Diversity of function and higher-order structure within HWE sensor histidine kinases

  1. Igor Dikiy
  2. Danielle Swingle
  3. Kaitlyn Toy
  4. Uthama R. Edupuganti
  5. Giomar Rivera-Cancel
  6. Kevin H. Gardner

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Abstract

Integral to the protein structure/function paradigm, oligomeric state is typically conserved along with function across evolution. However, notable exceptions such as the hemoglobins show how evolution can alter oligomerization to enable new regulatory mechanisms. Here we examine this linkage in histidine kinases (HKs), a large class of widely distributed prokaryotic environmental sensors. While the majority of HKs are transmembrane homodimers, members of the HWE/HisKA2 family can deviate from this architecture as exemplified by our finding of a monomeric soluble HWE/HisKA2 HK (EL346, a photosensing Light-Oxygen-Voltage (LOV)-HK). To further explore the diversity of oligomerization states and regulation within this family, we biophysically and biochemically characterized multiple EL346 homologs and found a range of HK oligomeric states and functions. Three LOV-HK homologs are primarily dimeric with differing structural and functional responses to light, while two Per-ARNT-Sim (PAS)-HKs interconvert between differentially active monomers and dimers, suggesting dimerization might control enzymatic activity for these proteins. Finally, we examined putative interfaces in a dimeric LOV-HK, finding that multiple regions contribute to dimerization. Our findings suggest the potential for novel regulatory modes and oligomeric states beyond those traditionally defined for this important family of environmental sensors.

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  1. eLife

    ###Reviewer #3:

    This study provides experimental evidence that, in contrast to a currently accepted view, some sensor histidine kinases exist in more than one oligomerization state and that a monomer-to-dimer transition might play a role in signal transduction. Such transition is well documented for eukaryotic signal transduction systems, but not in prokaryotes. Thus, the findings reported here open an avenue to a broader investigation of this phenomenon and its potential generalization.

    My only major comment is the inexpert level of bioinformatics analysis. While all specific concerns seem minor (listed in the corresponding section below), taken together they amount to a bigger problem, particularly with presentation. On the other hand, none of the shortcomings with the bioinformatics part seriously affect major conclusions of this study.

  2. eLife

    ###Reviewer #2:

    Manuscript Summary:

    The manuscript by Dikiy et al. extends previous investigations from the Gardner lab on the oligomeric states of histidine kinases containing photosensing LOV domains (LOV-HKs). The Gardner lab had previously characterized two dimeric and one monomeric LOV-HK from Erythrobacter litoralis. In the present study, they perform sequence analyses to identify soluble LOV- and PAS-domain containing HKs similar to the previously characterized monomeric LOV-HK EL346. They characterize the photocycle, oligomeric state, and autophosphorylation activity of several of these HKs. Finally, noting that one dimeric LOV-HK (RH376) has three small regions of sequence that are absent from the monomeric EL346, they delete these regions individually and in combination to generate a set of mutated RH376 proteins that they characterize.

    General Assessment:

    The results of this study are consistent with previous studies from the Gardner laboratory, indicating that functional LOV-HKs can exist as monomers, dimers, or mixtures of both. Perhaps unsurprisingly, the effects of deletions engineered to identify determinants of dimerization do not clearly align with any simple hypotheses and limited insights are gained. Overall, the study would benefit from greater precision in the writing of the manuscript, greater rigor in experimental design and analyses of data, and restraint in tempering conclusions to better align with the data.

    Major Comments:

    1. The introduction could be improved by more precise language (see details in Minor Comments).

    2. Details about the autophosphorylation assay should be provided. Specifically, the concentrations of proteins used in the assays need to be specified, unless the stated concentrations are the final concentrations in the assay, in which case this needs to be more clearly indicated. The extremely low concentration of ATP (3.6 uM) is problematic. Even for initial rate determinations, ADP generated during the reaction will likely inhibit phosphorylation under these conditions.

    3. Figure 1. Given the substantial domain rearrangements that are known to occur during signaling, it would be helpful to specify the signaling states depicted in the schematic structures.

    4. Line 231 subtitle and lines 257-258. This conclusion seems to be somewhat overstated given the small number of proteins examined. Within Table 2, one of three EL346-like LOV-HKs is monomeric and the same is true for the three LOV-HKs examined. This ratio of 4:2 dimers to monomers does not seem sufficient to conclude that LOV-HKs are generally dimeric.

