Fig. 8: CCT-like domains and alternative binding motifs expand complexity.
From: Kinase interaction analysis predicts actions of the WNK-OSR1/SPAK pathway
A Region of the human kinome tree containing WNK1-4, OSR1, SPAK, and NRBP1/2; all proteins contain CCT or CCT-like (CCTL) domains59. B Diagram depicting locations of CCT and CCTL domains in proteins. C Multiple sequence alignment of regions of CCT/CCTL domains that comprise motif binding pockets consisting of β-strand–loop–α-helix. Conserved binding residues indicated. D Comparison of crystal structure of OSR1 CCT:GRFQVT hexapeptide complex (left, PDB:2V3S) with AlphaFold3-derived models of OSR1 CCT (430–527), WNK1 CCTL1 (human, 482–597), and NRBP1 CCTL (436–535) bound to TSC22D1 RFxV motif (395–419)50,57,58,60. For NRBP1:TSC22D1, when using full-length proteins, differing models were obtained depending on the AlphaFold modeling server used. Both results can be found in the Supp. Fig. 9B. Identical results were obtained using truncated vs full-length proteins for OSR1 and WNK1. E, F Structural alignment of AlphaFold3 models in (D). Binding residues depicted as sticks. G Model of SPAK CCT:GRFQVT complex made by overlaying SPAK CCT structure (PDB: 7O86) onto the OSR1 CCT:GRFQVT complex structure. D477 (green) was mutated in the present study to inhibit binding while L491 (green) was mutated in previous study62. H, I 3xFLAG-tagged SPAK 50-545 ± D477Q or NRBP1 ± E488Q used to co-IP myc-NKCC2 1-170 and myc-TSC22D1, respectively, in HEK293T cells. Mean ± SEM of mutants relative to WT (n = 3, 95% confidence intervals shown). J Affinity determined by fluorescence anisotropy peptide competition assays (n = 3, see Fig. 1F and Supp. Fig. 3A. Asterisk (*) indicates results previously published19.
