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PDBsum entry 1i3z
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Signaling protein
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PDB id
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1i3z
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Contents |
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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Structural basis for the interaction of the free sh2 domain eat-2 with slam receptors in hematopoietic cells.
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Authors
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M.Morra,
J.Lu,
F.Poy,
M.Martin,
J.Sayos,
S.Calpe,
C.Gullo,
D.Howie,
S.Rietdijk,
A.Thompson,
A.J.Coyle,
C.Denny,
M.B.Yaffe,
P.Engel,
M.J.Eck,
C.Terhorst.
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Ref.
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EMBO J, 2001,
20,
5840-5852.
[DOI no: ]
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PubMed id
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Abstract
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The T and natural killer (NK) cell-specific gene SAP (SH2D1A) encodes a 'free
SH2 domain' that binds a specific tyrosine motif in the cytoplasmic tail of SLAM
(CD150) and related cell surface proteins. Mutations in SH2D1A cause the
X-linked lymphoproliferative disease, a primary immunodeficiency. Here we report
that a second gene encoding a free SH2 domain, EAT-2, is expressed in
macrophages and B lympho cytes. The EAT-2 structure in complex with a
phosphotyrosine peptide containing a sequence motif with Tyr281 of the
cytoplasmic tail of CD150 is very similar to the structure of SH2D1A complexed
with the same peptide. This explains the high affinity of EAT-2 for the pTyr
motif in the cytoplasmic tail of CD150 but, unlike SH2D1A, EAT-2 does not bind
to non-phosphorylated CD150. EAT-2 binds to the phosphorylated receptors CD84,
CD150, CD229 and CD244, and acts as a natural inhibitor, which interferes with
the recruitment of the tyrosine phosphatase SHP-2. We conclude that EAT-2 plays
a role in controlling signal transduction through at least four receptors
expressed on the surface of professional antigen-presenting cells.
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Figure 1.
Figure 1 The human EAT-2 gene. (A) Alignment of the human and
mouse EAT-2 nucleotide sequences. The coding region sequences of
the human (hEAT-2) and mouse (mEAT-2) EAT-2 cDNAs are compared.
Exon boundaries are indicated (bold font, identity of
nucleotides; regular font, difference of nucleotides). (B)
Genomic organization of the human EAT-2 gene. The human EAT-2
gene consists of four exons that present an overall organization
similar to that of the SH2D1A gene. The putative exon IIIA
represents part of exon III (see text).
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Figure 4.
Figure 4 EAT-2 binds exclusively to a phosphorylated peptide
(pY281) derived from the cytoplasmic tail of CD150. (A)
Fluorescence polarization analysis of the EAT-2 binding to a
phosphorylated pY281 peptide. Different concentrations of GST
-mouse EAT-2 (or GST -human SH2D1A) and an 11mer synthetic
peptide identical to amino acid residues 276 -287 of human CD150
(Sayos et al., 1998), tyrosine phosphorylated or not, were used.
Top panel: binding of GST -mouse EAT-2 to the pY281 (filled
triangles and continuous line) or the Y281 peptide (open squares
and dashed line). Bottom panel: binding of GST -human SH2D1A to
the pY281 (filled triangles and continuous line) or the Y281
peptide (open squares and dashed line). x-axis: protein
concentration (nM); y-axis: polarization units (mP). The table
summarizes the apparent dissociation constant (kD). (B) Hybrid
system analysis of the interaction between EAT-2 and the
cytoplasmic tail of CD150 in the presence or absence of fyn.
Dashed bars indicate the interaction between the EAT-2 (or
SH2D1A) full-length protein fused to a GAL4 DNA-binding domain
and the GAL4 DNA activation domain fused to the cytoplasmic tail
of the CD150 receptor. An empty pGAD424 vector was used as a
control (solid bars). The test was conducted in either the
presence or absence of fyn[420,531Y -F]. y-axis =  -galactosidase
(U/ml).
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The above figures are
reprinted
from an Open Access publication published by Macmillan Publishers Ltd:
EMBO J
(2001,
20,
5840-5852)
copyright 2001.
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Secondary reference #1
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Title
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Crystal structures of the xlp protein sap reveal a class of sh2 domains with extended, Phosphotyrosine-Independent sequence recognition.
