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PDBsum entry 2gnf
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Transferase/transferase inhibitor
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PDB id
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2gnf
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References listed in PDB file
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Key reference
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Title
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Structural analysis of protein kinase a mutants with rho-Kinase inhibitor specificity.
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Authors
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S.Bonn,
S.Herrero,
C.B.Breitenlechner,
A.Erlbruch,
W.Lehmann,
R.A.Engh,
M.Gassel,
D.Bossemeyer.
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Ref.
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J Biol Chem, 2006,
281,
24818-24830.
[DOI no: ]
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PubMed id
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Abstract
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Controlling aberrant kinase-mediated cellular signaling is a major strategy in
cancer therapy; successful protein kinase inhibitors such as Tarceva and Gleevec
verify this approach. Specificity of inhibitors for the targeted kinase(s),
however, is a crucial factor for therapeutic success. Based on homology
modeling, we previously identified four amino acids in the active site of
Rho-kinase that likely determine inhibitor specificities observed for Rho-kinase
relative to protein kinase A (PKA) (in PKA numbering: T183A, L49I, V123M, and
E127D), and a fifth (Q181K) that played a surprising role in PKA-PKB hybrid
proteins. We have systematically mutated these residues in PKA to their
counterparts in Rho-kinase, individually and in combination. Using four
Rho-kinase-specific, one PKA-specific, and one pan-kinase-specific inhibitor, we
measured the inhibitor-binding properties of the mutated proteins and identify
the roles of individual residues as specificity determinants. Two combined
mutant proteins, containing the combination of mutations T183A and L49I, closely
mimic Rho-kinase. Kinetic results corroborate the hypothesis that side-chain
identities form the major determinants of selectivity. An unexpected result of
the analysis is the consistent contribution of the individual mutations by
simple factors. Crystal structures of the surrogate kinase inhibitor complexes
provide a detailed basis for an understanding of these selectivity determinant
residues. The ability to obtain kinetic and structural data from these PKA
mutants, combined with their Rho-kinase-like selectivity profiles, make them
valuable for use as surrogate kinases for structure-based inhibitor design.
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Figure 1.
FIGURE 1. A, detail of the substitution positions in the
ATP binding site of PKA. B, the low molecular weight inhibitors
used in this study.
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Figure 9.
FIGURE 9. A, superposition of PKAR5-1077 (blue carbons) and
(1Q8W) PKAWT-HA1077 (gray carbon atoms). B, electron density map
(2F[o] - F[c] contoured at 1.5  ) of the inhibitor
binding pocket of PKAR5-1077.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2006,
281,
24818-24830)
copyright 2006.
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Secondary reference #1
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Title
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Protein kinase a in complex with rho-Kinase inhibitors y-27632, Fasudil, And h-1152p: structural basis of selectivity.
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Authors
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C.Breitenlechner,
M.Gassel,
H.Hidaka,
V.Kinzel,
R.Huber,
R.A.Engh,
D.Bossemeyer.
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Ref.
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Structure, 2003,
11,
1595-1607.
[DOI no: ]
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PubMed id
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Figure 6.
Figure 6. Comparison of HA-1077 and H-1152POverlay of
HA-1077 and H-1152P demonstrates the colocalization of the
isoquinoline atoms with respect to the surrounding residues.
Both inhibitor molecules form an H bond to the backbone amide of
Val123 in the hinge region. The position of the homopiperazine
rings, however, diverge by ca. 1.5 Å. Consequently, H bonds
between the homopiperazine nitrogen and Glu127 and Glu170 are
formed only in the PKA-1077 complex. The contact between C10 and
Thr183, which prevents as a steric clash a HA-1077-like
positioning of the H-1152P homopiperazine ring, is shown as a
red double arrow.
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The above figure is
reproduced from the cited reference
with permission from Cell Press
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