The Crystal Structure of Domain 1 of Receptor Protein-tyrosine Phosphatase μ

Receptor-like protein-tyrosine phosphatases (RPTPs) play important roles in regulating intracellular processes. We have been investigating the regulation and function of RPTPμ, a receptor-like PTP related to the Ig superfamily of cell adhesion molecules. Recently, the crystal structure of a dimer of the membrane proximal domain of RPTPα (RPTPα D1) was described (Bilwes, A. M., den Hertog, J., Hunter, T., and Noel J. P. (1996)Nature 382, 555–559). Within this crystal structure, the catalytic site of each subunit of the dimer is sterically blocked by the insertion of the N-terminal helix-turn-helix segment of the dyad-related monomer. It was proposed that dimerization would lead to inhibition of catalytic activity and may provide a paradigm for the regulation of the RPTP family. We have determined the crystal structure, to 2.3 Å resolution, of RPTPμ D1, which shares 46% sequence identity with that of RPTPα D1. Although the tertiary structures of RPTPα D1 and RPTPμ D1 are very similar, with a root mean square deviation between equivalent Cα atoms of 1.1 Å, the quaternary structures of these two proteins are different. Neither the catalytic site nor the N-terminal helix-turn-helix segment of RPTPμ D1 participates in protein-protein interactions. The catalytic site of RPTPμ D1 is unhindered and adopts an open conformation similar to that of the cytosolic PTP, PTP1B (Barford, D., Flint, A. J., and Tonks, N. K. (1994) Science 263, 1397–1404). We propose that dimerization-induced modulation of RPTP activity may not be a general feature of this family of enzymes. Receptor-like protein-tyrosine phosphatases (RPTPs) play important roles in regulating intracellular processes. We have been investigating the regulation and function of RPTPμ, a receptor-like PTP related to the Ig superfamily of cell adhesion molecules. Recently, the crystal structure of a dimer of the membrane proximal domain of RPTPα (RPTPα D1) was described (Bilwes, A. M., den Hertog, J., Hunter, T., and Noel J. P. (1996)Nature 382, 555–559). Within this crystal structure, the catalytic site of each subunit of the dimer is sterically blocked by the insertion of the N-terminal helix-turn-helix segment of the dyad-related monomer. It was proposed that dimerization would lead to inhibition of catalytic activity and may provide a paradigm for the regulation of the RPTP family. We have determined the crystal structure, to 2.3 Å resolution, of RPTPμ D1, which shares 46% sequence identity with that of RPTPα D1. Although the tertiary structures of RPTPα D1 and RPTPμ D1 are very similar, with a root mean square deviation between equivalent Cα atoms of 1.1 Å, the quaternary structures of these two proteins are different. Neither the catalytic site nor the N-terminal helix-turn-helix segment of RPTPμ D1 participates in protein-protein interactions. The catalytic site of RPTPμ D1 is unhindered and adopts an open conformation similar to that of the cytosolic PTP, PTP1B (Barford, D., Flint, A. J., and Tonks, N. K. (1994) Science 263, 1397–1404). We propose that dimerization-induced modulation of RPTP activity may not be a general feature of this family of enzymes. Numerous cellular events are regulated by reversible phosphorylation of tyrosine residues, including growth, differentiation, the cell cycle, cell-cell adhesion, and cell-matrix contacts (1Neel B.G. Tonks N.K. Curr. Opin. Cell Biol. 1997; 9: 193-204Crossref PubMed Scopus (741) Google Scholar). Phosphotyrosine levels are controlled by the coordinated and competing actions of protein-tyrosine kinases and protein-tyrosine phosphatases (PTPs). 1The abbreviations used are: PTP, protein-tyrosine phosphatases; RPTP, receptor protein-tyrosine phosphatase; D1, domain 1 (membrane proximal domain) of RPTPs; DTT, dithiothreitol; NCS, noncrystallographic symmetry. The PTPs comprise a diverse family of transmembrane receptor-like and nontransmembrane, cytosolic enzymes (1Neel B.G. Tonks N.K. Curr. Opin. Cell Biol. 1997; 9: 193-204Crossref PubMed Scopus (741) Google Scholar). Receptor-like PTPs are likely to play crucial roles in transducing transmembrane signals and evidence is accumulating to link these enzymes to the control of phenomena mediated by cell adhesion (1Neel B.G. Tonks N.K. Curr. Opin. Cell Biol. 1997; 9: 193-204Crossref PubMed Scopus (741) Google Scholar). By interacting with ligands through their extracellular