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PDBsum entry 1ksw
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Contents |
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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Mutant tyrosine kinases with unnatural nucleotide specificity retain the structure and phospho-Acceptor specificity of the wild-Type enzyme.
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Authors
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L.A.Witucki,
X.Huang,
K.Shah,
Y.Liu,
S.Kyin,
M.J.Eck,
K.M.Shokat.
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Ref.
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Chem Biol, 2002,
9,
25-33.
[DOI no: ]
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PubMed id
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Abstract
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The direct substrates of one protein kinase in a cell can be identified by
mutation of the ATP binding pocket to allow an unnatural ATP analog to be
accepted exclusively by the engineered kinase. Here, we present structural and
functional assessment of peptide specificity of mutant protein kinases with
unnatural ATP analogs. The crystal structure (2.8 A resolution) of c-Src (T338G)
with N(6)-(benzyl) ADP bound shows that the creation of a unique nucleotide
binding pocket does not alter the phospho-acceptor binding site of the kinase. A
panel of optimal peptide substrates of defined sequence, as well as a degenerate
peptide library, was utilized to assess the phospho-acceptor specificity of the
engineered "traceable" kinases. The specificity profiles for the
mutant kinases were found to be identical to those of their wild-type
counterparts.
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Figure 1.
Figure 1. Chemical Structures of A*TP Analogs Used in This
Study1: N^6-(benzyl) ATP; 2: N^6-(cyclopentyl) ATP. Definitions
of analog-sensitive (as) kinase mutants used in this study.
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Figure 2.
Figure 2. Comparison of Wild-Type and Analog-Specific c-Src
Crystal Structures(A) The crystal structure of c-Src-as1
superimposed on wild-type c-Src. c-Src-as1 is shown in gray, and
c-Src is in red. The rmsd for the overlay is 0.35 Å.(B)
The binding of the A*TP analog, N^6-(benzyl) ADP to the mutant
c-Src (T338G) kinase. The surface corresponding to the glycine
residue at the 338 position is colored red. The benzyl ring of
the A*TP analog projects into a pocket in the nucleotide binding
cleft. This pocket is made accessible by the c-Src (T338G) point
mutation. For clarity, the 11 residues that bind over the
nucleotide at the front of the nucleotide cleft are omitted from
the figure in order to more clearly show the surface at the back
of the nucleotide binding pocket where the 338 residue lies. The
omitted residues are c-Src 272–282.(C) The steric clash of the
wild-type c-Src threonine residue at the 338 position, shown in
red, with the N^6-(benzyl) ATP analog (blue). The gray surface
was built over the crystal structure of the mutant kinase
overlayed with the wild-type c-Src crystal structure, and the
surface was rendered over threonine 338 (red). The N^6-(benzyl)
ADP (blue) is superimposed on the AMP-PNP ligand (yellow).
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The above figures are
reprinted
by permission from Cell Press:
Chem Biol
(2002,
9,
25-33)
copyright 2002.
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