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Protein Sci. Jun 2006; 15(6): 1500–1505.
PMCID: PMC2242534

The crystal structure of human receptor protein tyrosine phosphatase κ phosphatase domain 1


The receptor-type protein tyrosine phosphatases (RPTPs) are integral membrane proteins composed of extracellular adhesion molecule-like domains, a single transmembrane domain, and a cytoplasmic domain. The cytoplasmic domain consists of tandem PTP domains, of which the D1 domain is enzymatically active. RPTPκ is a member of the R2A/IIb subfamily of RPTPs along with RPTPμ, RPTPρ, and RPTPλ. Here, we have determined the crystal structure of catalytically active, monomeric D1 domain of RPTPκ at 1.9 Å. Structural comparison with other PTP family members indicates an overall classical PTP architecture of twisted mixed β-sheets flanked by α-helices, in which the catalytically important WPD loop is in an unhindered open conformation. Though the residues forming the dimeric interface in the RPTPμ structure are all conserved, they are not involved in the protein–protein interaction in RPTPκ. The N-terminal β-strand, formed by βx association with βy, is conserved only in RPTPs but not in cytosolic PTPs, and this feature is conserved in the RPTPκ structure forming a β-strand. Analytical ultracentrifugation studies show that the presence of reducing agents and higher ionic strength are necessary to maintain RPTPκ as a monomer. In this family the crystal structure of catalytically active RPTPμ D1 was solved as a dimer, but the dimerization was proposed to be a consequence of crystallization since the protein was monomeric in solution. In agreement, we show that RPTPκ is monomeric in solution and crystal structure.

Keywords: crystal structure, protein tyrosine phosphatase κ, RPTPκ, catalytic phosphatase D1 domain

Reversible phosphorylation of tyrosine residues is a key regulatory mechanism in numerous eukaryotic cellular events such as proliferation, differentiation, gene expression, and migration (Neel and Tonks 1997). The 107 human PTPs (protein tyrosine phophatases) identified are classified into the phospho-tyrosine-specific, “classical” PTPs, and the dual-specificity PTPs that dephosphorylate phospho-tyrosine, -threonine, and -serine residues. The 38 classical PTP family members include receptor-like transmembrane forms and nontransmembrane cytosolic forms (Andersen et al. 2001; Alonso et al. 2004). These PTPs are divided into two major subfamilies: 17 nonreceptor (or cytoplasmic) PTPs and 21 receptor-like (transmembrane) PTPs. Transmembrane receptor-like PTPs contain a variable sized extracellular region, a single transmembrane segment, and an intracellular region containing either one membrane proximal PTP domain (D1), or a D1 domain and a second membrane distal PTP domain (D2). The first, membrane proximal, phosphatase domain, D1, harbors most, or in some cases, all of the catalytic activity, whereas D2 is highly conserved with little or no activity (Neel and Tonks 1997).

Receptor-like PTPs play an essential role in transducing transmembrane signals, and some of these are suggested to be involved to the control of phenomena mediated by cell adhesion (Neel and Tonks 1997; Schnekenburger et al. 2005). RPTPκ belongs together with RPTPμ, RPTPρ, and RPTPλ to the R2A/IIb subfamily of receptor protein tyrosine phosphatases (McAndrew et al. 1998; Andersen et al. 2001) (Fig. (Fig.1A).1A). RPTPκ possesses an extracellular region, a single transmembrane region, and two intracellular tandem catalytic domains (Jiang et al. 1993). The extracellular domain of the RPTPκ precursor protein contains an immunoglobulin-like domain and four fibronectin type III-like repeats, preceded by a signal peptide and a region of ~150 amino acids with similarity to the Xenopus A5 antigen, a putative neuronal recognition molecule (McAndrew et al. 1998). The purified extracellular domain of RPTPκ functions as a substrate for adhesion by cells expressing RPTPκ and induces aggregation of coated synthetic beads (Drosopoulos et al. 1999).

Figure 1.
(A) Structure-based sequence alignment of RPTPκ, RPTPλ, RPTPρ, and RPTPμ (PDB codes: RPTPμ, 1RPM; RPTPκ, 2C7S). Secondary structure elements were determined using the program ICM (Molsoft) using the nomenclature ...

