Structure determination and ligand interactions of the PDZ2b domain of PTP-Bas (hPTP1E): splicing-induced modulation of ligand specificity.

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.

[1]  M. Billeter,et al.  MOLMOL: a program for display and analysis of macromolecular structures. , 1996, Journal of molecular graphics.

[2]  David R Cooper,et al.  Molecular roots of degenerate specificity in syntenin's PDZ2 domain: reassessment of the PDZ recognition paradigm. , 2003, Structure.

[3]  Eric Oldfield,et al.  1H, 13C and 15N chemical shift referencing in biomolecular NMR , 1995, Journal of biomolecular NMR.

[4]  R. Liddington,et al.  Crystal structure of a PDZ domain , 1996, Nature.

[5]  T. Pawson,et al.  The Carboxyl Terminus of B Class Ephrins Constitutes a PDZ Domain Binding Motif* , 1999, The Journal of Biological Chemistry.

[6]  John H. Lewis,et al.  Crystal Structures of a Complexed and Peptide-Free Membrane Protein–Binding Domain: Molecular Basis of Peptide Recognition by PDZ , 1996, Cell.

[7]  Geerten W Vuister,et al.  Structure, dynamics and binding characteristics of the second PDZ domain of PTP-BL. , 2002, Journal of Molecular Biology.

[8]  W. Gronwald,et al.  Automated assignment of NOESY NMR spectra using a knowledge based method (KNOWNOE) , 2002, Journal of biomolecular NMR.

[9]  Emiko Suzuki,et al.  A multivalent PDZ-domain protein assembles signalling complexes in a G-protein-coupled cascade , 1997, Nature.

[10]  G. Bodenhausen,et al.  Natural abundance nitrogen-15 NMR by enhanced heteronuclear spectroscopy , 1980 .

[11]  Volkmar Lessmann,et al.  The Adenomatous Polyposis Coli-protein (APC) interacts with the protein tyrosine phosphatase PTP-BL via an alternatively spliced PDZ domain , 2000, Oncogene.

[12]  E. Cuppen,et al.  The zyxin-related protein TRIP6 interacts with PDZ motifs in the adaptor protein RIL and the protein tyrosine phosphatase PTP-BL. , 2000, European journal of cell biology.

[13]  D. Bredt,et al.  Solution structure and backbone dynamics of the second PDZ domain of postsynaptic density-95. , 2000, Journal of molecular biology.

[14]  W. Lim,et al.  Mechanism and role of PDZ domains in signaling complex assembly. , 2001, Journal of cell science.

[15]  Richard R. Ernst,et al.  Investigation of exchange processes by two‐dimensional NMR spectroscopy , 1979 .

[16]  M. Sheng,et al.  PDZ domains and the organization of supramolecular complexes. , 2001, Annual review of neuroscience.

[17]  U. Hellman,et al.  A Novel GTPase-activating Protein for Rho Interacts with a PDZ Domain of the Protein-tyrosine Phosphatase PTPL1* , 1997, The Journal of Biological Chemistry.

[18]  Kalle Gehring,et al.  Solution structure of the PDZ2 domain from cytosolic human phosphatase hPTP1E complexed with a peptide reveals contribution of the beta2-beta3 loop to PDZ domain-ligand interactions. , 2002, Journal of molecular biology.

[19]  C. Heldin,et al.  Cloning and characterization of PTPL1, a protein tyrosine phosphatase with similarities to cytoskeletal-associated proteins. , 1994, Journal of Biological Chemistry.

[20]  John Calvin Reed,et al.  FAP-1: a protein tyrosine phosphatase that associates with Fas. , 1995, Science.

[21]  K. Maekawa,et al.  Molecular cloning of a novel protein‐tyrosine phosphatase containing a membrane‐binding domain and GLGF repeats , 1994, FEBS letters.

[22]  T. Dittmar,et al.  The protein tyrosine phosphatase PTP-BL associates with the midbody and is involved in the regulation of cytokinesis. , 2003, Molecular biology of the cell.

[23]  R. Stocco,et al.  A novel protein-tyrosine phosphatase with homology to both the cytoskeletal proteins of the band 4.1 family and junction-associated guanylate kinases. , 1994, The Journal of biological chemistry.

[24]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.

[25]  H. Kalbitzer,et al.  Relax, a flexible program for the back calculation of NOESY spectra based on complete-relaxation-matrix formalism. , 1997, Journal of magnetic resonance.

