Conserved water networks in WNK1/SA. (A) CWN1 in Subunit A of uWNK1/SA (PDB file 6CN9). Conserved water network 1 (CWN1) in marine, 14 water molecules. Subunit A, cyan, Subunit B, green, Activation Loop, red, and Catalytic loop, yellow. (B) Three observed water networks (CWN1-3, black ovals) were identified by superposition of PDB 6CN9 and 5DRB. Colors are as in (A). (C) Overlay of waters in PDB files 6CN9 (marine) and 5DRB (yellow). (D) CWN2 is in the subunit interface (same coloring as in (A). N-terminal domains were superimposed.

Effects of PEG400 on the WNK1/SA dimer and space group. Monoclininc WNK1/SA/PEG400 (PDB in submission) with symmetry mate (pink) (A) and WNK1/SA (PDB 6CN9) (B) are superimposed (6CN9 in grey) (C). (D) Superposition of one subunit of WNK1/SA/PEG400 from (A) with WNK1/SA from (B) to show rotation that accompanies monomer conversion induced by PEG400. (E) Osmolyte induced conformational changes as a function of sequence. WNK1/SA (PDB file 6CN9) and WNK1/SA/PEG400, pink trace, pWNK1 (PDB 5W7T) and pWNK1/sucrose (Akella et al., 2020), orange trace, and pWNK1 and PKA (PDB file 1ATP), green trace.

PEG400-induced conformational change in the active site of WNK1/SA. (A) PEG400 induced conformational changes in WNK1/SA. WNK1/SA Sub A (cyan), Sub B (green), superimposed with WNK1/SA/PEG400 (pink). (B) Conformational changes in the active site in both the 3/10 chloride binding helix and the Activation Loop, same coloring as in (A). Residues E388 and K375 move significantly (red arrows), affecting the 3/10 helix, and the CWN1 binding pocket present in WNK1/SA. (C) Electron density of waters in WNK1/SA/PEG400. (D) Overlay of CWN1 with waters remaining in PEG400 (magenta), and waters remaining in pWNK1 (PDB file 5W7T) (green).

Residues in the AL-CL Cluster and positions mutated. (A) AL-CL Cluster in uWNK1 (PDB file 6CN9). Labels in WNK1 numbering (with WNK3 numbers in parenthesis). Cartoon coloring is the same as Figure 1A. Pan-kinase conserved catalytic residues (D349 and K351) are labelled in gray. AL-CL residue labels are colored to indicate mutant assay results: mutants more active than wild-type, red, similar to wildtype, green, and less active than wild-type, blue. (B) Sequence conservation in the AL-CL Cluster and neighboring residues. Pan-kinase conserved Catalytic Loop residues and pan-WNK Activation Loop phosphorylation sites are yellow, other colors indicate assay results, as in (A).

Effects of chloride on uWNK3 AL-CL Cluster mutant autophosphorylation at S308. (A) Wild-type uWNK3 . (B) uWNK3/E314Q. (C) uWNK3/E214A at 25 °C. (D) uWNK3/K236A (E) uWNK3/K307A (F) uWNK3/M301A. (G) uWNK3/Y346F. (H) uWNK3/D279N. Reactions run in 4 μM uWNK3, 30 °C (unless otherwise indicated), at chloride concentrations of 50 mM (purple), 150 mM (pink), and 250 mM (magenta). Bars indicate standard error from triplicate independent experiments.

Effects of osmolytes on uWNK3 AL-CL cluster mutants. ADP production is measured by ADP Glo® (Promega) with gOSR1 peptide as a substrate in the absence (grey) or presence (red) of 15% PEG400 15 min, 25°C. Luminescence is proportional to ATP consumption. Circles represent independent triplicated experiments.