RPTPκ is widely expressed in the spleen, prostate and ovary, brain, lung, skeletal muscle, heart, placenta, liver, kidney, and intestine (Yang et al. 1997; Shen et al. 1999), and the expression is induced by TGF-β and by high cell density (Yang et al. 1996). RPTPκ has been shown to stimulate cell motility and neurite outgrowth, and is required for both the anti-proliferative and the pro-migratory effects of TGF-β, suggesting a role in regulation, maintenance, and restitution of cell adhesions (Wang et al. 2005). These functions might be regulated through dephosphorylation of β- and γ-catenin at adherens junctions (Fuchs et al. 1996).

Expression of RPTPκ is absent or down-regulated in >20% of melanoma cell lines and in some unmanipulated melanoma biopsies (Novellino et al. 2003). Furthermore, the human RPTPκ gene lies in a region frequently deleted in hematological neoplasms, melanomas, ovary carcinomas, and many other solid tumors (Nakamura et al. 2003). RPTPκ plays a dual role as tumor suppressor and tumor promoter in mammary epithelial cells (Wang et al. 2005). RNA interference (RNAi) experiments showed that down-regulation of RPTPκ results in acceleration of cell cycle progression, enhancement of the cellular response to epidermal growth factor (EGF), and abrogates TGF-β-mediated anti-mitogenesis, whereas overexpression of RPTPκ results in inhibition of basal and EGF-induced proliferation and ERbB receptor signaling in cancer cells. Therefore, RPTPκ is a key regulator of EGFR tyrosine phosphorylation and function in human keratinocytes (Yang et al. 1996; Xu et al. 2005). Here, we report the crystal structure of the catalytic D1 domain of human RPTPκ at high resolution along with the self-association analysis using analytical ultracentrifugation (AUC).

Results and Discussion

It was suggested that RPTP activity was regulated by dimerization, and the first evidence for dimerization as a regulatory mechanism of RPTP activity came from studies with a chimeric protein consisting of the extracellular domain of the epidermal growth factor receptor (EGFR, a prototypical RPTK) and the intracellular domain of the RPTP CD45 (Desai et al. 1993). The crystal structure of the N-terminal membrane-proximal PTP domain of RPTPα (RPTP α-D1) provided structural support for dimerization-induced inhibition of RPTP activity (Bilwes et al. 1996). Nonetheless, CD45 structure shows that the cytoplasmic region of CD45 does not dimerize, and the dimeric interaction as observed for RPTPα D1 would be impossible given the D1–D2 intramolecular domain orientation (Nam et al. 2005).

Among the R2A/IIb subfamily of receptor protein tyrosine phosphatases the crystal structure of RPTPμ has been solved (Hoffmann et al. 1997). In the crystal structure of RPTPμ D1, the subunits associate to form a dimer with twofold symmetry. The dimerization was proposed to be a consequence of crystallization since the protein was monomeric in solution. The dimer also showed no obstruction to the catalytic site. However the crystal structure of RPTPα was shown to be a crystallographic dimer in which the active site of one domain is occluded by a helix-turn-helix wedge motif of its dyad-related partner (Bilwes et al. 1996). This occurs first, as a result of sterically blocking substrate access to the catalytic site, and second, by constraining the WPD loops in an open conformation that is unable to adopt the closed conformation necessary for catalysis. However, biochemical evidences suggest that the transmembrane region is essential for dimerization in vivo (Jiang et al. 2000); therefore, it has been proposed that the D1 domain wedge motif may only serve to stabilize the RPTPα dimeric state and occlude the related D1's active site, whereas the transmembrane region most likely provides the energetic driving force for dimerization.

RPTPκ D1 domain is a monomer

The receptor PTPκ D1 protein was purified and found to be active (data not shown). The homogeneity of the purified sample was analyzed using analytical ultracentrifugation using various buffers (Fig. (Fig.1B1B,,C).C). When the buffer 10 mM HEPES (pH 7.5), 25 mM NaCl without DTT was used, two main species were observed in almost equal amounts, giving s20,w values of 3.09S and 4.81S (Fig. (Fig.1B).1B). Upon conversion to a molecular weight distribution in SEDIT, the two peaks gave molecular weights of 33 kDa and 65 kDa (Fig. (Fig.1C).1C). Therefore the RPTPκ protein is present as a monomer and dimer and smaller amounts of higher species. In contrast, in buffer 50 mM HEPES (pH 7.5), 150 mM NaCl, 5 mM DTT, RPTPκ protein is 97% monomer, giving an s20,w value of 3.14S. Therefore it is clear that the presence of reducing agents and higher ionic strength prevent the self-association behavior of RPTPκ.

