Ligand binding represses bacterial histidine kinase activity by inhibiting its dimerization

  1. Gaurav D. Sankhe
  2. Jiawei Xing
  3. Merissa Xiao
  4. John Buglino
  5. Huilin Li
  6. Igor B. Zhulin
  7. Michael S. Glickman

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Abstract

Two component systems (TCS) mediate bacterial signal transduction in response to specific environmental conditions. The two components are the sensor kinase (SK), which senses the signal and autophosphorylates on a histidine residue, and a response regulator (RR), which is phosphorylated by the kinase and modifies gene expression. Despite intensive study, the mechanisms of signal sensing by sensor kinases are incompletely defined and the mechanisms by which SKs can sense multiple ligands are unclear. Mycobacterium tuberculosis PdtaS/PdtaR is a soluble TCS pair that participates in the Rip1 signal transduction cascade to control virulence by responding to copper and nitric oxide (NO). In contrast to paradigmatic ligand activated SKs, PdtaS is constitutively active without ligand and directly inhibited by Cu or NO, yet it remains unclear how such chemically diverse ligands are sensed. Here we show that PdtaS is a dimeric kinase that constitutively autophosphorylates in trans. Cu and NO both inhibit PdtaS phosphorylation by inhibiting dimerization. Phylogenetic analysis of the PdtaS family reveals conservation of the GAF/PAS dimer interface rather than the ligand binding pockets and mutations in the GAF dimer interface that alter dimerization impair multi-ligand sensing both in vitro and in M. tuberculosis cells. These results indicate that a single bacterial kinase can sense chemically diverse inputs through inhibition of dimerization dependent phosphorylation.

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    Reply to the reviewers

    ____Overall, we were encouraged by the comments of the reviewers, who mostly agreed that the study advance our understanding of two component system signaling mechanisms. The most substantive critique raised was the lack of mechanistic insight into the specific binding sites of Cu and NO on the PdtaS protein and the lack of examination of additional ligands such as cyclic di-GMP and zinc. We agree with this critique and cannot, and did not, make specific statements about the location of ligand binding. However, w____e draw a clear distinction in the manuscript between the functional effects of a chemical entity (ligand) on kinase activity and knowledge of the precise binding site of that ligand on the protein. As acknowledged, we did not determine the binding site. However, we do demonstrate the functional effect of the ligands, and these effects cannot occur without physical interaction between the ligand and the protein, so we believe the statement that the ligands are having this effect through binding is accurate, without knowledge of the precise location of that binding. ____

    Reviewer 1:

    • The primary concern pertains to ligand recognition by PdtaS. While PdtaS constitutive autophosphorylation is shown to be dependent on dimerization, there is no direct evidence of ligand binding. How Cu and NO inhibits PdtaS activity remains uncharacterized. Is it unclear if there are specific binding pockets inducing PdtaS conformational switch, if both substrates compete for a single binding pocket, or if Cu and NO inhibit dimerization by binding to the dimer interface. Similarly, it is unclear if NO does not covalently modify the key cysteine residues by S-nitrosylation, nor if Cu induces a distinct and reversible thiol-switch by site-specific oxidation that regulates PdtaS dimerization and activity.

    • __Response: Our discussion already contained this sentence: "Although we identify mutations with both positive and negative effects on dimer affinity which have effects of ligand inhibition of the kinase, this data does not identify specific molecular details on how dimerization is inhibited and whether Cu and NO both interact with the same regions of the dimer interface." We have added a sentence acknowledging that we do not determine whether NO is covalently modifying a thiol (line 343). __

    • Given the focus on ligand effects on PdtaS dimerization and activity, zinc and c-di-GMP should also be considered, as prior studies have suggested they may be sensed by PdtaS. Similarly, given the claim of multiligand sensing, it would be valuable to examine the combined effects of NO and Cu. Do they act additively, synergistically, or interfere with each other?

    • Response: We have added data to the manuscript examining the effect of c-di-GMP on kinase activity in combination with Cu. We do not observe a substantial synergistic effect. This new data is now Figure S3.

    • PdtaS variants and mutants are neither introduced nor adequately described. For example, in lines 144-150, PdtaS-H303Q and G443 are mentioned without citation, and their construction is not described in the Materials and Methods section. As a result, it is difficult to determine which experiments and constructs are specific to this manuscript. Please provide a detailed Materials and Methods section, and include as supplementary material a complete list of all strains, primers, and constructs used in this study, along with their origins.

    • __Response: We have added a section to the methods detailing the construction of the PdtaS mutant protein expression plasmids. __

    • References: Xing J et al 2023 is duplicated. Please correct in the text and in the references list.

    • Response: We have deleted the duplicate reference

    • Please provide molecular weights on gels (fig. 1C, D, E, 2A, 5C, D, 7A). Please provide incubation time for kinase reactions in figure legends (e.g. Fig 1C, D, E, ...).

