Mutagenesis of p38alpha 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{sub m} = 1.3 {sup o}C and D168G {Delta}T{sub m} = 3.8 {sup o}C), and two mutations significantly decreased the stability of p38{alpha} (F169R {Delta}T{sub m} = -3.2 {sup o}C and F169G {Delta}T{sub m} = -4.7 {sup o}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.
- Research Organization:
- Brookhaven National Lab. (BNL), Upton, NY (United States). National Synchrotron Light Source
- Sponsoring Organization:
- Doe - Office Of Science
- DOE Contract Number:
- DE-AC02-98CH10886
- OSTI ID:
- 930453
- Report Number(s):
- BNL-81205-2008-JA; TRN: US200904%%724
- Journal Information:
- Biochemistry, Vol. 46; ISSN 0006-2960
- Country of Publication:
- United States
- Language:
- English
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