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PDBsum entry 3c4x
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References listed in PDB file
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Key reference
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Title
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Structures of rhodopsin kinase in different ligand states reveal key elements involved in g protein-Coupled receptor kinase activation.
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Authors
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P.Singh,
B.Wang,
T.Maeda,
K.Palczewski,
J.J.Tesmer.
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Ref.
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J Biol Chem, 2008,
283,
14053-14062.
[DOI no: ]
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PubMed id
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Abstract
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G protein-coupled receptor (GPCR) kinases (GRKs) phosphorylate activated
heptahelical receptors, leading to their uncoupling from G proteins. Here we
report six crystal structures of rhodopsin kinase (GRK1), revealing not only
three distinct nucleotide-binding states of a GRK but also two key structural
elements believed to be involved in the recognition of activated GPCRs. The
first is the C-terminal extension of the kinase domain, which was observed in
all nucleotide-bound GRK1 structures. The second is residues 5-30 of the N
terminus, observed in one of the GRK1.(Mg2+)2.ATP structures. The N terminus was
also clearly phosphorylated, leading to the identification of two novel
phosphorylation sites by mass spectral analysis. Co-localization of the N
terminus and the C-terminal extension near the hinge of the kinase domain
suggests that activated GPCRs stimulate kinase activity by binding to this
region to facilitate full closure of the kinase domain.
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Figure 1.
FIGURE 1. Overview of GRK1 and its active site. a,
GRK1[535]-His[6] crystallized as a homodimer using a conserved
interface of the RH domain in all the crystal forms. Shown is
the most complete structure, that of crystal form I. The RH
terminal subdomain is colored magenta (helices  0-3 and
8-11), and the bundle
subdomain (helices 4- 7) is slate blue. The
small lobe of the kinase domain (yellow) is composed of six
β-strands (orange) and two -helices ( B and
C),
whereas the large lobe is primarily -helical. The ligand
(Mg^2+)[2]·ATP is drawn as spheres. Magnesium atoms are
colored black, carbons are white, nitrogens are blue, oxygens
are red, phosphates are orange, and chloride ions are cyan. The
extreme N-terminal region and the C-terminal extension of the
kinase domain are green. b, substrate complex of GRK1. Shown is
a  [A]-weighted |F[o]| -
|F[c]| omit map contoured at 4 , wherein ATP, Mg^2+,
and associated waters (green) were excluded from refinement
(crystal form I and chain B). Lys^216 (β1 sheet, orange
carbons) coordinates the - and β-phosphates.
Glu^332 (yellow carbons) coordinates both Mg^2+ atoms. c,
product complex of GRK1. Shown is a [A]-weighted |F[o]| -
|F[c]| omit map contoured at 5 , wherein ADP, Mg^2+,
and associated waters were excluded from refinement (crystal
form IV). d, the peptide-binding channel of GRK1. The molecular
surface of GRK1 is colored by its electrostatic potential from
-7 (red, acidic) to +7 (blue, basic) kT/e^-. The channel has a
strikingly basic character, explaining why GRK1 prefers acidic
substrates (48, 49) and how it can phosphorylate multiple
closely spaced Ser and Thr residues at the C terminus of Rho^*.
The channel is also wider in GRK1 than in nucleotide-bound PKB
(e), reflecting the more open conformation of the GRK1 kinase
domain of GRK1. As a result, the phosphoacceptor oxygen of the
modeled peptide is >4 Å from the  -phosphate of ATP,
which is too far for covalent chemistry to occur. A model of
residues 332-345 from the C terminus of Rho^*, is shown as a
stick model docked to the large lobe with Ser^338 in position to
be phosphorylated (position "+0"). Residues in the F- G loop
of the large lobe appear to obstruct the N-terminal end of the
peptide-binding site. e, the GSK3β peptide bound to PKB. The
PKB kinase domain (Protein Data Bank code 1O6L [PDB]
) is in its closed conformation. The channel is markedly acidic,
in line with a preference for basic substrates.
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Figure 3.
FIGURE 3. The phosphorylation sites of GRK1. a, the
RH-kinase core of GRK1. The structure corresponds to that of
crystal form I (with composite C-terminal extension; see Fig.
2). The Ser^5, Thr^8, Ser^21, Ser^488, and Thr^489
phosphorylation sites are drawn as stick models. The expected
position of the membrane plane is indicated. Top inset, the
Ser^488 and Thr^489 phosphorylation sites correspond to the AGC
kinase turn motif. Bottom inset, interaction of Thr(P)^8 with
the RH domain. Gln^73 and Glu^93 form direct hydrogen bonds,
whereas Lys^69 and Lys^90 complement the charge of the phosphate
moiety. These crystals grew at pH 4.35, and so either Glu^93 or
the phosphate group could be protonated. b, tandem mass
spectrometry spectra of phosphopeptides from GRK1[535]-His[6]
(Pool A, pretreated with 4 mM ATP and 2 mM MgCl[2]). Both Ser^5
and Thr^8 sites were identified in a single peptide. The Ser^5
site was also readily observed in endogenous GRK1, as were the
previously observed phosphorylation sites at Ser^21, Ser^488,
and Thr^489 (supplemental Fig. S7).
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2008,
283,
14053-14062)
copyright 2008.
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