Mitogen-activated protein kinases (MAPKs) fulfill essential biological functions 
and are key pharmaceutical targets. Regulation of MAPKs is achieved via a 
plethora of regulatory proteins including activating MAPKKs and an abundance of 
deactivating phosphatases. Although all regulatory proteins use an identical 
interaction site on MAPKs, the common docking and hydrophobic pocket, they use 
distinct kinase interaction motif (KIM or D-motif) sequences that are present in 
linear, peptide-like, or well folded protein domains. It has been recently shown 
that a KIM-containing MAPK-specific dual specificity phosphatase DUSP10 uses a 
unique binding mode to interact with p38α. Here we describe the interaction of 
the MAPK binding domain of DUSP16 with p38α and show that despite belonging to 
the same dual specificity phosphatase (DUSP) family, its interaction mode 
differs from that of DUSP10. Indeed, the DUSP16 MAPK binding domain uses an 
additional helix, α-helix 4, to further engage p38α. This leads to an additional 
interaction surface on p38α. Together, these structural and energetic 
differences in p38α engagement highlight the fine-tuning necessary to achieve 
MAPK specificity and regulation among multiple regulatory proteins.