Analysis of OSR1 kinase-peptide binding substrate interaction through computational protein docking software (893.37)

The oxidative‐stress‐responsive kinase 1 (OSR1) and the STE20/SPS1‐related proline/alanine‐rich kinase (SPAK) are key enzymes in a signaling cascade regulating the activity of Na + ‐K + ‐2Cl ‐ cotransporters (NKCC1‐2) and Na + ‐Cl ‐ cotransporter (NCC). Both kinases have a conserved carboxyl‐terminal (CCT) domain, which recognizes a unique peptide (Arg‐Phe‐ Xaa ‐Val) motif present in OSR1‐ and SPAK‐activating kinases [with‐no‐lysine kinase 1 (WNK1) and WNK4] as well as their substrates (NKCC1, NKCC2, and NCC). In this study, various modalities of the Rosetta Software Suite including flexible peptide docking and protein design were utilized to predict several constructed hexapeptides that would favorably bind to the CCT domain after individual residue mutation or whole hexapeptide design. These constructs are all derived from a crystal structure of the CCT domain of OSR1 in complex with a hexapeptide (Gly‐Arg‐Phe‐Gln‐Val‐Thr) derived from WNK4 (2v3s). Among the favorable point mutations include an arginine to histidine substitution at position 2 and a phenylalanine to tyrosine substitution at position 3 of the hexapeptide. In addition, the designed hexapeptides FRFEVT (Phe‐Arg‐Phe‐Glu‐Val‐Thr) and TRFDVT (Thr‐Arg‐Phe‐Asp‐Val‐Thr) were both predicted to bind to the CCT domain. The results of these computations were confirmed through the use of yeast two‐hybrid screening and frequencies of computationally derived motifs were calculated in known proteins sequence by searching the NCBI mouse protein database. Our study show that the Rosetta Molecular Sotware Suite can very precisely model the binding of the GRFQVT peptide to the CCT/PF2 domain of OSR1 and our in silico mutagenesis study shows that the nature of the residues required for binding might be more flexible than previously recognized. Grant Funding Source : RO1 DK0935010, RO1 GM074771