[26]  W. Gronwald,et al.  Computer assisted assignment of 13C or 15N edited 3D-NOESY-HSQC spectra using back calculated and experimental spectra. , 1999, Journal of magnetic resonance.

[27]  W. Lim,et al.  Unexpected modes of PDZ domain scaffolding revealed by structure of nNOS-syntrophin complex. , 1999, Science.

[28]  Joël Vandekerckhove,et al.  PIP(2)-PDZ domain binding controls the association of syntenin with the plasma membrane. , 2002, Molecular cell.

[29]  Jens Schneider-Mergener,et al.  Journal speciation , 1998, Nature Structural Biology.

[30]  M. Zimmer,et al.  EphrinB phosphorylation and reverse signaling: regulation by Src kinases and PTP-BL phosphatase. , 2002, Molecular cell.

[31]  M. Sheng,et al.  Postsynaptic Signaling and Plasticity Mechanisms , 2002, Science.

[32]  H. Hameister,et al.  Molecular cloning of a mouse epithelial protein‐tyrosine phosphatase with similarities to submembranous proteins , 1995, Journal of cellular biochemistry.

[33]  B. Wieringa,et al.  Identification and molecular characterization of BP75, a novel bromodomain‐containing protein , 1999, FEBS letters.

[34]  Wei Feng,et al.  PDZ7 of Glutamate Receptor Interacting Protein Binds to Its Target via a Novel Hydrophobic Surface Area* , 2002, The Journal of Biological Chemistry.

[35]  C. Zuker,et al.  Independent Anchoring and Assembly Mechanisms of INAD Signaling Complexes in Drosophila Photoreceptors , 2001, The Journal of Neuroscience.

[36]  R. Heumann,et al.  The protein kinase C‐related kinase PRK2 interacts with the protein tyrosine phosphatase PTP‐BL via a novel PDZ domain binding motif , 2001, FEBS letters.

[37]  Stuart K. Kim,et al.  LET-23 Receptor Localization by the Cell Junction Protein LIN-7 during C. elegans Vulval Induction , 1996, Cell.

[38]  N. Nomura,et al.  Characterization of a protein tyrosine phosphatase (RIP) expressed at a very early stage of differentiation in both mouse erythroleukemia and embryonal carcinoma cells , 1995, FEBS letters.

[39]  E. Cuppen,et al.  PDZ motifs in PTP-BL and RIL bind to internal protein segments in the LIM domain protein RIL. , 1998, Molecular biology of the cell.

[40]  Mingjie Zhang,et al.  Solution structure of the extended neuronal nitric oxide synthase PDZ domain complexed with an associated peptide , 1999, Nature Structural Biology.

[41]  A. Bax,et al.  Protein backbone angle restraints from searching a database for chemical shift and sequence homology , 1999, Journal of biomolecular NMR.

[42]  G. Kozlov,et al.  Solution structure of the PDZ2 domain from human phosphatase hPTP1E and its interactions with C-terminal peptides from the Fas receptor. , 2000, Biochemistry.

[43]  D. G. Davis,et al.  Long range hydrogen bond mediated effects in peptides nitrogen 15 nmr study of gramicidin s in water and organic solvents , 1984 .

[44]  Axel T. Brünger,et al.  Crystal structure of the hCASK PDZ domain reveals the structural basis of class II PDZ domain target recognition , 1998, Nature Structural Biology.

[45]  D. Banville,et al.  ZRP-1, a Zyxin-related Protein, Interacts with the Second PDZ Domain of the Cytosolic Protein Tyrosine Phosphatase hPTP1E* , 1999, The Journal of Biological Chemistry.

[46]  W. Gronwald,et al.  RFAC, a program for automated NMR R-factor estimation , 2000, Journal of biomolecular NMR.

[47]  S. Glaser,et al.  A general enhancement scheme in heteronuclear multidimensional NMR employing pulsed field gradients , 1994, Journal of biomolecular NMR.

[48]  J. Thornton,et al.  AQUA and PROCHECK-NMR: Programs for checking the quality of protein structures solved by NMR , 1996, Journal of biomolecular NMR.

[49]  Stuart K. Kim,et al.  The LIN-2/LIN-7/LIN-10 Complex Mediates Basolateral Membrane Localization of the C. elegans EGF Receptor LET-23 in Vulval Epithelial Cells , 1998, Cell.