domains, the intracellular catalytic activity of RPTPs may be regulated, hence controlling the levels of cellular phosphotyrosine. The receptor-like PTPs RPTPμ, RPTPκ, and RPTPλ, which have extracellular segments sharing structural similarities with Ig superfamily cell adhesion molecules, have been shown to mediate cell aggregation via homophilic interactions (2Brady-Kalnay S.M. Flint A.J. Tonks N.K. J. Cell Biol. 1993; 122: 961-972Crossref PubMed Scopus (242) Google Scholar, 3Sap J.Y Jiang Y.P. Friedlander D. Grumet M. Schlessinger J. Mol. Cell. Biol. 1994; 14: 1-9Crossref PubMed Google Scholar, 4Cheng J. Wu K. Armanini M.O. Rourke N. Dowbenko D. Lasky L.A. J. Biol. Chem. 1997; 272: 7264-7277Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). For RPTPμ, the homophilic binding site(s) reside on the immunoglobulin domain, whereas the intracellular segment consists of a juxtamembrane, cadherin-like domain, and two PTP domains (5Brady-Kalnay S.M. Tonks N.K. J. Biol. Chem. 1994; 269: 28472-28477Abstract Full Text PDF PubMed Google Scholar). A role for RPTPμ in regulating cell junctions and cytoskeletal organization is suggested by the finding that the RPTPμ is associated with cadherin-catenin complexes at adherens junctions (6Brady-Kalnay S.M. Rimm D.L. Tonks N.K. J. Cell Biol. 1995; 130: 977-986Crossref PubMed Scopus (287) Google Scholar). Similar results have been demonstrated for RPTPκ (7Fuchs M. Muller T. Lerch M.M. Ullrich A. J. Biol. Chem. 1996; 271: 16712-16719Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar) and RPTPλ (4Cheng J. Wu K. Armanini M.O. Rourke N. Dowbenko D. Lasky L.A. J. Biol. Chem. 1997; 272: 7264-7277Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The crystal structure of PTP1B in a complex with a phosphotyrosyl peptide substrate (8Jia Z. Barford D. Flint A.J. Tonks N.K. Science. 1995; 268: 1754-1758Crossref PubMed Scopus (559) Google Scholar), together with the results of a number of kinetic studies (9Denu J.M. Stuckey J.A. Saper M.A. Dixon J.E. Cell. 1996; 87: 361-364Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar), have revealed important insights into the catalytic mechanism of members of the PTP family. The signature motif, (I/V)HCXAGXXR(S/T)G, which defines this family of enzymes, contains the catalytically essential Cys and Arg residues. It forms a rigid cradle structure that coordinates binding of the phosphate moiety of the substrate and positions the cysteine residue to act as a nucleophile. This motif lies at the base of a cleft on the surface of the enzyme, which is defined at one end by the invariant residue Tyr46 and at the other by Phe182, which is in a loop containing the invariant general acid Asp181within the highly conserved motif WPD. It is the depth of the cleft, equivalent to the length of a phosphotyrosine side chain, that is a major determinant of the specificity of these enzymes for Tyr(P) substrates, since the side chains of Ser(P) and Thr(P) are too short to permit access of the phosphate to the catalytic cysteine. Binding of substrate induces a profound conformational change in the enzyme such that on engagement of Tyr(P) the WPD loop flips down onto the Tyr(P) side chain (8Jia Z. Barford D. Flint A.J. Tonks N.K. Science. 1995; 268: 1754-1758Crossref PubMed Scopus (559) Google Scholar). This creates a predominantly hydrophobic recognition pocket that surrounds and interacts with the phenyl ring of the Tyr(P) side chain and allows Asp181 to act as a catalytic acid. Catalysis proceeds via nucleophilic attack by the invariant Cys (Cys215) on the substrate phosphorous atom and protonation of the tyrosyl-leaving group of the substrate by Asp181resulting in formation of a cysteinyl-phosphate covalent intermediate. The reaction is then completed by hydrolysis of the covalent intermediate by a water molecule, possibly activated by Asp181 (10Denu J.M. Lohse D.L. Vijayalakshmi J. Saper M.A. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2493-2498Crossref PubMed Scopus (252) Google Scholar). Recently, a model for the regulation of RPTP activity was proposed by Bilwes et al. (11Bilwes M. den Hertog J. Hunter T. Noel J.P. Nature. 1996; 382: 555-559Crossref PubMed Scopus (292) Google Scholar) on the basis of their crystal structure of domain 1 of RPTPα. In contrast to the cytosolic PTPs, PTP1B andYersinia PTP, which are monomeric (12Barford D. Flint A.J. Tonks N.K. Science. 