presence of substrate (1 mM GTP) and s-site effector (1 mM dTTP in ITC experiments, and 5 mM dTTP in MST experiments), and they thus measure binding of ATP or dATP to the ATP cone. SEC analysis with 2 µM apo-PcNrdD and higher nucleotide concentrations (1 mM) was performed, confirming the presence of both dimers and tetramers in solution at different ratios depending on the addition of ATP or dATP. The SEC analysis, included as Figure 3 - figure supplement 1, confirms the existence of an equilibrium in solution.

• The effects of dATP must be presented more clearly. The authors did not observe a significant difference in oligomeric states between 50 or 100 uM dATP vs 50 uM dATP and 100 uM CTP. The former condition has dATP ~ 2x higher than the Kd and KL (Figure 1b) and therefore could be considered as "inhibited". On the other hand, NrdD should be fully active under the latter condition. The absence of difference in the oligomeric states between these two different conditions suggested to me that the oligomeric state does not regulate the NrdD activity. The authors seemed to indicate the same conclusion, but did not describe it clearly.

We agree that the oligomeric state most likely does not regulate the NrdD activity and hope to have explained this better in the revised version.

• Figure 3 legend mentioned a and b, but the figure was not labeled.

We have corrected this.

• The authors should triplicate the analysis and report the errors.

Five scans were added for each trace to increase the signal-to-noise level (included in figure legend).

(8) EPR characterization of Gly radical

• The amount of Gly radical must be quantified by EPR. The authors must report how much NrdD has Gly radical.

The concentration of NrdD (1 µM) in the activity assays is too low to be quantified by EPR. In the EPR experiment the glycyl radical content is given in the figure legend.

• The authors claim that the Gly radical environment was similar based on the doublet feature. However, the double feature comes from the hyperfine splitting with α proton whose orientation relative to the radical p-orbital would not be affected by the conformation or the environment. Thus, this conclusion is incorrect and must be removed.

We thank the reviewer for the clarifying comment and have removed our suggestion in the text.

(9) Gly711 should be shown in Fig. 6e to help readers understand the last paragraph on page 12.

The figure reference has been changed to Fig. 7, where this is shown more clearly. In Fig. 6e, inclusion of Gly711 would obscure other important information.

(10) GRD structure with dATP

The disorder of GRD in the presence of dATP does not agree with the formation of Gly radical under the same conditions. Gly radical is unlikely stable if it is extensively exposed to solvent. Most likely, the observed cryo-EM structures represent the conformation irrelevant to Gly radical formation.

We agree that the glycyl radical is unlikely to be stable if exposed to solvent. We believe that the GRD is not completely disordered but most likely made more mobile through rigid body movements of the domain to an extent that makes it invisible in the cryo-EM maps. It is most likely still in the vicinity of the active site, shielding the glycyl radical. Our new HDX-MS results show a small but tangible increase in mobility of the GRD in the presence of dATP compared to ATP. Of course the differences in dynamics remain to be confirmed. It is worth noting that the group of Catherine Drennan at MIT published a conference abstract more than a year ago that suggested a similar pattern of ordered/dynamic GRDs, based on crystal structures, though the details have not yet been published (https://doi.org/10.1096/fasebj.2022.36.S1.R3407).

We also agree that the cryo-EM structures do not show the GRD conformation relevant to Gly radical formation, as this has been shown spectroscopically for the GRE pyruvate formate lyase to require large conformational changes in the GRD and also the presence of the activase. However, revealing this conformation would be a completely different project. We postulate that inactivation proceeds by prevention of radical transfer to the substrate, not by prevention of its formation.

We have altered the wording in several places in the revised manuscript, including the title, to avoid using the term “disorder”, as this may imply (partial) unfolding, and we certainly do not wish to imply that.

(11) The difference between dATP and ATP binding

From the presented structures, it was not clear how the absence of 2'-OH affects the oligomeric state and the structure of the GRD. The low resolution of the ATP-bound structure precluded the comparison between the ATP and dATP-bound structures.

We agree that a detailed analysis of the differences between ATP- and dATP-bound structures requires higher resolution structures, particularly of the ATP-bound form. This will be the subject of future studies.

(12) Conclusion about the disordered GRD.

-The authors should describe the reason why the dATP binding affected the structure of GRD. The authors did not discuss why dATP binding affected the folding or mobility of GRD. Since this is the key conclusion of this manuscript and the authors are making this conclusion based on the absence of the ordered GRD structure (hence the negative results), the authors should carefully describe why the dATP binding does not allow the binding/folding of GRD in the position observed in the ATP-bound structure.