    • __Response: All of these incubation times are included in the materials and methods. We ____will add to the figure legends depending on journal style. We have added selected MW numbers to the MW markers in 1D,E, 5D,7A.

    __

    • Please indicate whether representative experiments are shown, and specify the number of replicates performed for each assay (e.g. Fig 1C, D, E, ...). This information is essential for assessing the reproducibility and robustness of the findings.

    • __Response: We are somewhat confused by this comment. For each claim made about quantitative effects, we include a quantitation panel that contains experimental replicates (1F, 2D, 4D, 5C, 7B). For MST graphs, we state the number of replicates for each time point. __

    • Please clarify the discrepancy in Figure 2A regarding the calcium concentration used. The results section (line 163) refers to 10 µM, whereas the figure legend (line 393) states 1 mM.

    • __Response: We have corrected the figure legend to 10____m __

    • Figure 2A should include zinc, as previous work by the authors has shown that zinc directly inhibits the kinase activity of PdtaS. It would also be informative to test c-di-GMP in Fig. 2, given that c-di-GMP has been described to binds PdtaS (PMID: 33772870), and that c-di-GMP binding at dimer interfaces has been demonstrated in transcription factors (e.g., PMID: 25171413).

    • __Response: We did not test zinc because in our prior studies the effects of Zn and Cu were identical. We have tested c-di-GMP as noted above (see new Fig S3). __

    • The interpretation in lines 206-207 is not convincing. PdtaS homologs may differ in ligand specificity, precluding the presence of a conserved ligand-binding cavity but not of a specific ligand binding cavity in the GAF/PAS domains. Functional divergence of the binding site can occur, and this possibility should be acknowledged.

    • Response: We are somewhat confused by this comment. This is the sentence in question: "____This analysis suggests that the PdtaS kinase family has evolved to conserve the dimerization interface, shown above to be important for autokinase activity, but that the putative ligand binding domains do not have a conserved ligand cavity, arguing against a specific ligand that binds in the GAF or PAS pocket in this family of histidine kinases." The sentence does not argue that there is no ligand binding in the GAF PAS cavity, only that the cavity is not conserved, and this argues against a single ligand. To clarify this point, we will insert the word "single" before "specific ligand"

    Reviewer 2:

    • The dimer model is consistent with trans phosphorylation, but I did not see model quality described, especially in the H303-ATP binding interface. Can the authors provide AlphaFold PAE and pLDDT scores?

    • __Response: We have added a SI figure (Figure S4) with this data. __

    • Although the effect of Cu and NO on the two mutant PdtaS is clear, why the WT activity in Fig. 2A is not also inhibited is not obvious to me, especially since WT dimerization is affected by Cu and NO (Fig. 2B, C). Is there also cis-autophosphorylation that masks reductions in trans phosphorylation? Is the WT signal saturated on this autorad?

    • __Response: This assay, as noted in the figure legend, was done with 10____m____M Cu. This dose is only mildly inhibitory to the wild type kinase, as demonstrated in Figure 5D-E, which clearly demonstrates Cu inhibition. We don't have an explanation for why the trans phosphorylation mutant pair is inhibited by lower doses of Cu. It is possible this reflects some cis-autophosphorylation, but the strong inhibition of trans autophosphorylation is consistent with our model. __

    • The two Cys residues in PdtaS were previously found to affect kinase activity. Here, the authors show they also modestly affect dimerization. Since ~1/3 of mycobacteriales have both Cys, a double mutant would have been interesting for the in vitro characterization (it is used in live bacteria in Fig. 7A) and might show a more pronounced effect (not critical).

    • __Response: Although we agree, we attempted to purify the double cysteine mutant from * coli* but unable to due to insolubility, so we were unable to test the protein. __

    • Although competition data and structural model clearly indicate trans phosphorylation, some cis-phosphorylation can probably not be ruled out, especially since the dimer mutant H67A shows some activity. Although that mutation does not seem to fully disrupt the dimer, the H67A activity could be indicative of some cis-phosphorylation.

    • __Response: The H67A mutant is a dimerization mutant that weakens, but does not completely disrupt the dimer. This mutant cannot be used to distinguish cis vs trans phosphorylation and therefore we cannot rule out a mixture of cis vs trans autophosphorylation. The data in figure 1 argues for trans phosphorylation being the dominant mechanism. __

    • The Cys residues destabilize the dimer, and mutating the Cys stabilizes it, even canceling out the effect of the chemical destabilizers Cu and NO. In Fig. 4A, it looks like all Cys are too far apart to form disulfides, but Cu2+ can cause formation of disulfides. Can the authors comment on the distance of the Cys and the likelihood that disulfides have a role in this mechanism? If this were plausible, thiol-to-disulfide ratios with and without Cu could be directly measured. Although a bit more of a stretch, NO could also contribute to disulfide formation through ROS, and disulfides could be a way by which these two disparate ligands have the shared effect on activation shown here.

