Water and chloride as allosteric inhibitors in WNK kinase osmosensing

Reviewer #1 (Public Review):

Summary:
This manuscript addresses the regulation of the osmosensing protein kinases, WNK1 and WNK3. Prior work by the authors has shown that these enzymes are activated by PEG400 or ethylene glycol and inhibited by chloride ion, and that activation is associated with a conformational transition from dimer to monomer. In X-ray structures of the WNK1/SA inactive dimer, a water-mediated hydrogen bond network was observed between the catalytic loop (CL) and the activation loop (AL), named CWN1. This led to the proposal that bound water may be part of the osmosensing mechanism.

The current study carries this work further, by applying PEG400 to Xtals of dimeric WNK1/SA. This results in a change in kinase conformation and space group, along with 4-9 fewer waters in CWN1 and the complete disappearance of another water cluster (CWN2) located at the dimer interface. Six conserved residues lining the CWN1 pocket in WNK3 are mutated to determine effects on activity and inhibition by chloride ion (measured by AL autophosphorylation) and monomer-dimer interconversion (light scattering).

The results show that two mutants (E314Q/A in WNK3) at a site central to the water cluster result in increased kinase activity (autophosphorylation), and increased SLS, interpreted as aggregation. Three sites (D279A, Y346F, M301A) inhibit kinase activity with varying effects on oligomerization - Y346A and M301A retain monomer-dimer ratios similar to WT while D279N promotes aggregation. K236A and K307A show activity and monomer:dimer ratios similar to WT. Selected mutants (E314Q, D279N, Y346F) and WT appear to retain osmosensitivity with comparable activation by PEG400.

The study concludes that osmolytes may activate the kinase by removing waters from the CWN1 and CWN2 clusters, suggesting that waters might be considered allosteric ligands that promote the inactive structure of WNKs. The differing effects of mutations may be ascribed to disruption of the water networks as well as inhibitory perturbations at the active site.

Strengths:
This study presents a novel and unique function for bound water, and its potential role to explain osmosensory regulation. The mechanism is innovative and the new structures and mutational data presented by the work will be useful for further investigations of the mechanisms that enable cells to respond to osmotic pressure.

Weaknesses:
Given that all mutants tested showed the same degree of activation by PEG400, it seemed possible that PEG400 might be an allosteric activator of WNK1/3 through direct binding interactions. Perhaps PEG400 eliminates CWN1/2 waters by inducing conformational changes so that water loss is an effect not a cause of activation. To address this it would be helpful to comment on whether new electron densities appeared in the X-ray structure of WNK1/SA/PEG400 that might reflect PEG400 interactions with chains A or B. It would also be helpful to discuss any experiments that might have been done in previous work to examine the direct binding of glycerol and other osmolytes to WNKs.

The study would benefit from a deeper discussion about how to reconcile the different effects of mutations. For example, wouldn't most or all of the mutations be expected to disrupt the water network, and relieve the proposed autoinhibition? This seemed especially true for some of the residues, like Y420(Y346), D353(D279), and K310(K236), which based on Fig 3 appeared to interact with waters that were removed by PEG400.

Alternatively, perhaps the waters in CWN2 are more important for maintaining the autoinhibited structure. This possibility would be useful to discuss, and perhaps comment on what may be known about the energetic contributions of bound water towards stabilizing dimers.

It would also be useful to comment on why aggregation of E319Q/A shouldn't inhibit kinase activity instead of activating it.

The X-ray work was done entirely with WNK1 while the mutational work was done entirely with WNK3. Therefore, a simple explanation for the disconnect between structure and mutations might be that WNK1 and WNK3 differ enough that predictions from the structure of one are not applicable to mutations of the other. It would be helpful to describe past work comparing the structure and regulation of WNK1 and WNK3 that support the assumption of their interchangeability.

Reviewer #2 (Public Review):

Summary:
This work tests the hypothesis that water coordination in WNK kinases is linked to allosteric control of activity. It is proposed that dimeric WNK is inactive and bound to some conserved water molecules, and that monomerization/activation involves departure of these waters. New data here include a crystal structure of monomeric WNK1 which shows missing waters compared to the dimeric structure, in support of the hypothesis. Mutant proteins of a different isozyme (WNK3) designed to disrupt water coordination were produced, and activity and quaternary structure were measured. The results with WNK3 do not clearly support or refute the hypothesis as there is no systematic correlation between mutations designed to disrupt water coordination and activity or quaternary structure.

Strengths:
The most interesting result presented here is that P1 crystals of WNK1 convert to P21 in the presence of PEG400 and still diffract (rather than being destroyed as the crystal contacts change, as one would expect). All of the assays for activity and osmolyte sensing are carried out well.

Weaknesses:
The rationale for using WNK3 for the mutagenesis study is that it is more sensitive to osmotic pressure than WNK1. I think that WNK1 would have been a better platform because of the direct correlation to the structural work leading to the hypothesis being tested. All of the crystallographic work is WNK1; it is not logical to jump to WNK3 without other practical considerations.

Osmolyte sensing was tested by measuring ATP consumption as a function of PEG400 (Figure 6). Data for the subset of mutants analyzed by this assay showed increasing activity. It is not clear why the same collection of mutant proteins analyzed in the experiments of Figure 5 was not also measured for osmolyte sensing in Figure 6.

The last set of data presented uses light scattering to test whether the WNK3 mutant proteins exhibit quaternary structural changes consistent with the monomer/dimer hypothesis. If they did, one would expect a higher degree of monomer for those that are activated by mutation, and a lower amount of monomer (like wt) for those that are not. Instead, one of the mutant proteins that showed the most chloride inhibition (Y346F) had a quaternary structure similar to the wt protein, and others have similar monomer/dimer mixtures but distinct chloride inhibition profiles (K307A and M301A). I don't see how the light scattering data contribute to this story other than to refute the hypothesis by showing a lack of correlation between quaternary structure, water binding, and activity. This is another reason why the disconnect between WNK1 and WNK3 could be a problem. All of the detailed structural work with WNK1 must be assumed with WNK3; perhaps the light scattering data are contradicting this assumption?