Mutagenesis of p38α MAP kinase establishes key roles of Phe169 in function and structural dynamics and reveals a novel DFG-OUT state

In order to study the role of Phe169 in p38 alpha MAP kinase structure and function, wild-type p38 alpha and five p38 alpha DFG motif mutants were examined in Vitro for phosphorylation by MKK6, kinase activity toward ATF2 substrate, thermal stability, and X-ray crystal structure. All six p38 alpha variants were efficiently phosphorylated by MKK6. However, only one activated p38 alpha mutant (F169Y) possessed measurable kinase activity (1% compared to wild-type). The loss of kinase activity among the DFG mutants may result from an inability to correctly position Asp168 in the activated form of p38 alpha. Two mutations significantly increased the thermal stability of p38 alpha (F169A Delta T-m = 1.3 degrees C and D168G Delta T-m = 3.8 degrees C), and two mutations significantly decreased the stability of p38 alpha (F169R Delta T-m) = -3.2 degrees C and F169G Delta T-m) = -4.7 degrees C). Interestingly, X-ray crystal structures of two thermally destabilized p38 alpha-F169R and p38 alpha-F169G mutants revealed a DFG-OUT conformation in the absence of an inhibitor molecule. This DFG-OUT conformation, termed alpha-DFG-OUT, is different from the ones previously identified in p38 alpha crystal structures with bound inhibitors and postulated from high-temperature molecular dynamics simulations. Taken together, these results indicate that Phe169 is optimized for p38 alpha functional activity and structural dynamics, rather than for structural stability. The alpha-DFG-OUT conformation observed for p38 alpha-F169R and p38 alpha-F169G may represent a naturally occurring intermediate state of p38 alpha that provides access for binding of allosteric inhibitors. A model of the local forces driving the DFG IN-OUT transition in p38 alpha is proposed.