    • __Response: As stated above, we are not able to comment about whether direct modification of these cysteines is occurring. We do not believe the proximity of the cysteines would allow disulfide formation. __

    • The interdomain mutation Arg261Ala is quite nice and shows a specific effect on activity, but not dimerization, indicating that this interdomain ion bond somehow transfers the dimerization signal from the GAF to the PAS domain. Were there any other interdomain bonds? For completeness, was the basal autophosphorylating or phosphotransfer activity to PdtaR affected by the 261 mutation?

    • __Response: We did not detect any other bonds in our modeling. The basal level of autophosphorylation of the R261A protein compared to WT is visible at the lower end of the Cu inhibition curve in 6E and is comparable. We did not observe a difference in autophosphorylation at 0 Cu in the gels supporting this curve. __

    • First sentence in the Discussion, wording: The study investigates kinase activation, not signal sensing in a strict sense.

    • __Response: We have edited this sentence. __

    • Although this is primarily a biochemical, mechanistic study, one or two sentences on the biological significance of PdtaS/R in M. tuberculosis in the Introduction would be nice

    • __Response: We believe these sentences in the introduction already satisfies this request: "PdtaS and PdtaR were implicated in the Rip1 pathway of * tuberculosis* signal transduction by a genetic suppressor screen in which inactivation of either PdtaS or PdtaR reverted the copper and nitric oxide sensitivity of M. tuberculosis lacking rip1. Copper and NO directly inhibit the kinase activity of PdtaS, an inhibition that requires the N terminal GAF and PAS domains ____[39]____, indicating that the GAF-PAS are necessary to transmit the inhibitory signal to the kinase domain." We would also note that although much of the data is biochemical, we test the in vivo relevance of our model using M. tuberculosis strains carrying PdtaS mutations

    __

    Reviewer 3

    • Limited characterization and validation of dimerization measurements: (a) while MST is an established technique, the central thesis relies heavily on dimerization measurements using this single method. Given the importance of this finding, at least one additional orthogonal approach would strengthen the conclusions significantly. Analytic size exclusion chromatography (SEC) could be a very simple, accessible and reliable approach to address this core mechanistic question. By choosing the right size resolution separation matrix, the authors should be able to separate complete monomers, from partial complexes (e.g. dimers only held through the kinase domain) and full dimers (the species the authors expect for the constitutively active wt protein). Ready advantage of having the wt protein can be taken, as well as several dimerization mutants (C53A, C57A, H67A), and presence/absence of cognate ligands (NO, Cu). For necessary reference standards, a dilution series should be able to reveal the elution position for wt monomers (and if this approach reveals to be difficult, mild chaotropic conditions can always be attempted, often times also pH shifts can do the job). Other techniques can point in the same direction as SEC, such as SAXS (best coupled to a SEC, or SEC-SAXS), native polyacrylamide gel electrophoresis, and/or dynamic light scattering.
    • __Response: We appreciate the reviewers' careful suggestions for additional experimental approaches. Although logical, we are unable to undertake them at this time and further exploration will hopefully be stimulated by our study. __

    (b) More importantly, additional techniques should be chosen such that a clear distinction can be made between two different scenarios, namely: that only the sensory domains (PAS/GAF) undergo ligand-triggered dissociation; or, instead, that the entire protein dissociates into separate monomers (i.e. including the kinase domains). This seems like an extremely important distinction, so that the proposed kinase-regulation mechanism is well understood/described. The first scenario would be less "disruptive" wrt previous paradigms (sensory domain dissociation could well be linked to a conformational rearrangement that allosterically inhibits kinase auto-phosphorylation).

    • __Response: We agree that this is an important distinction. We would note here that our prior data (Buglino et al eLife 2021) demonstrated that the isolated kinase domain of PdtaS is not inhibited by copper or NO, indicating that the effect of these ligands both requires the GAF-PAS and that the kinase dimer itself is not sensitive to ligand induced inhibition. This result does not directly address the reviewer's question, which is whether there is localized inhibition of dimerization in the GAF-PAS dimer, which, via an allosteric mechanism, inhibits phosphorylation by the kinase domain, which we have shown is in trans, without actual dissociation. We are not aware of a technique that could distinguish what would presumably be a type of allosteric localized dimer disruption from full dissociation. Our data clearly indicates that the kinase inhibition effect is mediated by the dimer dissociation effect on the GAF/PAS and full characterization of the effects of that on the kinase domain will await further studies outside of this paper. __
    1. R261A mechanistic inconsistency: The manuscript shows that the R261A mutant has attenuated copper inhibition in vitro, albeit remaining functional in vivo (Figure 7B). While the authors acknowledge this suggests their "interdomain coupling model is incomplete or compensated by other mechanism in vivo," this significant discrepancy undermines confidence in the proposed mechanism and deserves more thorough investigation and/or discussion.
    • __Response: We thank the reviewer for this comment, which relates to the R261A mutant. We disagree that this result "undermines confidence in the proposed mechanism". We rigorously interrogated our in vitro findings by genetic complementation in M. tuberculosis cells using epitope tagged proteins and these results largely confirmed the model in that C53A, C53A/C57A, and H67A all inactivated signaling, as predicted from our model. R261 is the exception and, as we discuss, it indicates that we do not completely understand the in vivo determinants of coupling between the GAF-PAS dimer and the kinase domain, which is dependent on R261A in vitro. __

