Fig. 1: Contributions to OSR1 and SPAK CCT domain binding by residues within and around core RFxV/I and RxFxV/I motifs.
From: Kinase interaction analysis predicts actions of the WNK-OSR1/SPAK pathway
A Domains of SPAK (STK39) and OSR1 (OXSR1). Conserved C-terminal (CCT) domains interact with R-F-x-V/I and R-x-F-x-V/I motifs. B Crystal structure of the complex of OSR1 CCT and a WNK1-derived hexapeptide, GRFQVT (PDB ID: 2V3S). The peptide interacts partially as a terminal β-strand (β-strand addition), causing sidechains to alternate direction, suggesting adjacent residues do not strongly influence each other’s binding. C Motif numbering scheme. D Peptide array setup. Peptides are covalently linked to the membrane. Each spot represents a single position within the base sequence that was varied to every amino acid. Primary anti-His6 antibody detects bound CCT. Fluorophore-labeled secondary antibody detects primary. E Quantification of peptide array spot intensities. Original array images in Supp. Fig. 1. Asterisk (*) above red residue indicates position varied for every amino acid. Caret (^) below residue indicates site in base peptide that deviates from wild-type sequence. The number to the right indicates the motif position mutated. Top (blue) row: hWNK4 (human WNK4) 1012–1024 G1215A. Middle (orange) rows: wild-type hWNK4 1012–1024. Bottom (purple) rows: hWNK1 1253–1265 R1257A (R-x-F-x-V/I motif; wild-type hWNK1 sequence contains both motifs, R-R-F-x-V/I). See main text for detailed explanations. n = 2 arrays per CCT domain, normalized by the mean intensity of the array. Ranges reported in Supp. Fig. 2. F Affinity determined by fluorescence anisotropy peptide competition with titrated, unlabeled peptides displacing 25 nM labeled peptide (NH3+-NLVGRF-[DAP-FAM]-VSPVPE-COO−] [diaminopropionic acid (DAP)]. SPAK/OSR1 CCTs are constant at 1.5/3.0 μM. Red letters mutated relative to wild-type sequence, and blue letter is position 0. The top group is hWNK4 1012–1024, the bottom is hWNK1 1253–1265. Affinity reported as inhibition dissociation constant (Ki, µM). Upper/lower limits of the 95% confidence interval are indicated. Fold-change decrease in affinity is a fold-change increase in Ki relative to wild type. Goodness of fit reported as R-squared (R2). n = 3. Curves and fluorescent probe binding in Supp. Fig. 3. GraphPad Prism 10, one-site fit to the Ki model used for data analysis. Asterisk (*) indicates previously published19.
