Mitogen-activated protein kinase kinase 7 (MAP2K7) is an indispensable kinase of the c-jun N-terminal kinase signal cascade and is rigorously regulated via phosphorylation. To investigate the regulatory mechanism of the inactive non-phosphorylated state of MAP2K7, the crystal structures of the wild-type and C218S mutant were solved. The wild-type apo-structure revealed an unprecedented auto-inhibition form that occluded the ATP site. This closed form was configured by the n-sigma(x) interaction of Cys218, a non-conserved residue among the MAP2K family kinases, with Gly145 in the glycine-rich loop. The interaction was unaltered in the presence of an ATP analog, whereas the C218S mutation precluded the closed configuration. These structural insights are potentially valuable for drug discovery of highly selective MAP2K7 inhibitors. (C) 2016 Elsevier Inc. All rights reserved.