    Insufficient evidence about signal integration: While the authors argue this mechanism enables "integration of multiple inputs into the kinase without the constraints of specific ligand recognition" (lines 342-344), this appears conceptually flawed to me. The ligands (Cu and NO) must still be specifically sensed and bound somewhere on the protein to trigger dimerization disruption - the mechanism simply uses dimerization modulation as the output rather than the more typical allosteric conformational changes. The conservation pattern (interface > binding sites) may reflect selective pressure to maintain dimerization capability across the family, while individual species evolved different ligand specificities. The authors should clarify that their mechanism represents a novel output mode for ligand sensing rather than an alternative to specific ligand recognition, and discuss how this distinction affects their evolutionary interpretation.

    • Response: We thank the reviewer for this comment. The issue raised is the difference between ligand "recognition" and "sensing" with the former implying a specific binding site (which we acknowledge above and in the paper that we do not identify) and the functional output modified by ligands. Our data supports that dimerization is an important mechanism of sensing, but we do not claim that the dimer interface is the binding site for the ligands. We would note that the following sentences were in the reviewed version of the paper and we believe clearly make the exact distinction that the reviewer requests: __ __Abstract: "These results indicate that a single bacterial kinase can sense chemically diverse inputs through inhibition of dimerization dependent phosphorylation"

    Line 110: "Ligand binding pockets of GAF and PAS domains can bind a wide variety of ligands[38], but it remains to be determined whether multi-ligand sensing by PdtaS represents a manifestation of specific chemical recognition by the GAF-PAS domains or some other mechanism."

    Line 119: "Mutations in the GAF dimer interface that alter dimerization also impair multi-ligand __sensing __of Cu and NO in vitro and in M. tuberculosis cells. Our findings establish a mechanism of multi ligand sensing through alteration of sensor oligomeric state."

    Line 330: "Taken together, these data are consistent with a model in which modulation of dimer affinity is the sensing mechanism of the mycobacterial clade of PdtaS kinases, rather than specific recognition of Cu or NO by the ligand binding pockets of the GAF or PAS domains."

    __We have edited one instance (line 337) in the discussion where the use of "recognition" might have been misconstrued. __

    Minor Comments

    1. The manuscript could better explain why PdtaS is described as "constitutively active" - the distinction between showing autophosphorylation activity in vitro versus true constitutive activity could be clearer. Can the authors show or refer to evidence of live constitutive PdtaR phosphorylation by PdtaS? (e.g. PhosTag electrophoresis gels of whole protein extracts and Western blotting revealed by anti-PdtaR; the use of NO and Cu can easily be used as inhibitors in such experimental setup).

    • Response: We thank the reviewer for this question. Our basis for claiming that the kinase is constitutively active, both for autophosphorylation and phosphotransfer to PdtaR, is the following:

    • __In the work of others and our prior work, PdtaS autophosphorylates without added ligand, which is contrary to most histidine kinases which are ligand activated. __

    • __PtdaS phosphorylates PdtaR without added ligand in vitro (see Figure 5C of this paper) __

    • __In terms of in vivo demonstration of PdtaR phosphorylation, this is very challenging in all response regulators given the unstable nature of the aspartate phosphorylation. We have been unsuccessful in visualizing PdtaR phosphorylation in vivo using phostag or western blotting. However, we note that our prior work demonstrated that mutation of the phosphoacceptor residue in PdtaR (D65A) phenocopied loss of both PdtaS and PdtaR (Buglino et al eLife 2021). __

    • Figure 5D shows some gel quality issues and also limited detail in the legend to know what exactly each panel represents and labels' definitions (e.g. "Ca" on the first lane, etc). The difference between wt and mutant is not clearcut to me, difficult from these data alone to derive a reliable Ki. Furthermore, the control gel on the bottom for the wt (I believe this is a cold control gel to see loaded quantities of protein on each lane?), seems to have less protein in the higher Cu concentrations.