As mentioned in our response to point 4 in this reviewer’s Public Review, it is difficult to propose a direct structural mechanism for transmission of the allosteric signal from the a-site in the ATP-cone to the active site and GRD given that the ATP-cones and linker are disordered in the dATP-bound dimers and that the linker cannot be completely modelled even in the dATP-bound tetramers. Our first hypotheses were that the ATP-cone might work by a steric occlusion mechanism, but the reality appears more complex. Most likely dATP binding causes a change in the dynamics of the linker region and NxN flap that directly affects substrate binding and simultaneously causes higher mobility of the GRD, given that all are part of a connected system. The structures determined in the presence of dATP and CTP show that CTP cannot bind in the absence of an ordered NxN flap. We hope that future structural studies of NrdDs from other organisms may shed further light on this mechanism.

• The authors should test if the dATP inhibition is reversible for PcNrdD. If dATP binding induces dissociation of GRD from the active site and makes GRD flexible, Gly radical would most likely be quenched by formate or other components in the assay solution. If dATP inhibition is reversible, it is hard to believe that Gly radical dissociates completely from the active site.

As-purified PcNrdD contains dATP and can after removal of bound nucleotides bind substrate in presence of ATP. The as-purified PcNrdD protein contained 30% nucleotide contamination. After precipitation, HPLC analysis identified a major peak corresponding to dATP/dADP. Purification conditions were optimised to remove the nucleotides and we have added this information to the purification description.

(13) Functional support for the observed structures.

Similar to X-ray crystallography, cryo-EM is a highly selective method that requires the selection of particles that can be analyzed with sufficient resolution. This means that the analysis could be biased towards the protein conformations stable on the cryo-EM grid. Consequently, testing the structural observations by functional characterization of mutant enzymes is critical. However, the authors did not perform such functional characterizations and made conclusions purely based on the structural observations.

We acknowledge this limitation. We constructed several mutations located at the tetrameric interface between the ATP-cone and the core protein based on the cryo-EM structure of dATP loaded NrdD. Unfortunately, these mutant proteins were unstable and led to protein cleavage.

(14) Other minor points:

• In the introduction, the authors stated "The presence and function of the ATP-cone domain distinguish anaerobic RNRs from the other members of the large glycyl radical enzyme (GRE) family that are otherwise structurally and mechanistically related (Backman et al., 2017)." This statement is misleading because GREs are functionally diverse.

We have removed the words “and mechanistically” to reduce ambiguity.

• p. 12, e.g. should be removed.

We are not sure what is meant here. Does the reviewer mean p. 21 “The interactions are mostly hydrophobic but are reinforced by several H-bonds, e.g. between Gln3D-Gln458A, Ser53D–Gln458A, Arg11D-Asp468A, the main chain amide of Ile12D and Tyr557A.”?

Reviewer #3 (Recommendations For The Authors):

Overall, the work presents an impressive and in-depth structural view of the conformational changes stemming from the interactions of (d)ATP allosteric effector molecules that are interrelated to RNR function. The manuscript is written clearly and provides a solid overview of RNR chemistry. The cryo-EM data show striking differences between ATP and dATP bound forms, though in select regions, the resolution is not good enough for strong interpretations of the finer details.

(1) In cryo-EM structures, dATP appears to shift the oligomerization equilibrium from nearly all dimeric forms (absence of dATP) to a mixture of both dimeric and tetrameric species (presence of dATP). The examination of the oligomeric composition in solution using the GEMMA - a mass spectral technique - showed somewhat similar trends, though given the magnitude of the differences, it was less compelling. Have the authors considered a complementary solution technique, such as analytical SEC or dynamic light scattering that could provide further support for the change in oligomerization as observed in the cryo-EM?

SEC analysis with 2 µM apoPcNrdD and higher nucleotide concentrations (1 mM) was performed, confirming the presence of both dimer and tetramer in solution at different ratios depending on the addition of ATP or dATP. The SEC analysis, included as Figure 3 - figure supplement 1, confirms the existence of an equilibrium in solution.

(2) The protein as isolated from the final SEC shows a predominant peak corresponding to aggregate protein. It would be helpful if the authors ran an analytical SEC on the protein sample that is more refined to see how much soluble dimer/tetramer vs. aggregate protein there is. This could impact the kinetic and thermodynamic analysis of effector interactions. Further, the second major peak is labeled as 'monomer'. Is the protein isolated as a monomer and then forms dimer upon effector binding? It is unclear. The authors should consider presenting the SEC standards for the given column and buffer condition so that a reasonable estimate of the oligomerization status of the isolated protein can be assigned.

Can the reviewer possibly have believed that Figure 1 - supplementary figure 2a shows PcNrdD rather than PcNrdG? The figure supplement corresponds to the as-isolated SEC analysis of the activase (PcNrdG), which shows the presence of two main peaks of aggregates and monomer. The monomeric peak was reinjected and showed no presence of further aggregation states. Currently it is not known which oligomeric state the activase harbours upon binding to PcNrdD and glycyl radical formation. None of the other SEC figures in the MS has any predominant peak corresponding to aggregated protein.

