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PDBsum entry 2zgd
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De novo protein
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PDB id
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2zgd
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References listed in PDB file
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Key reference
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Title
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Asparagine beta-Hydroxylation stabilizes the ankyrin repeat domain fold.
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Authors
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L.Kelly,
M.A.Mcdonough,
M.L.Coleman,
P.J.Ratcliffe,
C.J.Schofield.
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Ref.
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Mol Biosyst, 2009,
5,
52-58.
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PubMed id
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Abstract
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Ankyrin repeats (ARs) are one of the most common structural motifs among
eukaryotic proteins. Recent analyses have shown that factor inhibiting
hypoxia-inducible factor (FIH) catalyses the hydroxylation of highly conserved
Asn-residues within ankyrin repeat domains (ARDs). However, the effect of
Asn-hydroxylation on ARD structure is unknown. Supporting the proposal that
FIH-mediated ARD hydroxylation is ubiquitous we report that consensus ARD
proteins are FIH substrates both in vitro and in vivo. X-ray diffraction
analyses revealed that hydroxylation does not alter the archetypical ARD
conformation in the crystalline state. However, other biophysical analyses
revealed that hydroxylation significantly stabilizes the ARD fold in solution.
We propose that intracellular protein hydroxylation is much more common than
previously thought and that one of its roles is stabilization of localized
regions of ARD folds.
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Secondary reference #1
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Title
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Asparaginyl hydroxylation of the notch ankyrin repeat domain by factor inhibiting hypoxia-Inducible factor.
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Authors
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M.L.Coleman,
M.A.Mcdonough,
K.S.Hewitson,
C.Coles,
J.Mecinovic,
M.Edelmann,
K.M.Cook,
M.E.Cockman,
D.E.Lancaster,
B.M.Kessler,
N.J.Oldham,
P.J.Ratcliffe,
C.J.Schofield.
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Ref.
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J Biol Chem, 2007,
282,
24027-24038.
[DOI no: ]
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PubMed id
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Figure 5.
Structure of Asn-hydroxylated N1 ARD.A, stereoview ribbon
representation from the 2.35 Å resolution crystal
structure of N1 ARD (OH); six ARs (numbered 2–7) are observed
in the N1 ARD (OH) structure. The hydroxylated Asn-1945 (AR 2)
and unhydroxylated Asn-2012 (AR 4) are yellow ball-and-stick
representations. B, stereoview of electron density around
Asn-1945 showing the pro-S configuration of the hydroxylated
Asn-1945 β-carbon. 2F[o] - F[c] map (cyan mesh) contoured to
1σ and an F[o] - F[c] OMIT map (dark blue mesh) contoured to
5σ (produced by omitting the hydroxyl group from the
calculation). This region of the β-hairpin loop has a type I
β-turn (Asp-1943, Ala-1944, Asn-1945, and Ile-1946 at the i, i
+ 1, i + 2, and i + 3 positions, respectively). Asn-1945 makes
three hydrogen bonds (dashed black lines); the nitrogen and
oxygen of the side chain amide form hydrogen bonds to the
backbone carbonyl oxygen of Ala-1975 and NH of Asp-1977, and the
β-hydroxy group hydrogen bonds with an Asp-1943 carboxylate
oxygen. C, ribbon representation of the Asn-1945 hydroxylated
mN1-(1898–2105) (blue) and the MAML-1 (green)·human
N1-(1873–2127) (purple)·CSL
(yellow/beige/orange)·DNA (blue) complex (Protein Data
Bank code 2F8X) structures (24) superimposed.
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Figure 6.