    • __Response: The calcium lane is the divalent ion control as in the other figures. The legend of this figure refers to figure 1, which is more detailed and the identical assay. As noted in the methods, the phosphorylation signal is normalized to the total protein in the lower gel, so the lower amount of protein in these lanes in incorporated into the quantitation, which itself is derived from triplicate experiments, as noted in the legend. __

    Enough experimental detail should be included on figure legends so that the experiments are self-explanatory.

    • __Response: This is a journal style question that will be addressed depending on the identity of the eventual journal. __

    Lines 184-185 : they only refer to the fact that c-di-GMP binds to the GAF domain of PdtaS, yet the paper by Hariharan et al 2021 also shows that it activates PdtaS's autokinase activity. This should be double-checked and taken into account for the discussion of cognate ligands' effects.

    • Response: As noted above, we have added a supplemental figure (S3) that examines the effect of c-di-GMP on autokinase activity. Hariharan reported activation of PdtaS by c-di-GMP (Figure 6A of that publication). We do not see similar activation and do not observe an effect of cDG with Cu. Line 233: "Dimerization separation of function mutations" title is unclear

    • Response: Edited The structural model source (AlphaFold3) should be mentioned in the main text, not just figure legends. AF3-predicted models should be illustrated according per-residue pLDDT reliability indices (typically with a color ramp).

    • __Response: The paper contains this sentence:____ "____We performed bioinformatic analyses of the conservation of PdtaS across the Actinomycetota phyla and mapped this conservation onto the predicted full length PdtaS dimer structure predicted using AlphaFold". We have added the specific AlphaFold model (3) to this sentence. __
    • __We have also added an SI figure containing the pLDDT data (Figure S4). __
  2. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    The manuscript by Sankhe and collaborators, investigates the mechanism by which the Mycobacterium tuberculosis two-component system PdtaS/PdtaR senses copper and nitric oxide. The authors demonstrate that PdtaS is a constitutively active histidine kinase that autophosphorylates in trans, and that ligand-triggered inhibition occurs through disruption of dimerization rather than typical allosteric conformational changes of the dimeric species. Through phylogenetic analysis, mutagenesis, and biochemical assays, they show that conservation occurs primarily at the dimer interface rather than putative ligand binding sites, supporting a novel mechanism of multi-ligand sensing through modulation of oligomeric state. The experimental design is generally sound, with appropriate controls and multiple lines of evidence supporting the main conclusions. The trans-autophosphorylation experiments are particularly elegant and convincing. While there are some mechanistic concerns that should be addressed (particularly around R261A, and the actual dimerization extent/effect), the core findings are significant, and the work represents an important contribution to understanding bacterial signal transduction.

    Major Comments:

    1. Limited characterization and validation of dimerization measurements: (a) while MST is an established technique, the central thesis relies heavily on dimerization measurements using this single method. Given the importance of this finding, at least one additional orthogonal approach would strengthen the conclusions significantly. (b) More importantly, additional techniques should be chosen such that a clear distinction can be made between two different scenarios, namely: that only the sensory domains (PAS/GAF) undergo ligand-triggered dissociation; or, instead, that the entire protein dissociates into separate monomers (i.e. including the kinase domains). This seems like an extremely important distinction, so that the proposed kinase-regulation mechanism is well understood/described. The first scenario would be less "disruptive" wrt previous paradigms (sensory domain dissociation could well be linked to a conformational rearrangement that allosterically inhibits kinase auto-phosphorylation). Analytic size exclusion chromatography (SEC) could be a very simple, accessible and reliable approach to address this core mechanistic question. By choosing the right size resolution separation matrix, the authors should be able to separate complete monomers, from partial complexes (e.g. dimers only held through the kinase domain) and full dimers (the species the authors expect for the constitutively active wt protein). Ready advantage of having the wt protein can be taken, as well as several dimerization mutants (C53A, C57A, H67A), and presence/absence of cognate ligands (NO, Cu). For necessary reference standards, a dilution series should be able to reveal the elution position for wt monomers (and if this approach reveals to be difficult, mild chaotropic conditions can always be attempted, often times also pH shifts can do the job). Other techniques can point in the same direction as SEC, such as SAXS (best coupled to a SEC, or SEC-SAXS), native polyacrylamide gel electrophoresis, and/or dynamic light scattering.
    2. R261A mechanistic inconsistency: The manuscript shows that the R261A mutant has attenuated copper inhibition in vitro, albeit remaining functional in vivo (Figure 7B). While the authors acknowledge this suggests their "interdomain coupling model is incomplete or compensated by other mechanism in vivo," this significant discrepancy undermines confidence in the proposed mechanism and deserves more thorough investigation and/or discussion.
    3. Insufficient evidence about signal integration: While the authors argue this mechanism enables "integration of multiple inputs into the kinase without the constraints of specific ligand recognition" (lines 342-344), this appears conceptually flawed to me. The ligands (Cu and NO) must still be specifically sensed and bound somewhere on the protein to trigger dimerization disruption - the mechanism simply uses dimerization modulation as the output rather than the more typical allosteric conformational changes. The conservation pattern (interface > binding sites) may reflect selective pressure to maintain dimerization capability across the family, while individual species evolved different ligand specificities. The authors should clarify that their mechanism represents a novel output mode for ligand sensing rather than an alternative to specific ligand recognition, and discuss how this distinction affects their evolutionary interpretation.