(3) More details are needed for the ITC section. The ITC methods are not clear. What is the exact composition of the ligand solution being titrated into the protein solution? It is unclear how the less-than-unity binding stoichiometry was determined and what it means. Is the n value for the monomer, dimer, or tetramer forms? It is concerning that n < 1 is observed for dATP binding in the ITC whereas there are 3 dATP bound/subunit in the cryo-EM. For completeness, titration of a buffer into protein solution (no ligand) should be conducted and presented to demonstrate that the heats produced in Figure 2 correspond to the ligand only (and not a buffer mismatch).

ITC experiments were performed in the presence of 1 mM GTP (c-site) and 1 mM dTTP (ssite). Unlike other parameters in ITC analyses, the N value is usually the least accurate of all fitted parameters and strongly depends on the concentration of the active protein in the sample. N values described in the current study are in the same range as values reported for ATP-cones in other RNRs and NrdR (Rozman Grinberg & al 2018a, 2018b, 2022 McKethan and Spiro 2013). The results most likely reflect two high-affinity binding sites for dATP and one high affinity binding site for ATP. Different nucleotide concentrations were used in the cryoEM and ITC experiments.

(4) It is intriguing that the binding of dATP doesn't quell the glycyl radical. In fact, it appears that, as the authors suggest, the amount of glycyl radical might be increased in these samples. However, the cryo-EM data indicates that the GRD is disordered. It is unclear how these would be correlated, as one would not expect a disordered structural element to maintain such a potent oxidant.

As already written above, we do not wish to imply that the GRD is completely or even highly disordered, just that its dynamics increase in the presence of dATP. Otherwise we completely agree that a very exposed Gly radical is incompatible with its stability. It could be that the amount of disorder is exaggerated somewhat by the vitrification process in cryo-EM. We have tried to reword some of the text to emphasise higher mobility rather than disorder.

It has been difficult to propose a direct structural mechanism for transmission of the allosteric signal from the a-site in the ATP-cone to the active site and GRD given that the ATP-cones and linker are disordered in the dATP-bound dimers and that the linker can not be completely modelled even in the dATP-bound tetramers. We initially thought that a steric occlusion mechanism might be at play, but the reality appears more complex. Most likely dATP binding causes a change in the dynamics of the linker region and NxN flap that directly affects substrate binding and simultaneously causes higher mobility of the GRD, given that all are part of a connected system. The structures determined in the presence of dATP and CTP show that CTP cannot bind in the absence of an ordered NxN flap. We hope that future structural studies of NrdDs from other organisms may shed further light on this mechanism.

(5) It is a bit difficult to keep track of the myriad of structural information and differences amongst the various nucleotide-dependent conditions. It would be useful for the authors to add a summary figure that depicts the various oligomers, orientations, and (dis)ordered structural elements with cartoon representations.

Thank you for this suggestion. It has been added as Figure 11.

(6) The mechanism by which (d)ATP binding changes the (dis)ordering of select loops based on the current cryo-EM data is unclear (even the authors agree). The addition of molecular dynamics (MD) simulations on two different structures to reveal the network or structural communication would be a great addition to the work and validate the structural data.

We have discussed this with a colleague who is an expert in MD. Their advice was that such simulations would be very difficult given that some amino-acids are missing in both of the relevant starting structures (ATP-CTP and dATP-CTP dimer) and could give very variable results. Thus we chose to do complementary experiments with hydrogen-deuterium exchange mass spectrometry (HDX-MS) instead. The results are included in the revised manuscript.

Minor points

(1) There are some conflicting reports as to whether P. copri is considered a human 'pathogen'. According to Yeoh, et al Scientific Reports 2022, P. copri is one of the predominant microbes in the human gut and is linked to a positive impact on metabolism. Perhaps the addition of a citation that provides support for it as a pathogen would clarify the statement on p. 3.

We have added a recent reference (Nii T, Maeda Y, Motooka D, et al. (2023) Genomic repertoires linked with pathogenic potency of arthritogenic Prevotella copri isolated from the gut of patients with rheumatoid arthritis. Ann Rheum Dis 82: 621-629. doi: 10.1136/annrheumdis-2022-222881).

(2) In Figure 3, the number of dimers/tetramers for dATP (100 uM) does not add up to 100.

What is the other 2%?

Thank you for pointing this out - it has been corrected.

(3) The data in Figures 5C and D do show slight changes that could be fit and interpreted as a 'weak' interaction. Thus, the statement on p 9 "where dATP-loaded PcNrdD could bind neither GTP nor CTP" should be changed to indicate that the interactions are weak (or that the nucleotides weakly associate).

The text and the figure have been changed according to the reviewer’s suggestion.