Notch-FIH interactions.A, the FIH homodimer (molecule A
(pink) and molecule B (green)) in complex with N1-(1930–1949)
(yellow ball-and-stick representation bound to A, blue bound to
B) with the double-stranded β-helix core (blue in A and
raspberry in B) and Fe(II) (orange sphere). B, stereoview ribbon
representation of the
FIH·Fe(II)·2OG·N1-(1930–1949) dimer
structure complexed with the N1 peptide to 2.4 Å
resolution in yellow ball-and-stick representation and the σ[A]
weighted composite OMIT mF[o] - DF[c] difference electron
density contoured to 3σ (blue mesh) created by omitting the
Notch1 atoms from the calculation. Electron density was apparent
for residues 1936–1945 of the N1-(1930–1949) peptide (close
up view; supplemental Fig. S3). C, the FIH active site (FIH
(salmon with residues in white ball-and-stick representation),
2OG (green), N1-(1930–1949) (yellow), and Fe(II) (orange
sphere)). The pro-S C–H bond (gray) modeled at the Cβ
position of N1 Asn-1945 is positioned for oxidation. D,
ball-and-stick representation of the superimposed peptides when
bound to FIH. yellow, N1-(1930–1949); cyan, N1-(1997–2016);
white, HIF1αCAD. E, close up of superimposed structures:
FIH·N1-(1930–1949) (yellow),
FIH·N1-(1997–2016) (cyan), and FIH·HIF1αCAD
(white) showing the binding of the conserved leucine (N1
Leu-1937/2004 and HIF1αCAD Leu-795) in a hydrophobic pocket
(yellow-green surface) formed by residues from molecules A (dark
green) and B(orange) of the FIH dimer. F, superimposition of the
target Asn of N1-(1930–1949) when bound to FIH and the
equivalent Asn in the N1 ARD structure emphasizes the
conformational changes between them.
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The above figures are
reproduced from the cited reference
with permission from the ASBMB
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Secondary reference #2
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Title
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Posttranslational hydroxylation of ankyrin repeats in ikappab proteins by the hypoxia-Inducible factor (hif) asparaginyl hydroxylase, Factor inhibiting hif (fih).
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Authors
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M.E.Cockman,
D.E.Lancaster,
I.P.Stolze,
K.S.Hewitson,
M.A.Mcdonough,
M.L.Coleman,
C.H.Coles,
X.Yu,
R.T.Hay,
S.C.Ley,
C.W.Pugh,
N.J.Oldham,
N.Masson,
C.J.Schofield,
P.J.Ratcliffe.
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Ref.
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Proc Natl Acad Sci U S A, 2006,
103,
14767-14772.
[DOI no: ]
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PubMed id
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Figure 1.
Fig. 1. Interactions between p105 and FIH. (A) Interaction
of [^35S]methionine-labeled p105 polypeptides with extract from
control cells (–) or cells induced with doxycycline (DOX) to
express FLAG-FIH (+). Anti-FLAG immunoprecipitations;
coprecipitating products are visualized by autoradiography.
Residues 650–684 are essential for FIH interaction. (B)
Alignment of residues 667–690 (fourth ankyrin repeat) of p105
with the ARD of FEM1  and UACA as well as the
HIF-1  hydroxylation site
([*]). (C) Interaction between FIH and transfected full-length
PK-tagged p105, Asn-678 mutant p105 (N678A), in 293T cells
exposed to DMOG (+) or vehicle control (–). EV, empty vector
control. Anti-PK immunoprecipitates; coprecipitating FIH is
detected by anti-FIH mAb. (D) Coimmunoprecipitation of
endogenous FIH with p105 from HeLa cell extract. p105 IP,
antibody to the C terminus of p105; CON IP, preimmune serum.
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Figure 2.
Fig. 2. Recombinant p105 and I  B promote decarboxylation
of 2-OG in assays of FIH activity. 2-OG decarboxylation is
measured by ^14CO[2] formation. (A) Reactions containing the
indicated GST-tagged proteins: HIF-1 775–826 (HIF-1 C-terminal activation
domain); p105 ARD 537–809 (p105 ARD); p105 ARD Asn-678 mutant
(p105 ARD(N678A)). Wild type (p105 ARD) but not the N678A mutant
promoted 2-OG decarboxylation. Higher background was observed
with p105 ARD(N678A) because of less pure preparation of
protein. (B) Experiments comparing p105 ARD with full-length I
B
and
I B
proteins. I B , but
not I B , promotes 2-OG
decarboxylation.
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