    Minor Comments

    1. The manuscript could better explain why PdtaS is described as "constitutively active" - the distinction between showing autophosphorylation activity in vitro versus true constitutive activity could be clearer. Can the authors show or refer to evidence of live constitutive PdtaR phosphorylation by PdtaS? (e.g. PhosTag electrophoresis gels of whole protein extracts and Western blotting revealed by anti-PdtaR; the use of NO and Cu can easily be used as inhibitors in such experimental setup).
    2. Figure 5D shows some gel quality issues and also limited detail in the legend to know what exactly each panel represents and labels' definitions (e.g. "Ca" on the first lane, etc). The difference between wt and mutant is not clearcut to me, difficult from these data alone to derive a reliable Ki. Furthermore, the control gel on the bottom for the wt (I believe this is a cold control gel to see loaded quantities of protein on each lane?), seems to have less protein in the higher Cu concentrations.
    3. Enough experimental detail should be included on figure legends so that the experiments are self-explanatory.
    4. Lines 184-185 : they only refer to the fact that c-di-GMP binds to the GAF domain of PdtaS, yet the paper by Hariharan et al 2021 also shows that it activates PdtaS's autokinase activity. This should be doube-checked and taken into account for the discussion of cogante ligands' effects.
    5. Line 233: "Dimerization separation of function mutations" title is unclear
    6. The structural model source (AlphaFold3) should be mentioned in the main text, not just figure legends. AF3-predicted models should be illustrated according per-residue pLDDT reliability indices (typically with a color ramp).
    7. Ensure consistent reporting of replicate numbers across all experiments.

    Significance

    General assessment:

    The study provides elegant trans-autophosphorylation experiments and strong phylogenetic support for dimerization interface conservation. However, it relies heavily on MST as the sole method for measuring dimerization -the main finding in terms of novelty- and shows mechanistic inconsistencies (R261A functional in vivo despite attenuated inhibition in vitro).

    Scientific Advance:

    While the authors overstate novelty by claiming to bypass "specific ligand recognition" (ligands must still bind specifically to trigger dimerization disruption), the identification of dimerization modulation as the inhibitory output mechanism represents a meaningful advance. The work establishes an important framework for understanding how M. tuberculosis senses multiple host-derived stresses and may inform studies of other inhibitory sensor kinases.

    Target Audience:

    I believe this work will be of great interest to bacterial signaling researchers and M. tuberculosis pathogenesis specialists, with broader appeal to the microbiology community studying two-component systems and host-pathogen interactions. The dimerization-based mechanism may also attract structural biologists studying multi-domain sensor architectures.

  3. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    Summary

    This study explores the activation mechanism of a two-component system in M. tuberculosis, PdtaS/R. PdtaS/R is known to sense Cu and NO. Here, the authors show that PdtaS autophosphorylates in trans upon dimerization. PdtaS is constitutively active, and Cu and NO binding inactivate the kinase by preventing dimerization. The dimerization interface, but not the ligand binding domain, is conserved in PdtaS orthologs, and disruption of the dimer interface also disrupts ligand sensing in vitro and in live M. tuberculosis.

    Major comments

    This is an interesting and thorough analysis of the (in)activation mechanism of a TCS. Although much work has been done on such systems, this TCS is quite interesting as it is a rarer cytosolic, soluble system, because it has been shown to sense two chemically very different ligands- Cu and NO (and apparently also cdi-GMP)- and because it is constitutively active and inactivated by ligands, which is more unusual. A main strength of the paper is the identification of a range of mutants with specific effects on dimerization, activity, auto and substrate phosphorylation and GAF-PAS interactions to probe and parse the contribution of different aspects of the mechanism. The flow of the experiments is logical, and the data are generally clear, even though TCS phosphorylation is short-lived and can be tricky to capture. The heterodimer mixing experiments using WT, phosphoreceptor His-, and ATP binding mutants are clear and conclusively show trans phosphorylation. The control ruling out dimerization defects of the mutants is useful, and bioinformatic analysis of the GAF and PAS domains shows surprisingly clearly the conservation of the dimer interface, not the ligand binding site. The experiments showing the effects of dimerization on the activity of PdtaS are conclusive, with mutations showing stronger (Cys) or weaker (His67, already shown in a previous paper) dimerization and the expected effects on PdtaS activity. Testing some key mutants in live bacteria is another nice feature of the study that shows that in vitro findings (mostly) carry over to live bacteria, which is not always the case, and often just not tested. In sum, this is a solid, straightforward study on the activation mechanism of a more unusual M. tuberculosis TCS.

