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PDBsum entry 1q7x
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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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Structure determination and ligand interactions of the pdz2b domain of ptp-Bas (hptp1e): splicing-Induced modulation of ligand specificity.
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Authors
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N.Kachel,
K.S.Erdmann,
W.Kremer,
P.Wolff,
W.Gronwald,
R.Heumann,
H.R.Kalbitzer.
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Ref.
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J Mol Biol, 2003,
334,
143-155.
[DOI no: ]
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PubMed id
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Abstract
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Two versions of the PDZ2 domain of the protein tyrosine phosphatase
PTP-Bas/human PTP-BL are generated by alternative splicing. The domains differ
by the insertion of five amino acid residues and their affinity to the tumour
suppressor protein APC. Whereas PDZ2a is able to bind APC in the nanomolar
range, PDZ2b shows no apparent interaction with APC. Here the solution structure
of the splicing variant of PDZ2 with the insertion has been determined using 2D
and 3D heteronuclear NMR experiments. The structural reason for the changed
binding specificity is the reorientation of the loop with extra five amino acid
residues, which folds back onto beta-strands two and three. In addition the
side-chain of Lys32 closes the binding site of the APC binding protein and the
two helices, especially alpha-helix 2, change their relative position to the
protein core. Consecutively, the binding site is sterically no longer fully
accessible. From the NMR-titration studies with a C-terminal APC-peptide the
affinity of the peptide with the protein can be estimated as 540(+/-40)microM.
The binding site encompasses part of the analogous binding site of PDZ2a as
already described previously, yet specific interaction sites are abolished by
the insertion of amino acids in PDZ2b. As shown by high-affinity chromatography,
GST-PDZ2b and GST-PDZ2a bind to phosphatidylinositol 4,5-bisphosphate (PIP(2))
micelles with a dissociation constant K(D) of 21 microM and 55 microM,
respectively. In line with these data PDZ2b binds isolated, dissolved PIP(2) and
PIP(3) (phosphatidylinositol 3,4,5-trisphosphate) molecules specifically with a
lower K(D) of 230(+/-20)microM as detected by NMR spectroscopy. The binding site
could be located by our studies and involves the residues Ile24, Val26, Val70,
Asn71, Gly77, Ala78, Glu85, Arg88, Gly91 and Gln92. PIP(2) and PIP(3) binding
takes place in the groove of the PDZ domain that is normally part of the APC
binding site.
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Figure 4.
Figure 4. NOE contacts per residue in PDZ2b of PTP-Bas. The secondary structure is symbolised by arrows
(b-strands) and lanyards (a-helices). The position of the insertion is marked in grey.
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Figure 6.
Figure 6. NMR structure of PDZ2b. (A) Ribbon representation of the lowest-energy structure of PDZ2b of PTP-Bas.
The insertion is highlighted in orange. (B) Backbone representations of the 20 lowest-energy structures from the struc-
ture calculation. (C) Electrostatic potential of PDZ2b; negatively charged regions are coloured red, positively charged
regions are blue. All molecules are shown in the same orientation. The Figure was prepared using the program
MOLMOL.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2003,
334,
143-155)
copyright 2003.
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