Quality of the model

We solved the monomeric structure of the receptor RPTPκ D1 domain, and it has been refined to 1.9 Å, low R-factor values, and satisfactory geometry (Table (Table1;1; Fig. Fig.2).2). The structure of RPTPκ determined here contains residues Met865 to Phe1156 of the catalytic D1 domain. The entire structure was very well defined in the electron density. The asymmetric unit contains one protein molecule as well as an acetate moiety bound to the active-site cysteine mimicking binding of a phosphate moiety. The crystal packing showed no obstruction to the catalytically important WPD loop, which is distinct to the RPTPα.

Figure 2.
Three-dimensional structure of RPTPκ. (Red) α-Helices; (green) β-strands; (magenta) 310-helices. The helix-turn-helix segment (α1′, α2′) and the β-strand formed by association of βx ...
Table 1.
Crystallographic data and refinement statistics

Overall structure and active-site properties

The overall structure of RPTPκ resembles closely the structure of RPTPμ, which has 79.9% sequence identity and 1.05 Å RMSD. The main secondary structure elements were found to be conserved in both structures, containing overall classical PTP architecture of twisted mixed β-sheets flanked by α-helices. While the catalytically important WPD (Trp1049–Ala1058) loop conformation is similar to RPTPμ in an unhindered open conformation, it is more open than the cytosolic PTP1B (PDB). As in all tyrosine phosphatases the active-site cysteine (Cys1083) is located in the well-conserved phosphate binding loop, and an acetate molecule bound to it might mimic binding of a phosphate moiety. In PTP1B residues Met258 and Gly259 form an open cleft, allowing direct access of substrates to the active site, whereas in RPTPα, bulkier residues are found in this position causing steric hindrance, and they are key determinants of substrate selectivity (Peters et al. 2000; Iversen et al. 2001). In RPTPκ, Ile1123 and Asn1124 are present in the corresponding positions, and Asn1124 seems to be likely to accommodate the phosphopeptides with a small amino acid in the pY+1 position.

Secondary structure comparisons with other PTPs

In RPTPμ, though the crystal packing shows no obstruction of the catalytic sites, the interaction between the two dimeric subunits are hydrophobic and mediated by Thr1025 and Ile1027 from one monomer and Ile1050 and Glu 1052 from another (Hoffmann et al. 1997). It is interesting to note that the corresponding conserved residues Thr1025, Ile 1027, Ile 1050, and Glu 1052 in RPTPκ are not involved in the protein–protein interaction, but they are solvent-exposed in the structure. Similar to other PTPs, RPTPκ also lacks the second tyrosine pocket seen in PTP1B formed by Arg24, Arg254, and Glu262. The helix-turn-helix wedge, which is involved in dimerization in RPTPα, is not well conserved among the RPTPs but the α1 and α2 helices are conserved between the RPTPμ and RPTPκ except the turn (Fig. (Fig.1),1), which forms a small distortion in the helix α2′. The N-terminal β-strand, formed by βx association with βy, is conserved between RPTPμ, RPTPκ, and RPTPα, and most of the RPTPs. This feature was suggested to distinguish RPTPD1s from the cytosolic PTPs, and this is conserved in the RPTPκ structure forming a β-strand at the N-terminal (Fig. (Fig.11).


The structure of RPTPα showed the dimeric form of RPTP, which led investigators to propose an interesting suggestion that the regulation of the RPTP could be occurring through ligand-induced dimerization (Jiang et al. 1999). However, the crystal structure of RPTPμ dimeric conformation showed that neither the catalytic site nor the N-terminal helix-turn-helix is involved in protein–protein interactions similar to RPTPα. Combining the RPTPμ structural data along with the biochemical evidence, Hoffmann et al. (1997) suggested that the regulation through dimerization could be specific to the RPTPα subfamily and their close homologs but not a general mechanism for all RPTPs. Our results clearly show that the difference in buffer condition can induce multimeric forms of phosphatase D1 domain in solution. The RPTPκ monomer D1 domain is the first monomeric crystal structure in this family that is similar to RPTPμ, and the regulation through dimerization proposed for RPTPα is not observed in RPTPκ. However crystal structures represent only a particular conformation of a molecule, and therefore the regulatory mechanism of RPTPκ in cells remains to be studied.

