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PDBsum entry 3mh2
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References listed in PDB file
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Key reference
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Title
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Mutagenesis of p38alpha map kinase establishes key roles of phe169 in function and structural dynamics and reveals a novel dfg-Out state.
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Authors
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M.Bukhtiyarova,
M.Karpusas,
K.Northrop,
H.V.Namboodiri,
E.B.Springman.
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Ref.
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Biochemistry, 2007,
46,
5687-5696.
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PubMed id
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Abstract
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In order to study the role of Phe169 in p38alpha MAP kinase structure and
function, wild-type p38alpha and five p38alpha 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 p38alpha variants were
efficiently phosphorylated by MKK6. However, only one activated p38alpha 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 p38alpha. Two mutations
significantly increased the thermal stability of p38alpha (F169A DeltaTm = 1.3
degrees C and D168G DeltaTm = 3.8 degrees C), and two mutations significantly
decreased the stability of p38alpha (F169R DeltaTm = -3.2 degrees C and F169G
DeltaTm = -4.7 degrees C). Interestingly, X-ray crystal structures of two
thermally destabilized p38alpha-F169R and p38alpha-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 p38alpha crystal structures with bound inhibitors and postulated
from high-temperature molecular dynamics simulations. Taken together, these
results indicate that Phe169 is optimized for p38alpha functional activity and
structural dynamics, rather than for structural stability. The alpha-DFG-OUT
conformation observed for p38alpha-F169R and p38alpha-F169G may represent a
naturally occurring intermediate state of p38alpha that provides access for
binding of allosteric inhibitors. A model of the local forces driving the DFG
IN-OUT transition in p38alpha is proposed.
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