    Minor comments

    The dimer model is consistent with trans phosphorylation, but I did not see model quality described, especially in the H303-ATP binding interface. Can the authors provide AlphaFold PAE and pLDDT scores?

    Although the effect of Cu and NO on the two mutant PdtaS is clear, why the WT activity in Fig. 2A is not also inhibited is not obvious to me, especially since WT dimerization is affected by Cu and NO (Fig. 2B, C). Is there also cis-autophosphorylation that masks reductions in trans phosphorylation? Is the WT signal saturated on this autorad?

    Related: Although competition data and structural model clearly indicate trans phosphorylation, some cis-phosphorylation can probably not be ruled out, especially since the dimer mutant H67A shows some activity. Although that mutation does not seem to fully disrupt the dimer, the H67A activity could be indicative of some cis-phosphorylation.

    Some kinetic experiments would have been useful to gauge the timescale of these mechanisms (but not critical).

    The two Cys residues in PdtaS were previously found to affect kinase activity. Here, the authors show they also modestly affect dimerization. Since ~1/3 of mycobacteriales have both Cys, a double mutant would have been interesting for the in vitro characterization (it is used in live bacteria in Fig. 7A) and might show a more pronounced effect (not critical).

    The Cys residues destabilize the dimer, and mutating the Cys stabilizes it, even canceling out the effect of the chemical destabilizers Cu and NO. In Fig. 4A, it looks like all Cys are too far apart to form disulfides, but Cu2+ can cause formation of disulfides. Can the authors comment on the distance of the Cys and the likelihood that disulfides have a role in this mechanism? If this were plausible, thiol-to-disulfide ratios with and without Cu could be directly measured. Although a bit more of a stretch, NO could also contribute to disulfide formation through ROS, and disulfides could be a way by which these two disparate ligands have the shared effect on activation shown here.

    The interdomain mutation Arg261Ala is quite nice and shows a specific effect on activity, but not dimerization, indicating that this interdomain ion bond somehow transfers the dimerization signal from the GAF to the PAS domain. Were there any other interdomain bonds? For completeness, was the basal autophosphorylating or phosphotransfer activity to PdtaR affected by the 261 mutation?

    First sentence in the Discussion, wording: The study investigates kinase activation, not signal sensing in a strict sense.

    Although this is primarily a biochemical, mechanistic study, one or two sentences on the biological significance of PdtaS/R in M. tuberculosis in the Introduction would be nice

    Christoph Grundner

    Significance

    This is a mechanistic study on the (in)activation mechanism of an M. tuberculosis TCS with some unusual features: The kinase, PdtaS, is constitutively active and ligand binding inactivates it. It is a soluble system that binds multiple ligands, fewer of which have been described to date. While trans-phosphorylation and regulation by dimerization are not conceptually new, they were also not a given in this more unusual TCS. The authors identified relevant mutants at several steps of the activation mechanism to specifically probe the effect of dimerization, interdomain communication etc., and test their relevance in live bacteria, which goes beyond what comparable biochemical studies typically do. The authors have previously published some aspects of the PdtaS/R activation mechanism (inhibition by Cu and NO, Cys mutants). The conserved dimer interface suggests that many of the PdtaS orthologs are similarly regulated, and that different ligands can converge on this dimerization-dependent activation. Thus, the study could be relevant for the whole family and the range of ligands they likely sense. The study summarizes the findings in the idea that PdtaS activity relies on dimerization to react to divergent ligands rather than specific ligand binding to the GAF and PAS domains. This statement is perhaps too strong and that rather than either/or, it is likely both. Overall, this is a thorough biochemical study that reveals aspects of a more non-typical TCS activation mechanism that are of high interest to the Mtb and bacterial signaling field.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    In this manuscript the authors aim to characterize the mechanism regulating PdtaS activity, a histidine kinase of Mycobacterium tuberculosis responding to nitric oxide (NO) and copper ions (Cu). The PdtaS/PdtaR two-component system is atypical, with PdtaS being cytoplasmic and phosphorylating PdtaR in the absence of signal(s). Here, the authors characterize key residues involved in PdtaS dimerization necessary for PdtaS activity on PdtaR, in vitro and in M. tuberculosis. They show that both NO and Cu inhibit dimerization and thus PdtaS autokinase activity. They propose a model where changes in dimer affinity serve as the sensing mechanism allowing to integrate multiple signals without relying on specific ligand binding.