Materials and methods


A sequence encompassing the catalytic domain of RPTPκ (residues Met865–Phe1156 of GenBank entry gi|18860902) was amplified by PCR and subbcloned into a pET-21a-derived vector. The vector includes a TEV-cleavable (*) N-terminal 6xHis tag (MHHHHHHSSGVDLGTENLYFQ*SM).

Expression and purification

Escherichia coli BL21(DE3) cells transformed with the expression constructs were grown at 37°C in Luria-Bertani medium containing 100 μg/mL kanamycin until the OD600 reached 0.3 and then transferred to 18°C. Protein expression was induced at an OD600 of 0.8 using 1 mM isopropyl-thio-β-D-galactopyranoside. Cells were harvested after 3 h by centrifugation at 4000g for 10 min and then lysed in 50 mM HEPES (pH 7.5), 500 mM NaCl, 1 mM PMSF, and 0.5 mM TCEP using an EmulsiFlex high-pressure homogenizer, and the cell extract was centrifuged at 60,000g for 30 min at 4°C. The supernatant was loaded onto 5 mL of the Nickel-sepharose affinity resin and washed with 10 volumes of loading buffer (50 mM HEPES at pH 7.5, 500 mM NaCl, 5 mM imidazole, 0.5 mM TCEP, 5% glycerol) and 10 volumes of wash buffer (50 mM HEPES at pH 7.5, 500 mM NaCl, 20 mM imidazole, 0.5 mM TCEP, 5% glycerol), then eluted with elution buffer (50 mM HEPES at pH 7.5, 500 mM NaCl, 250 mM imidazole, 0.5 mM TCEP, 5% glycerol) at 0.8 mL/min. The eluted protein was further purified by gel filtration S75 column equilibrated in 10 mM HEPES (pH 7.5), 25mM NaCl, 5 mM DTT. The purified proteins were homogeneous, as assessed by SDS-PAGE and electrospray mass spectrometry, which also confirmed the predicted mass of the proteins. Protein was concentrated to 7–10 mg/mL using a 10-kDa cutoff concentrator (Vivascience).

Analytical ultracentrifugation (AUC)

Sedimentation velocity was carried out on RPTPκ, using a Beckman Coulter Optima XLI analytical ultracentrifuge. Two different concentrations of RPTPκ were run, 1.2 mg/mL and 2.8 mg/mL, with the 1.2 mg/mL sample being run in two different buffers: (1) 10 mM HEPES (pH 7.5), 25 mM NaCl, and (2) 50 mM HEPES (pH 7.5), and 150 mM NaCl, 5 mM DTT. Samples were run at 50,000 rpm at a temperature of 10°C, and scans were taken using absorbance optics at 2-min intervals, with a detection wavelength of 297 nm. Data were analyzed using the c(s) model of SEDFIT (Schuck 2000). Subsequent sedimentation coefficient values obtained from these peaks were converted to s20,w values (corrected to conditions of standard temperature and buffer) in SEDNTERP.

Crystallization, data collection, structure solution, and refinement

Crystals were obtained using the vapor diffusion method and a protein concentration of 10 mg/mL by mixing 100 nL of the concentrated protein with 100 nL of a well solution containing 0.20 M NaNO3, 20.0% PEG 3350, 10.0% ethylene glycol. The crystal belongs to the space group P43212 with unit cell dimensions a,b = 91.37 Å and c = 108.45 containing one molecule in the asymmetric unit. The RPTPκ data set was collected at the beamline X10 at SLS to a resolution of 1.9 Å. Data collection statistics and cell parameters are listed in Table Table1.1. The structures were solved with molecular replacement using Phaser with the human protein phosphates RPTPμ (PDB ID 1RPM) as a search model. Iterative rounds of restrained refinement with TLS against maximum likelihood targets using Refmac5 were interspersed by manual rebuilding of the model using Coot. The structure was deposited in the Protein Data Bank (PDB) with accession number 2C7S.


We thank the crystallography group for collecting diffraction data and the Biotechnology Group for providing the expression vector. The Structural Genomics Consortium is a registered charity (no. 1097737) funded by the Wellcome Trust, GlaxoSmithKline, Genome Canada, the Canadian Institutes of Health Research, the Ontario Innovation Trust, the Ontario Research and Development Challenge Fund, and the Canadian Foundation for Innovation.


Reprint requests to: Stefan Knapp, Structural Genomics Consortium, University of Oxford, Botnar Research Centre, Oxford OX3 7LD, UK; e-mail: ku.ca.xo.cgs@ppank.nafets; fax: +44-1865-737231.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.062128706.


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