1994; 263: 1397-1404Crossref PubMed Scopus (688) Google Scholar, 13Stuckey J.A. Schubert H.L. Fauman E.B. Zhang Z.-Y. Dixon J.E. Saper M.A. Nature. 1994; 370: 571-575Crossref PubMed Scopus (379) Google Scholar), the crystal structure of domain 1 (D1) of RPTPα revealed a homodimeric organization that would result in an inhibited PTP (8Jia Z. Barford D. Flint A.J. Tonks N.K. Science. 1995; 268: 1754-1758Crossref PubMed Scopus (559) Google Scholar, 11Bilwes M. den Hertog J. Hunter T. Noel J.P. Nature. 1996; 382: 555-559Crossref PubMed Scopus (292) Google Scholar). The dimer results from a structural segment at the N terminus of the RPTPα D1 catalytic domain, consisting of a turn connecting helices α1′ and α2′ that wedges into the dyad-related catalytic site. The insertion of residues of the helix-turn-helix segment of one subunit into the catalytic site of the dyad-related subunit would be predicted to inhibit catalytic activity. This occurs first, as a result of sterically blocking substrate access to the catalytic site, and second, by constraining the WPD loop in an open conformation that is unable to adopt the closed conformation necessary for catalysis (8Jia Z. Barford D. Flint A.J. Tonks N.K. Science. 1995; 268: 1754-1758Crossref PubMed Scopus (559) Google Scholar). Distinct features of tertiary structure of RPTPα D1 that are absent from PTP1B are thought to facilitate this dimeric interaction. These include first, a two-residue insertion in the loop connecting α1′ and α2′, that inserts into the dyad-related catalytic site, and second, a β-strand (βx), immediately N-terminal to α1′ that forms a distinctive two-strand β-sheet with βy. The β-sheet between βx and βy, together with conserved residues within α1′ and α2′, contribute to defining the conformation of the helix-turn-helix segment of RPTPα D1 (11Bilwes M. den Hertog J. Hunter T. Noel J.P. Nature. 1996; 382: 555-559Crossref PubMed Scopus (292) Google Scholar). Interestingly, a sequence analysis of the PTP family suggested, on the basis of residue conservations, that the tertiary structural features of RPTPα D1 would be shared with D1s of other RPTPs but not cytosolic PTPs, such as PTP1B, or D2 of RPTPs (11Bilwes M. den Hertog J. Hunter T. Noel J.P. Nature. 1996; 382: 555-559Crossref PubMed Scopus (292) Google Scholar). The crystal structure of RPTPα D1 lead Bilwes et al. (11Bilwes M. den Hertog J. Hunter T. Noel J.P. Nature. 1996; 382: 555-559Crossref PubMed Scopus (292) Google Scholar) to propose a dimerization model for the regulation of RPTPα and, as a result of the sequence similarity with other RPTP D1s, it was suggested that this model could provide a paradigm for the regulation of RPTPs generally (11Bilwes M. den Hertog J. Hunter T. Noel J.P. Nature. 1996; 382: 555-559Crossref PubMed Scopus (292) Google Scholar). The model proposes that ligand binding to the extracellular segment would modulate dimerization and hence the catalytic activity of the PTP domain. We have been pursuing an investigation of the function of the Ig superfamily cell adhesion molecule related-phosphatase RPTPμ. At present little is known about the regulation of RPTPμ activity. Although it associates through its intracellular segment with the cadherin-catenin complex (6Brady-Kalnay S.M. Rimm D.L. Tonks N.K. J. Cell Biol. 1995; 130: 977-986Crossref PubMed Scopus (287) Google Scholar), the effects on PTP activity remain unclear. We have initiated a structural analysis of RPTPμ to provide further insights into the mechanisms for control of its enzymatic activity. In this regard, the possibility that the structure of RPTPα is indicative of a general mechanism for ligand-induced inhibition of RPTP activity is exciting. Here, we describe the crystal structure of domain 1 of human RPTPμ and show that although it is present in the crystal as a dimer, unlike RPTPα D1, the active site of RPTPμ is in an open, uninhibited conformation. Therefore our data indicate that regulation of RPTP activity by dimerization is not a general feature of these enzymes. Residues 874–1168 of human RPTPμ D1 were cloned into a modified version of the pET28a vector (Novagen Inc.) 2D. Barford and N. Hanlon, unpublished data. that incorporates an N-terminal hexa-His tag immediately preceding Gly874. The protein was overexpressed in Escherichia coli (strain B834) grown at 18 °C for 16 h. Protein purification was achieved with the following steps. Bacterial cells were lysed in 25 mm Tris·HCl (pH 8.0), 500 mmNaCl, 20 mm imidazole, 