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PDBsum entry 3f6x
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References listed in PDB file
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Key reference
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Title
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Tuning a three-Component reaction for trapping kinase substrate complexes.
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Authors
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A.V.Statsuk,
D.J.Maly,
M.A.Seeliger,
M.A.Fabian,
W.H.Biggs,
D.J.Lockhart,
P.P.Zarrinkar,
J.Kuriyan,
K.M.Shokat.
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Ref.
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J Am Chem Soc, 2008,
130,
17568-17574.
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PubMed id
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Note: In the PDB file this reference is
annotated as "TO BE PUBLISHED". The citation details given above have
been manually determined.
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Abstract
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The upstream protein kinases responsible for thousands of phosphorylation events
in the phosphoproteome remain to be discovered. We developed a three-component
chemical reaction which converts the transient noncovalent substrate-kinase
complex into a covalently cross-linked product by utilizing a dialdehyde-based
cross-linker, 1. Unfortunately, the reaction of 1 with a lysine in the kinase
active site and an engineered cysteine on the substrate to form an isoindole
cross-linked product could not be performed in the presence of competing
cellular proteins due to nonspecific side reactions. In order to more
selectively target the cross-linker to protein kinases in cell lysates, we
replaced the weak, kinase-binding adenosine moiety of 1 with a potent protein
kinase inhibitor scaffold. In addition, we replaced the o-phthaldialdehyde
moiety in 1 with a less-reactive thiophene-2,3-dicarboxaldehyde moiety. The
combination of these two structural modifications provides for cross-linking of
a cysteine-containing substrate to its corresponding kinase in the presence of
competing cellular proteins.
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