    5. Lines 270-274 and Fig. 3b. How do you know that the plateau is indicative of phosphatase activity rather than a simple equilibrium due to the presence of ADP in the reaction mixture (either as a contaminant in the ATP or generated during the reaction)? A minimum of 3 replicates should be shown with error bars. Which data from the two-trials were used to reach the conclusion of a 1.5-fold difference in activity? More rigorous statistics should be employed.

    6. Lines 274-279 and Fig. 3b. It is not clear from the description of the assay in the Methods section what concentrations of HKs were used in the assays. If concentrations were not similar for all proteins assayed, differences in rates are likely to result from different amounts of ADP generated during the reaction.

    7. Lines 278-279. It is a big leap to conclude that monomer-dimer transitions may be a regulatory strategy based on the observation of different rates of autophosphorylation. What concentrations of monomer and dimer proteins were used in the assays? And if the oligomeric state is used as a regulatory strategy, how? Do you envision some mechanism that regulates the oligomeric state and this in turn regulates autophosphorylation? (This is eventually addressed in the discussion. Perhaps the statement about a regulatory strategy should be withheld until the Discussion>)

    8. The sequence of the loop in DHp and CA domains of HKs has been used to predict cis- vs. trans- mechanisms of autophosphorylation. Please comment on the loops in the LOV-HKs. Presumably all monomeric HKs would have loops consistent with a cis- autophosphorylation mechanism. Are they similar in monomeric and dimeric LOV-HKs?

    9. Fig. 4. What are "monomer-1/dimer-1" and "monomer-2/dimer-2"? Why is there such a large difference in the activities observed for -1 and -2? Also, the y-axis in the graph in Fig. 4b appears to be mislabeled as "Concentration".

    10. Fig. 6. A minimum of 3 independent activity assays should be shown and statistical tests should be applied to determine the significance of the observed differences, especially given the large variations in the data.

    11. Lines 330-332 and Fig. S4. The absorbance profiles clearly differ between the proteins. How much variation would be necessary to claim that a protein was non-functional? Indeed, in the next sentence, it is acknowledged that flavin binding is adversely affected. If so, then what is meant by "the deletions do not perturb the folding and function of the LOV domain"?

    12. Lines 368-369. What experiments address the sufficiency of either RH1 or RH3 for dimerization? The rationale for this statement is not clear.

    13. Fig. S6. It is not conventional to introduce new data within the Discussion. Perhaps this figure should be moved to the Results.

  3. eLife

    ###Reviewer #1:

    The main objective of this study was to investigate a possible relationship between oligomerization and regulation in histidine kinases. To this end the authors identified novel LOV and PAS sensor kinases based on sequence homology searches with HK EL346, a soluble monomeric HK that senses blue light through a LOV domain. To study the monomer-dimer transition as a possible regulatory mechanism they try to "monomerize" a dimeric LOVHK, named RH376, by deleting three regions that could be determinants of the oligomeric state. Nevertheless, the authors found that none of these deletions disrupt the dimeric state of the protein. The conclusion of the work appears to be that multiple domains contribute to dimerization and function of HKs.

    This manuscript is experimentally well done and well written. First the authors show that Non-Lov PAS-HKs show a mix of monomers and dimers, both of which are active. Then, the study is focused in the LOV HKRH376 and in deletions RH1-RH3 and a double mutant RH1+3. RH1 and RH2 are active dimers while RH3 remains largely dimeric and is inactive. Finally, the double mutant is an inactive monomer. The major conclusions of this manuscript are that multiple regions determine oligomerization in this family of HKs and light-induced conformational changes have a complex relationship with autophosporylation and do not appear to be restricted to the oligomerization state. In summary, I found that the data, although technically sound, don´t provide mechanistic insights in the regulatory mechanism(s) of sensor kinases.

  4. eLife

    ##Preprint Review

    This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript. Michael T Laub (Massachusetts Institute of Technology) served as the Reviewing Editor.

    ###Summary:

    This study provides evidence that some sensor histidine kinases may exist in more than one oligomerization state and that a monomer-to-dimer transition might play a role in signal transduction. The results are consistent with and extend prior work from this lab and will be of interest to those studying two-component signal transduction.

  5. Version published to 10.1101/2020.07.08.194225 on bioRxiv