Biomol NMR Assign (2007) 1:151–153 DOI 10.1007/s12104-007-9043-y
ARTICLE
Sequence-specific 1H, 13C, and 15N backbone assignment of the 28 kDa PDZ2/PDZ3 tandem domain of the protein tyrosine phosphatase PTP-BL Christian P. Fetzer Æ Janelle Sauvageau Æ Gerd Kock Æ Carsten Berghaus Æ Jan-Amade´ Bangert Æ Markus Dicks Æ Rolf Heumann Æ Kai S. Erdmann Æ Raphael Stoll
Received: 27 April 2007 / Accepted: 14 July 2007 / Published online: 3 August 2007 Ó Springer Science+Business Media B.V. 2007
Abstract Protein tyrosine phosphatase-basophil like (PTP-BL) represents a large multi domain non-transmembrane scaffolding protein that contains five PDZ domains. Here we report the backbone assignments of the PDZ2/ PDZ3 tandem domain of PTP-BL. These assignments now provide a basis for the detailed structural investigation of the interaction between the PDZ domains 2 and 3 of PTPBL. It will lead to a better understanding of the proposed scaffolding function of this tandem domain in multi-protein complexes assembled by PTB-BL. Keywords Backbone resonance assignments PDZ2 PDZ3 PDZ tandem domain PTP-BL
Biological context PTP-BL (Protein tyrosine phosphatase-basophil like) is the mouse homologue of human PTP-Bas. Protein tyrosine phosphatase-basophil like (PTP-BL) represents a large multi domain non-transmembrane scaffolding protein with different functional properties. PTP-BL has been impli-
Christian P. Fetzer, Janelle Sauvageau and Gerd Kock contributed equally to this work. J. Sauvageau G. Kock C. Berghaus J.-A. Bangert M. Dicks R. Stoll (&) Biomolecular NMR, Faculty of Chemistry and Biochemistry, Ruhr-University of Bochum, NC 4/72, 44780 Bochum, Germany e-mail:
[email protected] C. P. Fetzer R. Heumann K. S. Erdmann Biochemistry II, Faculty of Chemistry and Biochemistry, Ruhr-University of Bochum, 44780 Bochum, Germany
cated in the regulation of several cellular processes such as cytokinesis and actin-cytoskeletal rearrangement (Erdmann 2003). The N-terminal part of the protein consists of a KIND (kinase non-catalytic C-lobe) domain, followed by a FERM (Four point one, Ezrin, Radixin, Moesin) domain, that is involved in the association of proteins to the plasma membrane. In addition, PTP-BL contains five PDZ (PSD95, Disc large, Zonula occludens) domains and a C-terminal protein tyrosine phosphatase domain (Erdmann 2003). Recently, the structures of PDZ2b of PTP-Bas and PDZ2 of PTP-BL have been solved (Kachel et al. 2003; Walma et al. 2004). PDZ domains share a globular fold, consisting of two a helices and 6 b strands, that form two anti parallel b sheets (Doyle et al. 1996; Moiras Cabral et al. 1996). PDZ domains are well known to mediate protein-protein interactions and are crucial for the assembly of a number of multiprotein complexes (Sheng and Sala 2001). Therefore, it is of great interest to further characterize binding properties of the PDZ domain module of PTP-BL. Hitherto, a large number of PTP-BL interacting proteins are known. For example, it was shown that PDZ1 of PTP-Bas binds to IjBa, that only one of the two splicing variants of PDZ2 is interacting with the tumour suppressor protein adenomatous polyposis coli, that PDZ3 associates with the protein kinase PRK2, and that one of the proteins that interacts with PDZ4 of PTP-BL is CRIP2, a LIM-only protein (for a review see Erdmann 2003). PDZ domains 2, 3 and 5 are also capable of binding the phospholipid phosphatidylinositol 4,5-bisphosphate (Zimmermann et al. 2002). Here we report the 1H, 15N, and 13C backbone resonance assignments of the 28 kDa PTP-BL PDZ2/PDZ3 tandem domain which encompasses 252 residues. These data will provide the basis to analyse a potential cross-talk between individual PDZ domains within the multi-PDZ domain protein PTP-BL.
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Methods and experiments Protein expression and purification The coding region of the PDZ2/PDZ3 tandem domain of PTP-BL comprising amino acid residues 1342–1593 was cloned in the bacterial expression vector pGEX2T (GE Healthcare). Details of the cloning procedure will be published elsewhere. The expression of the GST-fusion protein was performed in an Escherichia coli BL21DE3 expression strain. The uniformly 13C/15N and 15N isotopically enriched protein samples were prepared by growing the bacteria in minimal media containing 15NH4Cl either with or without 13C6-glucose. Purification of the GSTtagged protein was achieved by binding to Glutathione Sepharose (GE Healthcare) and subsequent cleavage with Thrombin protease (GE Healthcare). Thrombin protease was removed using HiTrapTMBenzamidine FF (GE Healthcare). The purity of the protein samples was checked by SDS-PAGE.