    Major comments:

    • The primary concern pertains to ligand recognition by PdtaS. While PdtaS constitutive autophosphorylation is shown to be dependent on dimerization, there is no direct evidence of ligand binding. How Cu and NO inhibits PdtaS activity remains uncharacterized. Is it unclear if there are specific binding pockets inducing PdtaS conformational switch, if both substrates compete for a single binding pocket, or if Cu and NO inhibit dimerization by binding to the dimer interface. Similarly, it is unclear if NO does not covalently modify the key cysteine residues by S-nitrosylation, nor if Cu induces a distinct and reversible thiol-switch by site-specific oxidation that regulates PdtaS dimerization and activity.
    • Since there is currently no direct evidence of ligand binding or residue modification, the conclusions drawn-particularly in the title and discussion-should be presented more cautiously, unless further structural, biochemical, or genetic data substantiate ligand binding.
    • Given the focus on ligand effects on PdtaS dimerization and activity, zinc and c-di-GMP should also be considered, as prior studies have suggested they may be sensed by PdtaS. Similarly, given the claim of multiligand sensing, it would be valuable to examine the combined effects of NO and Cu. Do they act additively, synergistically, or interfere with each other?
    • OPTIONAL. A significant limitation is the exclusive consideration of a single PdtaS conformation-the autophosphorylation-competent state. Histidine kinases typically cycle through at least three distinct enzymatic activities: phosphatase, autokinase, and phosphotransfer. Each of these functions relies on specific conformational states, which are often modulated by ligand binding. It is therefore important to investigate whether PdtaS also possesses phosphatase activity. Do ligands such as NO and Cu influence this activity-increasing phosphatase function, or simply inhibiting autophosphorylation and/or phosphotransfer? Moreover, does the monomeric or dimeric form of PdtaS exhibit phosphatase activity? In addition, the stability of phosphorylated PdtaR should be addressed, as it is crucial for understanding the overall dynamics and output of the signaling cascade.

    Minor comments:

    • PdtaS variants and mutants are neither introduced nor adequately described. For example, in lines 144-150, PdtaS-H303Q and G443 are mentioned without citation, and their construction is not described in the Materials and Methods section. As a result, it is difficult to determine which experiments and constructs are specific to this manuscript. Please provide a detailed Materials and Methods section, and include as supplementary material a complete list of all strains, primers, and constructs used in this study, along with their origins.
    • References: Xing J et al 2023 is duplicated. Please correct in the text and in the references list.
    • Please provide molecular weights on gels (fig. 1C, D, E, 2A, 5C, D, 7A).
    • Please provide incubation time for kinase reactions in figure legends (e.g. Fig 1C, D, E, ...).
    • Please indicate whether representative experiments are shown, and specify the number of replicates performed for each assay (e.g. Fig 1C, D, E, ...). This information is essential for assessing the reproducibility and robustness of the findings.
    • Please clarify the discrepancy in Figure 2A regarding the calcium concentration used. The results section (line 163) refers to 10 µM, whereas the figure legend (line 393) states 1 mM.
    • Figure 2A should include zinc, as previous work by the authors has shown that zinc directly inhibits the kinase activity of PdtaS. It would also be informative to test c-di-GMP in Fig. 2, given that c-di-GMP has been described to binds PdtaS (PMID: 33772870), and that c-di-GMP binding at dimer interfaces has been demonstrated in transcription factors (e.g., PMID: 25171413).
    • I am not convinced by the interpretation line 206-207. PdtaS homologs can have different ligand specificity, impling the conservation of a ligand cavity in the GAF/PAS domains.
    • The interpretation in lines 206-207 is not convincing. PdtaS homologs may differ in ligand specificity, precluding the presence of a conserved ligand-binding cavity but not of a specific ligand binding cavity in the GAF/PAS domains. Functional divergence of the binding site can occur, and this possibility should be acknowledged.

    Significance

    The manuscript demonstrates that PdtaS autokinase activity occurs in trans and that homodimerization is critical for its constitutive activity. While these findings extend previous work by the authors (PMID: 34003742)-which had already identified several key residues in PdtaS, including cysteines essential for NO and Cu sensing-they represent incremental advances. The mechanistic model proposed remains speculative without substantial additional experimental validation. The conclusions rely heavily on structural predictions from AlphaFold, representing only a single conformation corresponding to the autokinase-competent state. Crucially, the manuscript does not provide direct evidence for the mechanism of NO and Cu sensing. It also excludes the possibility of direct ligand binding-including untested candidates such as zinc and c-di-GMP-to a specific pocket, without experimentally addressing the hypothesis. These gaps significantly limit the mechanistic insight and overall impact of the study.

  5. Version published to 10.1101/2025.05.18.654591 on bioRxiv