2 mm benzamidine, 0.01 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 1 μg/ml aprotinin, 1 μg/ml pepstatin, 1 μg/ml DNase, 5 μg/ml lysozyme and the cleared lysate loaded onto a nickel-nitrilotriacetic acid-agarose column (Novagen Inc.) equilibrated in 50 mmTris·HCl (pH 8.0), 500 mm NaCl, 2 mmbenzamidine, and 20 mm imidazole. RPTPμ D1 was eluted using 50 mm imidazole. Peak fractions were dialyzed against a buffer of 25 mm Tris·HCl (pH 8.0), 50 mmNaCl, 2 mm DTT, 2 mm EDTA, 2 mmbenzamidine. Precipitated impurities where removed by centrifugation and the filtered solution loaded onto a Mono Q column (Pharmacia Biotech Inc.) equilibrated in dialysis buffer. RPTPμ D1 was collected in the flow-through, brought to 1.0 m ammonium sulfate, and applied to a phenyl TSK column (TosoH Corp.) equilibrated in 1.0m ammonium sulfate, 25 mm Tris·HCl (pH 8.0), 50 mm NaCl, 2 mm EDTA, 2 mm DTT, 2 mm benzamidine. The enzyme was eluted by applying a linear gradient of 300 ml to a buffer of 25 mm Tris·HCl (pH 8.0), 50 mm NaCl, 2 mm EDTA, 2 mmDTT, 2 mm benzamidine. Peak fractions were loaded onto a Superdex 75 gel filtration column (Pharmacia Biotech Inc.). The protein, at greater that 99% purity, was concentrated to 7.5 mg/ml and crystallized at 4 °C in conditions of 750 mm sodium citrate (pH 5.5), 2 mm DTT, 0.5 mm EDTA. 100 μl of RPTPμ D1 at 7.5 mg/ml was loaded onto a Superdex 75 prep gel filtration column (Pharmacia Biotech Inc.) equilibrated with 50 mm Tris·HCl (pH 7.5), 200 mm NaCl and run at a flow rate of 1 ml/min. X-ray crystallographic data were collected to 2.3-Å resolution and processed using DENZO and SCALEPACK (14Otwinowski Z. Sawyer L. Isaacs N. Bailey S. Data Collection and Processing. Science and Engineering Research Council, Daresbury Laboratory, Warrington, UK1993Google Scholar). The structure was determined by molecular replacement using the CCP4 integrated version of AMoRe (15Navaza J. Acta Crystallogr. Sec. A. 1994; 50: 157-163Crossref Scopus (5030) Google Scholar, 16Collaborative Computer Project No. 4 Acta Crystallogr. Sec. D. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar) using a polyalanine model of RPTPα (11Bilwes M. den Hertog J. Hunter T. Noel J.P. Nature. 1996; 382: 555-559Crossref PubMed Scopus (292) Google Scholar), including side chains of residues identical with those of RPTPμ, as a search model. Crystallographic refinement was performed using X-PLOR (17Brunger A.T. X-PLOR, Version 3.1. Yale University Press, New Haven, CT1992Google Scholar), applying strict noncrystallographic symmetry (NCS) restraints. Calculated phases using the model were improved by several cycles of combined solvent flattening (solvent content 0.50) and NCS averaging using the PHASES package (18Furey W. Swaminathan S. Am. Crystallogr. Assoc. Meet. Summ. 1990; 18: 73Google Scholar). Iterative rounds of simulated annealing refinement, combined with NCS averaging and solvent flattening, were applied to improve the quality of the maps and the protein model. During the final stages of refinement, the NCS restraints were removed. Analysis of the coordinates and electron density maps was performed with O (19Jones T.A. Zou J. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sec. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar). The final model consists of residues 879–1156 of RPTPμ D1 that are visible within the electron density and 340 water molecules (TableI). The structure of RPTPα D1 consists of residues 877–1156 (RPTPμ numbering (20Gebbink M. Van Etten I. Hateboer G. Suijkerbuijk R. Beijersbergen R. Van Kessel A. Moolenar W. FEBS Lett. 1991; 290: 123-130Crossref PubMed Scopus (110) Google Scholar)).Table ICrystallographic data and refinement statisticsCrystallographic data: Space groupP21 Cell parametersa = 95.54 Å, b = 36.29 Å, c = 95.38 Å, β = 95.66° Z2 X-ray sourcePX 7.2, SRS, Daresbury Resolution50 to 2.3 Å Observations (n)112,065 Unique reflections (complete)26,733 (89.5%) R where are the and mean of to 2.3 Å of reflections Protein atoms molecules R c and c are the and structure of Å R where are the and mean of R c and c are the and structure of h. in a In the crystal structure of RPTPμ D1, the to a dimer with symmetry It is likely that the dimerization of RPTPμ D1 is a of since studies revealed that the protein was monomeric with an molecular of in concentrated of mg/ml This is in contrast to RPTPα D1, which forms and in solution at protein of mg/ml (11Bilwes M. den Hertog J. Hunter T. Noel J.P. Nature. 