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PDBsum entry 1syk
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References listed in PDB file
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Key reference
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Title
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Crystal structure of the e230q mutant of camp-Dependent protein kinase reveals an unexpected apoenzyme conformation and an extended n-Terminal a helix.
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Authors
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J.Wu,
J.Yang,
N.Kannan,
Madhusudan,
N.H.Xuong,
L.F.Ten eyck,
S.S.Taylor.
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Ref.
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Protein Sci, 2005,
14,
2871-2879.
[DOI no: ]
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PubMed id
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Abstract
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Glu230, one of the acidic residues that cluster around the active site of the
catalytic subunit of cAMP-dependent protein kinase, plays an important role in
substrate recognition. Specifically, its side chain forms a direct salt-bridge
interaction with the substrate's P-2 Arg. Previous studies showed that mutation
of Glu230 to Gln (E230Q) caused significant decreases not only in substrate
binding but also in the rate of phosphoryl transfer. To better understand the
importance of Glu230 for structure and function, we solved the crystal structure
of the E230Q mutant at 2.8 A resolution. Surprisingly, the mutant preferred an
open conformation with no bound ligands observed, even though the crystals were
grown in the presence of MgATP and the inhibitor peptide, IP20. This is in
contrast to the wild-type protein that, under the same conditions, prefers the
closed conformation of a ternary complex. The structure highlights the
importance of the electrostatic surface not only for substrate binding and
catalysis, but also for the mechanism for closing the active site cleft. This
surface mutation clearly disrupts the recognition and binding of substrate
peptide so that the enzyme prefers an open conformation that cannot trap ATP.
This is consistent with the reinforcing concepts of conformational dynamics and
the synergistic binding of ATP and substrate peptide. Another unusual feature of
the structure is the observation of the entire N terminus (Gly1-Thr32) assumes
an extended alpha-helix conformation. Finally, based on temperature factors,
this mutant structure is more stable than the wild-type C-subunit in the apo
state.
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Figure 1.
Figure 1. Structural environment of Glu230. The D-helix,
F-helix, catalytic loop, P+1 loop, and part of the inhibitory
peptide IP20 are shown in ribbon diagram. Glu230 is located at
the C-terminal end of the F-helix. P-2 Arg from IP20 is rendered
as ball-and-sticks. Dotted lines represent interactions between
Glu230 and its environment.
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Figure 4.
Figure 4. Conservation of the IP20-binding site. Structures
of the wild-type apoenzyme (PDB ID 1J3H [PDB]
; yellow sticks), the E230Q mutant (red sticks), and the ternary
complex of wild-type protein with MgADP-aluminum fluoride and
substrate peptide SP20 (PDB ID 1L3R [PDB]
; dark thin sticks) were superimposed. Some of the negatively
charged residues involved in recognition of the arginines in the
substrate peptide are shown in sticks. SP20 from the ternary
structure (1L3R) is shown in ribbon diagram with P-site Ser,
P-2, P-3, and P-6 Args rendered as ball-and-sticks. Replacement
of Glu230 by Gln did not change its conformation, while its
binding partner Arg133 is disordered.
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The above figures are
reprinted
by permission from the Protein Society:
Protein Sci
(2005,
14,
2871-2879)
copyright 2005.
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