NMR spectroscopy Typically, NMR samples contained up to 1 mM of protein in 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.4 mM KH2PO4, 0.02% NaN3, pH 7.4 and protease inhibitor
Fig. 1 1H–15N HSQC spectrum of the PDZ2/PDZ3 tandem domain of PTP-BL at 298 K and 600 MHz. Residue specific assignment of backbone 1H and 15 N frequencies is indicated. Signals connected by horizontal lines correspond to side chain amide groups of asparagines and glutamine residues
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(complete, mini, EDTA-free; Roche). All NMR spectra were acquired at 298 K on a Bruker DRX600 spectrometer. Backbone assignments were obtained from three dimensional HNCA, HN(CO)CA, CBCA(CO)NH, and HNCO spectra (Grzesiek and Bax 1992). All spectra were processed with NMRPipe (Delaglio et al. 1995) and analysed with NMRView (Johnson and Blevins 1994).
Assignments and data deposition The 1H–15N HSQC of the PDZ2/PDZ3 tandem domain of PTP-BL is shown in Fig. 1. The overall backbone assignments have been completed to approximately 99% for Ca and 87% for amide proton resonances which could be detected in the spectra. The following consecutive residues have either been not assigned due to spectral overlap or are absent from the spectra: G1 to K10, S37-G42, and N156S158. For the PDZ2 domain, 96% of the Ca and 88% of the amide protons have been assigned. In the PDZ3 domain, 99% of the Ca and 91% of the amide resonances could be identified. The proline rich linker between PDZ2 and PDZ3 consists of 44 residues, of which 15 residues could be assigned. The remaining residues could not be identified either because of spectral overlap or absent resonances in the spectra. Therefore, 45% of the Ca and 42% of the linker proton amide resonances could be assigned. The chemical
Sequence-specific 1H,
13
C, and
15
N backbone assignment of the 28 kDa PDZ2/PDZ3 tandem domain
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Tim Czopka for expert technical assistance. We would also like to thank Miche`le Auger for stimulating discussions and continuous support. This work was supported by a DFG grant (ER291/2-1) to R.S and K.S.E. Part of the work was financially supported by the European Comission in the framework of the project INTCHEM (MESTCT-2005-020681). M. D. is supported by a fellowship from the HansBoeckler-Stiftung. R. S. gratefully recognizes generous support from the BMBF, FCI, DFG (SFB 642, A6 as well as ER291/2-1), Proteincenter (NRW center of excellence), and RUBiospek. We are grateful to Prof. Dr. von Kiedrowski for providing generous access to the DRX600.
References Fig. 2 Combined 1H and 15N NMR chemical shift differences between the isolated PDZ2 domain and PDZ2 as part of the PDZ2/ PDZ3 tandem domain plotted versus the amino acid sequence of PDZ2 (Walma et al. 2002, 2004; Kachel et al. 2003). The combined 1 H and 15N chemical shift differences were calculatedffi according to rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 2 Dd15 N 2 Dd1 HN þ the following equation: Ddobs ¼ 5
shift assignment of the backbone 1H, 15N, and 13C NMR resonances for the uncomplexed PDZ2/PDZ3 tandem domain construct (apo-PDZ2/PDZ3) has been deposited in the BioMagResBank (http://www.bmrb.wisc.edu) under accession number BMRB-15199. Figure 2 shows the combined 1H and 15N NMR chemical shift differences between the isolated PDZ2 domain and the PDZ2 domain as part of the PDZ2/PDZ3 tandem construct plotted versus the amino acid sequence of PDZ2 (Walma et al. 2002, 2004; Kachel et al. 2003). Only minor to medium chemical shift differences can be detected. These have the tendency to cluster near the carboxyterminus and opposite to the canonical peptide binding cleft of PDZ2. Obviously, there are relatively few interactions between these two domains. This is compatible with the linker between PDZ2 and PDZ3 that consists of 44 amino acids. Notwithstanding, these assignments now provide a basis for the detailed investigation of the interactions between the PDZ domains 2 and 3 of PTP-BL with their natural ligands that will shed light on the proposed scaffolding function of this tandem domain in multi-protein complexes assembled by PTB-BL.
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Acknowledgements We are extremely grateful to Gregor Barchan, Martin Gartmann, Hans-Jochen Hauswald, Sabine Laerbusch, and
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