1996; 382: 555-559Crossref PubMed Scopus (292) Google Scholar). The of RPTPμ D1 is from that of RPTPα D1, since the dimer of RPTPμ D1 not include the N-terminal helix-turn-helix segment or the catalytic site analysis of the catalytic site of RPTPμ D1. The interactions between the two dimeric of RPTPμ are hydrophobic in and mediated by residues and from one subunit and residue and the of from the These residues are not conserved within other RPTPs Within RPTPμ D1, the catalytic site is unhindered and adopts an open conformation similar to that of the of PTP1B (12Barford D. Flint A.J. Tonks N.K. Science. 1994; 263: 1397-1404Crossref PubMed Scopus (688) Google Scholar). Residues of RPTPμ, equivalent to those of the RPTPα helix-turn-helix that inserts into the related catalytic site, are to solvent that RPTPμ D1 as a monomeric with an molecular of used are: and c of RPTP D1s in the that the dimer of RPTPα and RPTPμ. residues of the Tyr(P) recognition loop of the catalytic site. residues from through to the WPD loop of the catalytic site. Residues of RPTPμ D1 and RPTPα D1 that interactions at their dimer are and The residues that the dimer of RPTPμ D1 are conserved the whereas residues of the RPTPα D1 are conserved within the helix-turn-helix but conserved within the catalytic site. residues are in on and highly conserved residues In contrast to the in quaternary structure between RPTPα and RPTPμ, the tertiary structures of domain 1 of these two RPTPs are very similar with a root mean square deviation between equivalent Cα atoms of 1.1 Å The major in tertiary structure are present within surface the tertiary of the N-terminal helix-turn-helix segment and N-terminal and associated β-sheet with βy, of RPTPμ are identical to that of the equivalent structural of RPTPα. These features RPTP D1s from the catalytic domains and N-terminal of cytosolic PTPs and are important in the dimeric organization of RPTPα D1. Interestingly, and RPTPα D1, which are present within the helix-turn-helix segment that inserts into the dyad-related catalytic site, with equivalent residues of RPTPμ D1 and The Cα atoms of these residues by Å although this of the chain is of within the N-terminal helix-turn-helix that contribute to the dimer of RPTPα D1 is the residues in this dimeric is conserved between RPTPμ D1 and RPTPα D1. catalytic site residues that the dimer of RPTPα D1 are invariant or highly conserved between RPTPs it is that invariant catalytic site residues would have the to with of the diverse N-terminal helix-turn-helix segments within the RPTP family. this were it would that RPTP may as the catalytic of RPTP would not between the helix-turn-helix segments of other The structure of D1 of RPTPα determined by Bilwes et M. den Hertog J. Hunter T. Noel J.P. Nature. 1996; 382: 555-559Crossref PubMed Scopus (292) Google Scholar) the of a dimer in which a helix-turn-helix segment on the N-terminal side of one catalytic domain the catalytic site of the PTP domain in the This the to propose the possibility that ligand-induced dimerization of RPTPs in general may lead to inhibition of activity. used this structural model to the of an J. Schlessinger J. A. Cell. 1993; Full Text PDF PubMed Scopus Google Scholar). of of the RPTP, from or cells to engagement of in cell be by of containing the catalytic domain of In the of the molecule in which the extracellular and transmembrane segments of were with those of the was in the of It was proposed that in the of dimerization of the is with inhibition of PTP activity. a of ligand binding on the catalytic activity of the not been results in the structure of RPTPα to other We show that the N-terminal helix-turn-helix of RPTPμ D1 not formation of an inhibited dimeric similar to that of RPTPα D1 (11Bilwes M. den Hertog J. Hunter T. Noel J.P. Nature. 1996; 382: 555-559Crossref PubMed Scopus (292) Google Scholar), and the dimeric structure of RPTPα D1 may not the for members of the RPTP family. may be a feature of a of such as RPTPα and its for which sequence similarity in those residues at the N-terminal helix-turn-helix dimer results not the possibility that in the of the membrane the of RPTPμ may an inhibited the we not of RPTPμ D1 in unlike that for RPTPα D1, and that within the RPTPμ D1 which would the of RPTPμ in the inhibition of catalytic is not that the N-terminal helix-turn-helix of RPTPμ D1 not play a role in regulating the activity of this