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PDBsum entry 2etz
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References listed in PDB file
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Key reference
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Title
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Molecular details of itk activation by prolyl isomerization and phospholigand binding: the nmr structure of the itk sh2 domain bound to a phosphopeptide.
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Authors
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E.V.Pletneva,
M.Sundd,
D.B.Fulton,
A.H.Andreotti.
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Ref.
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J Mol Biol, 2006,
357,
550-561.
[DOI no: ]
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PubMed id
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Abstract
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The Src homology 2 (SH2) domain of interleukin-2 tyrosine kinase (Itk) is a
critical component of the regulatory apparatus controlling the activity of this
immunologically important enzyme. To gain insight into the structural features
associated with the activated form of Itk, we have solved the NMR structure of
the SH2 domain bound to a phosphotyrosine-containing peptide (pY) and analyzed
changes in trans-hydrogen bond scalar couplings ((3h)J(NC')) that result from pY
binding. Isomerization of a single prolyl imide bond in this domain is
responsible for simultaneous existence of two distinct SH2 conformers. Prolyl
isomerization directs ligand recognition: the trans conformer preferentially
binds pY. The structure of the SH2/pY complex provides insight into the ligand
specificity; the BG loop in the ligand-free trans SH2 conformer is pre-arranged
for optimal contacts with the pY+3 residue of the ligand. Analysis of (3h)J(NC')
couplings arising from hydrogen bonds has revealed propagation of structural
changes from the pY binding pocket to the CD loop containing conformationally
heterogeneous proline as well as to the alphaB helix, on the opposite site of
the domain. These findings offer a structural framework for understanding the
roles of prolyl isomerization and pY binding in Itk regulation.
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Figure 2.
Figure 2. NMR structure of the Itk SH2 domain bound to the
Ac-ADpYEPP-NH[2] phosphopeptide. (a) The 20 lowest energy
structures of the Itk SH2/ADpYEPP complex. The peptide ligand is
shown in red and the SH2 domain in turquoise. Backbone heavy
atoms within the secondary structural elements over the entire
SH2 sequence were used for superpositions. (b) Ribbon diagram of
the energy minimized average structure of the Itk
SH2/phosphopeptide complex. (The view is identical with that
shown in (a).) Regular secondary structural elements and loop
regions are labeled. The location of Pro287 within the CD loop
is indicated. The phosphopeptide ligand (Ac-ADpYEPP-NH[2]) is
red and the phosphotyrosine (pY) and proline residue three
positions C-terminal (pY+3) are labeled. In both (a) and (b) the
first two residues of the phosphopeptide (AD) are not included
for clarity. No NOEs are observed between this region of the
peptide and the Itk SH2 domain.
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Figure 3.
Figure 3. (a) Superposition of the minimized average
structures of the cis SH2 domain (orange), the trans SH2 domain
(light blue) and the phospholigand-bound SH2 domain (dark blue).
The phospholigand is shown in red and the pY and pY+3 residues
are labeled. The BG loop is indicated and the arrow shows the
shift in position of the alpha carbon atom of Leu329 that
accompanies isomerization of Pro287 from the cis to trans
conformations. The structure of the BG loop for the
phospholigand-bound SH2 domain (dark blue) is similar to that of
BG loop in the ligand-free trans domain (light blue). (b)
Superposition of the lowest energy structures of the Itk cis SH2
domain (20 structures), the trans SH2 domain (20 structures) and
the SH2/phosphopeptide complex (20 structures). Colors
correspond to those in (a). The ensemble of structures indicates
that the BG loop in the cis SH2 structure (orange) adopts a
range of conformations that differ from the conformational
preferences of both the trans SH2 domain and the phospholigand
bound SH2 domain. For both (a) and (b) backbone heavy atoms
within the secondary structural elements were used for
superpositions.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2006,
357,
550-561)
copyright 2006.
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Secondary reference #1
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Title
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Structural characterization of a proline-Driven conformational switch within the itk sh2 domain.
