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PDBsum entry 3idb
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References listed in PDB file
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Key reference
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Title
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Novel isoform-Specific interfaces revealed by pka riibeta holoenzyme structures.
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Authors
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S.H.Brown,
J.Wu,
C.Kim,
K.Alberto,
S.S.Taylor.
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Ref.
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J Mol Biol, 2009,
393,
1070-1082.
[DOI no: ]
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PubMed id
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Abstract
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The cAMP-dependent protein kinase catalytic (C) subunit is inhibited by two
classes of functionally nonredundant regulatory (R) subunits, RI and RII. Unlike
RI subunits, RII subunits are both substrates and inhibitors. Because RIIbeta
knockout mice have important disease phenotypes, the RIIbeta holoenzyme is a
target for developing isoform-specific agonists and/or antagonists. We also know
little about the linker region that connects the inhibitor site to the
N-terminal dimerization domain, although this linker determines the unique
globular architecture of the RIIbeta holoenzyme. To understand how RIIbeta
functions as both an inhibitor and a substrate and to elucidate the structural
role of the linker, we engineered different RIIbeta constructs. In the absence
of nucleotide, RIIbeta(108-268), which contains a single cyclic nucleotide
binding domain, bound C subunit poorly, whereas with AMP-PNP, a non-hydrolyzable
ATP analog, the affinity was 11 nM. The RIIbeta(108-268) holoenzyme structure
(1.62 A) with AMP-PNP/Mn(2+) showed that we trapped the RIIbeta subunit in an
enzyme:substrate complex with the C subunit in a closed conformation. The
enhanced affinity afforded by AMP-PNP/Mn(2+) may be a useful strategy for
increasing affinity and trapping other protein substrates with their cognate
protein kinase. Because mutagenesis predicted that the region N-terminal to the
inhibitor site might dock differently to RI and RII, we also engineered
RIIbeta(102-265), which contained six additional linker residues. The additional
linker residues in RIIbeta(102-265) increased the affinity to 1.6 nM, suggesting
that docking to this surface may also enhance catalytic efficiency. In the
corresponding holoenzyme structure, this linker docks as an extended strand onto
the surface of the large lobe. This hydrophobic pocket, formed by the
alphaF-alphaG loop and conserved in many protein kinases, also provides a
docking site for the amphipathic helix of PKI. This novel orientation of the
linker peptide provides the first clues as to how this region contributes to the
unique organization of the RIIbeta holoenzyme.
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Figure 3.
Fig. 3. RIIβ holoenzyme assumes a closed confirmation: (a)
shows three holoenzyme structures with the trapped transition
state that is present in the RIIβ holoenzyme structure
(middle). The RIIα holoenzyme (left) is in an open form whereas
the RIIβ and RIα complexes (right) are in a closed
conformation. The color designations are as follows: E230^C,
E170^C, and E203^C are shown in red and provide the acidic
docking surface for the P − 3 and P − 2 arginines; the P + 1
pocket (L198^C and P202^C) is in white and provides the
hydrophobic site for the P + 1 residue; Y330^C in the C-tail is
yellow; the glycine rich loop is in pink; AMP-PNP is in black.
The temperature factors analysis of the C subunit in three
holoenzyme structures is shown in (b). Shown on the left is
RIIα(90–400):C in the absence of ATP (2QVS), with the
disordered/mobile regions underlined in red; in the middle is
RIIβ(108–268):C:AMP-PNP; on the right is
RIα(91–244):C:AMP-PNP (1U7E).
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Figure 6.
Fig. 6. Variation in positioning of the region that lies
N-terminal to the inhibitor site: (a) Highlighted N-terminal
binding regions of PKI (dark teal), RIα (light green), and
RIIβ (red) demonstrate differential binding between inhibitors.
RIIβ and PKI dock to the C-lobe, while RIα docks to the
C-terminal tail and N-lobe. Surface mesh representation is shown
for the highly conserved P − 3 to P + 1 region. Details of
RIIβ binding peptide are highlighted. (b) The sequence and
detailed structural comparison of the inhibitor peptides of PKI,
RIα, and RIIβ. (c) The αF–αG loop (tan) creates a
hydrophobic pocket that recognizes different substrates (red).
Tyr235^C and Phe239^C are highly conserved residues in this
pocket. The region of RIIβ that lies N-terminally to the
inhibitor site docks as a strand to this pocket (left) while the
amphipathic helix of PKI (right) docks to the same surface. In
both cases, the peptides dock against Phe239^C. In
RIIβ(108–268), this site is unoccupied (middle) and the side
chain of Phe239^C is rotated away from Tyr235^C. In the two
RIIβ holoenzymes, one can see how Tyr247^C in the G-helix
interfaces with the P + 1 Val and with the Tyr226^R in the PBC
of RIIβ.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2009,
393,
1070-1082)
copyright 2009.
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