Sequence Determinants of a Specific Inactive Protein Kinase Conformation

•Only some protein kinases have been shown to adopt specific inactive conformations•Two residues were found that confer sensitivity to ligands stabilizing the DFG-out inactive form•Kinases in two distinct families were sensitized to these ligands•Structural evidence is given of mutant Erk2 in the DFG-out inactive conformation

SummaryOnly a small percentage of protein kinases have been shown to adopt a distinct inactive ATP-binding site conformation, called the Asp-Phe-Gly-out (DFG-out) conformation. Given the high degree of homology within this enzyme family, we sought to understand the basis of this disparity on a sequence level. We identified two residue positions that sensitize mitogen-activated protein kinases (MAPKs) to inhibitors that stabilize the DFG-out inactive conformation. After characterizing the structure and dynamics of an inhibitor-sensitive MAPK mutant, we demonstrated the generality of this strategy by sensitizing a kinase (apoptosis signal-regulating kinase 1) not in the MAPK family to several DFG-out stabilizing ligands, using the same residue positions. The use of specific inactive conformations may aid the study of noncatalytic roles of protein kinases, such as binding partner interactions and scaffolding effects.

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