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Authors
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R.J.Mallis,
K.N.Brazin,
D.B.Fulton,
A.H.Andreotti.
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Ref.
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Nat Struct Biol, 2002,
9,
900-905.
[DOI no: ]
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PubMed id
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Figure 1.
Figure 1. NMR structures of the cis and trans Itk SH2
conformers. a, Stereo view of 20 low energy structures of the
cis (coral) and trans (turquoise) conformations of the Itk SH2
domain. Backbone heavy atoms within the secondary structural
elements over the entire sequence were used for superpositions.
b, Ribbon diagrams of the energy minimized average structures of
the cis (left) and trans (right) conformers. Secondary
structural elements and ligand-binding pockets are labeled in
(a,b) according to standard nomenclature for SH2 domains8. Pro
287 is labeled in each structure. c, Sequence of the Itk SH2
domain and sequence alignment of the CD loop regions in the SH2
domains of several tyrosine kinases. The residues that give rise
to nondegenerate chemical shifts2 are bold and underlined, and
Pro 287 is labeled. d, Solvent-accessible surface plot of the
cis conformer. The residues that give rise to dual resonances
because of Pro cis-trans isomerization are highlighted in white.
The trans conformer shows a similar contiguous surface for the
heterogeneous residues (data not shown). e, Overlay of the
energy minimized average structures of the cis (coral) and trans
(turquoise) conformers. Expanded views of the CD loop (left),
the central  -sheet
(right) and the BG loop regions (middle) are shown. All
structures are rendered using MolMol31.
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Figure 3.
Figure 3. Hydrophobic packing involving residues in the CD loop
of the cis SH2 structure provides stabilization energy. a,
Overlay of 20 lowest energy structures including the CD loop,
the central -sheet
and  A
of the cis (left) and trans (right) conformers. Side chains of
Leu 254 and Pro 287 are yellow. His 291 and Glu 250 are also
labeled. b, Overlay of 20 lowest energy structures (rotated
clockwise with respect to (a)) showing the CD loop of the cis
(left) and trans (right) conformers. Side chains of Ile 282, Ala
281 and Cys 288 are labeled and shown in yellow. Additional side
chains are included without labels for clarity. c,
Three-dimensional 13C-edited NOESY experiment showing
through-space proximity between the  -methyl
protons of Ile 282 and one of the -methylene
protons of Cys 288. The NOE is observed only for the cis
conformer (left panel). The total number of NOEs unique to the
cis and trans structures is shown in Table 1. d,
Three-dimensional 15N-edited TOCSY experiment illustrating the
nondegenerate resonance frequencies for the Cys 288 -methylenes
in the cis conformer (left). The same protons resonate at a
single frequency in the trans conformer (right). e, Expansion of
the 1H-15N HSQC spectra showing the amide signal of Gly 260
(6260) in the cis and trans forms. Left, unmodified, reduced Itk
SH2 domain. Middle, spectrum acquired following reaction of the
Itk SH2 domain with glutathione disulfide (GSSG) (20 mM GSSG, pH
7.4, 40 min, 25 °C). Right, spectrum acquired following reaction
with methyl methane thiosulfonate (MMTS) (5 mM MMTS, pH 7.4, 20
min). The percentage of SH2 domain in the cis conformation in
each of these experiments as measured by peak volumes of Gly 260
(cis) and Gly 260 (trans) was 48, 5 and 32% for the reduced,
GSSG-treated and MMTS-treated proteins, respectively. The
backbone amide resonance of Cys 288 was monitored over the
course of both reactions and the reactions were allowed to
proceed until no further chemical shift changes occurred. The
completeness of the S-glutathiolation reaction was also assessed
by separation of the domain with nondenaturing isoelectric
focusing (IEF) gel electrophoresis over a pH range of 3.5 -10 as
described^33.
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The above figures are
reproduced from the cited reference
with permission from Macmillan Publishers Ltd
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