The discrepancies in amino acid sequence of the phosphate-binding loop lead to distinctive binding modes of a covalent inhibitor for MAP2K1 and MAP2K6: Structural insights for producing selective inhibitors

Mitogen-activated protein kinase kinase 6 (MAP2K6) plays a crucial role in activating the p38 MAPK pathway, and dysregulation of this pathway is associated with serious diseases including autoimmune diseases. 5Z-7-oxozeaenol (5Z7O), a covalent-binding inhibitor, inhibits MAP2K6 approximately ten times more strongly than MAP2K1, a common off-target kinase of MAP2K6. Here, we determined the crystal structure of the 5Z7O-MAP2K6 complex and carefully compared it with that of the 5Z7O-MAP2K1 complex previously reported. The binding orientation of 5Z7O is slightly different between the MAP2K1 and MAP2K6 complexes, resulting in different hydrogen-bond networks and thereby the higher potency of 5Z7O for MAP2K6 than MAP2K1. 5Z7O formed hydrogen bonds with the arginine residue in the catalytic HRD motif of MAP2K6 and asparagine residue in the solvent-accessible region but not with the corresponding residues of MAP2K1. Structural comparison implied that these differences in hydrogen bonding were attributable to differences in the phosphate-binding loop (P-loop) between MAP2K6 and MAP2K1. Molecular dynamics simulation revealed the above-mentioned and further structural features of MAP2K1 and MAP2K6. These distinct structural features are potentially useful for producing selective inhibitors for MAP2K1 and MAP2K6.