Water and chloride as allosteric inhibitors in WNK kinase osmosensing
Osmotic stress and chloride regulate the autophosphorylation and activity of the WNK1 and WNK3 kinase domains. The kinase domain of unphosphorylated WNK1 (uWNK1) is an asymmetric dimer possessing water molecules conserved in multiple uWNK1 crystal structures. Conserved waters are present in two netw...
Saved in:
| Main Authors: | , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
eLife Sciences Publications Ltd
2024-11-01
|
| Series: | eLife |
| Subjects: | |
| Online Access: | https://elifesciences.org/articles/88224 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1850158896515121152 |
|---|---|
| author | Liliana R Teixeira Radha Akella John M Humphreys Haixia He Elizabeth J Goldsmith |
| author_facet | Liliana R Teixeira Radha Akella John M Humphreys Haixia He Elizabeth J Goldsmith |
| author_sort | Liliana R Teixeira |
| collection | DOAJ |
| description | Osmotic stress and chloride regulate the autophosphorylation and activity of the WNK1 and WNK3 kinase domains. The kinase domain of unphosphorylated WNK1 (uWNK1) is an asymmetric dimer possessing water molecules conserved in multiple uWNK1 crystal structures. Conserved waters are present in two networks, referred to here as conserved water networks 1 and 2 (CWN1 and CWN2). Here, we show that PEG400 applied to crystals of dimeric uWNK1 induces de-dimerization. Both the WNK1 the water networks and the chloride-binding site are disrupted by PEG400. CWN1 is surrounded by a cluster of pan-WNK-conserved charged residues. Here, we mutagenized these charges in WNK3, a highly active WNK isoform kinase domain, and WNK1, the isoform best studied crystallographically. Mutation of E314 in the Activation Loop of WNK3 (WNK3/E314Q and WNK3/E314A, and the homologous WNK1/E388A) enhanced the rate of autophosphorylation, and reduced chloride sensitivity. Other WNK3 mutants reduced the rate of autophosphorylation activity coupled with greater chloride sensitivity than wild-type. The water and chloride regulation thus appear linked. The lower activity of some mutants may reflect effects on catalysis. Crystallography showed that activating mutants introduced conformational changes in similar parts of the structure to those induced by PEG400. WNK activating mutations and crystallography support a role for CWN1 in WNK inhibition consistent with water functioning as an allosteric ligand. |
| format | Article |
| id | doaj-art-63414f7a5ce84bc2a7aa7faabfbedafe |
| institution | OA Journals |
| issn | 2050-084X |
| language | English |
| publishDate | 2024-11-01 |
| publisher | eLife Sciences Publications Ltd |
| record_format | Article |
| series | eLife |
| spelling | doaj-art-63414f7a5ce84bc2a7aa7faabfbedafe2025-08-20T02:23:44ZengeLife Sciences Publications LtdeLife2050-084X2024-11-011210.7554/eLife.88224Water and chloride as allosteric inhibitors in WNK kinase osmosensingLiliana R Teixeira0Radha Akella1John M Humphreys2Haixia He3Elizabeth J Goldsmith4https://orcid.org/0000-0001-8102-5012Department of Biophysics, The University of Texas Southwestern Medical Center, Dallas, United StatesDepartment of Biophysics, The University of Texas Southwestern Medical Center, Dallas, United StatesDepartment of Biophysics, The University of Texas Southwestern Medical Center, Dallas, United StatesDepartment of Biophysics, The University of Texas Southwestern Medical Center, Dallas, United StatesDepartment of Biophysics, The University of Texas Southwestern Medical Center, Dallas, United StatesOsmotic stress and chloride regulate the autophosphorylation and activity of the WNK1 and WNK3 kinase domains. The kinase domain of unphosphorylated WNK1 (uWNK1) is an asymmetric dimer possessing water molecules conserved in multiple uWNK1 crystal structures. Conserved waters are present in two networks, referred to here as conserved water networks 1 and 2 (CWN1 and CWN2). Here, we show that PEG400 applied to crystals of dimeric uWNK1 induces de-dimerization. Both the WNK1 the water networks and the chloride-binding site are disrupted by PEG400. CWN1 is surrounded by a cluster of pan-WNK-conserved charged residues. Here, we mutagenized these charges in WNK3, a highly active WNK isoform kinase domain, and WNK1, the isoform best studied crystallographically. Mutation of E314 in the Activation Loop of WNK3 (WNK3/E314Q and WNK3/E314A, and the homologous WNK1/E388A) enhanced the rate of autophosphorylation, and reduced chloride sensitivity. Other WNK3 mutants reduced the rate of autophosphorylation activity coupled with greater chloride sensitivity than wild-type. The water and chloride regulation thus appear linked. The lower activity of some mutants may reflect effects on catalysis. Crystallography showed that activating mutants introduced conformational changes in similar parts of the structure to those induced by PEG400. WNK activating mutations and crystallography support a role for CWN1 in WNK inhibition consistent with water functioning as an allosteric ligand.https://elifesciences.org/articles/88224waterosmosensorWNK1WNK3chloride ioncrystallography |
| spellingShingle | Liliana R Teixeira Radha Akella John M Humphreys Haixia He Elizabeth J Goldsmith Water and chloride as allosteric inhibitors in WNK kinase osmosensing eLife water osmosensor WNK1 WNK3 chloride ion crystallography |
| title | Water and chloride as allosteric inhibitors in WNK kinase osmosensing |
| title_full | Water and chloride as allosteric inhibitors in WNK kinase osmosensing |
| title_fullStr | Water and chloride as allosteric inhibitors in WNK kinase osmosensing |
| title_full_unstemmed | Water and chloride as allosteric inhibitors in WNK kinase osmosensing |
| title_short | Water and chloride as allosteric inhibitors in WNK kinase osmosensing |
| title_sort | water and chloride as allosteric inhibitors in wnk kinase osmosensing |
| topic | water osmosensor WNK1 WNK3 chloride ion crystallography |
| url | https://elifesciences.org/articles/88224 |
| work_keys_str_mv | AT lilianarteixeira waterandchlorideasallostericinhibitorsinwnkkinaseosmosensing AT radhaakella waterandchlorideasallostericinhibitorsinwnkkinaseosmosensing AT johnmhumphreys waterandchlorideasallostericinhibitorsinwnkkinaseosmosensing AT haixiahe waterandchlorideasallostericinhibitorsinwnkkinaseosmosensing AT elizabethjgoldsmith waterandchlorideasallostericinhibitorsinwnkkinaseosmosensing |