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Authors
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F.Poy,
M.B.Yaffe,
J.Sayos,
K.Saxena,
M.Morra,
J.Sumegi,
L.C.Cantley,
C.Terhorst,
M.J.Eck.
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Ref.
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Mol Cell, 1999,
4,
555-561.
[DOI no: ]
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PubMed id
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Figure 1.
Figure 1. Structure of SAP and the Location of Missense
Mutations Identified in XLP Patients(A) Ribbon diagram showing
the SAP/SLAM pY281 complex. The bound phosphopeptide is shown in
a stick representation (yellow). Selected SAP residues that form
the binding site are shown in blue. Elements of secondary
structure are labeled using the standard SH2 domain nomenclature
([8]). Note that the pY −3 to pY −1 residues of the peptide
make a parallel β sheet interaction with strand βD; the side
chains of these peptide residues make hydrophobic contacts with
Tyr-50, Ile-51, and Tyr-52 in strand βD, and with Leu-21. Thr
(pY −2) in the peptide hydrogen bonds with Glu-17 and with a
buried water molecule. The phosphotyrosine is coordinated in a
manner similar to that observed in the N-terminal domain of
SHP-2, and as in SHP-2, the phosphate group is rotated
“above” the plane of the phosphotyrosine ring.
Interestingly, arginine 13 (at position αA2), which is
conserved in almost all SH2 domains and usually contributes to
phosphotyrosine coordination, does not participate in phosphate
binding in the SAP complex. Instead, arginine 55 (βD6) hydrogen
bonds with the phosphate group. C-terminal to phosphotyrosine,
Val(pY +3) binds in a mostly hydrophobic cleft.(B) Point
mutations identified in XLP patients cluster along the
peptide-binding site and at the back of the domain. Mutations
that would be expected to directly disrupt the
phosphotyrosine-binding pocket are shown in green, and those
that would disrupt C-terminal interactions in magenta. The
remaining mutations (gold) are remote from the peptide-binding
surface and may destabilize the folded protein (see text).(C)
Structure-based sequence comparisons of human SAP, murine EAT-2,
and other SH2 domains. Elements of secondary structure are
indicated above the alignment. Numbering corresponds to human
SAP. The black diamonds indicate the mutations illustrated in
(B).
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Figure 2.
Figure 2. Structure and Comparisons of the SAP/SLAM Y281
Complex(A) Surface representation of the SAP domain with the
bound nonphosphorylated peptide shown in green. Hydrophobic
residues at the −1 and −3 positions of the peptide
intercalate with hydrophobic and aromatic residues on the
surface of the domain (see also [D] and Figure 1A). C-terminal
to phosphotyrosine, Val+3 is buried in a mostly hydrophobic
groove.(B) Superposition of the phosphorylated and
nonphosphorylated peptides shows that they adopt an essentially
identical conformation. An alpha-carbon trace of the domain is
shown in gray.(C) Superposition of the unliganded domain (blue)
and the phosphopeptide (yellow) and nonphosphorylated peptide
complexes (green). In the absence of bound peptide, the EF and
BG loops fold inward to close the hydrophobic +3 binding groove.
The conformation of the phosphotyrosine-binding pocket is
essentially the same in all structures. In the unliganded
structure, a sulfate ion occupies the position of the phosphate
group in the phosphopeptide complex.(D) Detail of the
phosphotyrosine-binding pocket in the SLAM/pY281 complex. Red
spheres represent ordered water molecules. The pY281 peptide is
shown in yellow. Thin cyan lines indicate potential hydrogen
bonds. Note that Arg-13 is poorly ordered and does not
participate in phosphotyrosine coordination.(E) Detail of the
phosphotyrosine-binding pocket in the nonphosphorylated
SLAM/Y281 complex. Red spheres represent ordered water
molecules. The Y281 peptide is shown in green. Thin cyan lines
indicate potential hydrogen bonds. Note that Arg-32 organizes an
extensive network of hydrogen bonds in spite of the lack of
phosphorylation of Tyr-281.
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The above figures are
reproduced from the cited reference
with permission from Cell Press
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