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• W2026671374 abstract "Protein-tyrosine phosphatase 1B (PTP1B) has recently received much attention as a potential drug target in type 2 diabetes. This has in particular been spurred by the finding that PTP1B knockout mice show increased insulin sensitivity and resistance to diet-induced obesity. Surprisingly, the highly homologous T cell protein-tyrosine phosphatase (TC-PTP) has received much less attention, and no x-ray structure has been provided. We have previously co-crystallized PTP1B with a number of low molecular weight inhibitors that inhibit TC-PTP with similar efficiency. Unexpectedly, we were not able to co-crystallize TC-PTP with the same set of inhibitors. This seems to be due to a multimerization process where residues 130–132, the DDQ loop, from one molecule is inserted into the active site of the neighboring molecule, resulting in a continuous string of interacting TC-PTP molecules. Importantly, despite the high degree of functional and structural similarity between TC-PTP and PTP1B, we have been able to identify areas close to the active site that might be addressed to develop selective inhibitors of each enzyme. Protein-tyrosine phosphatase 1B (PTP1B) has recently received much attention as a potential drug target in type 2 diabetes. This has in particular been spurred by the finding that PTP1B knockout mice show increased insulin sensitivity and resistance to diet-induced obesity. Surprisingly, the highly homologous T cell protein-tyrosine phosphatase (TC-PTP) has received much less attention, and no x-ray structure has been provided. We have previously co-crystallized PTP1B with a number of low molecular weight inhibitors that inhibit TC-PTP with similar efficiency. Unexpectedly, we were not able to co-crystallize TC-PTP with the same set of inhibitors. This seems to be due to a multimerization process where residues 130–132, the DDQ loop, from one molecule is inserted into the active site of the neighboring molecule, resulting in a continuous string of interacting TC-PTP molecules. Importantly, despite the high degree of functional and structural similarity between TC-PTP and PTP1B, we have been able to identify areas close to the active site that might be addressed to develop selective inhibitors of each enzyme. Protein-tyrosine phosphatases (PTPs) 1The abbreviations used are: PTPprotein-tyrosine phosphataseTCT cell1The abbreviations used are: PTPprotein-tyrosine phosphataseTCT cell are key regulators of signal transduction processes (1Hunter T. Cell. 2000; 100: 113-127Abstract Full Text Full Text PDF PubMed Scopus (2231) Google Scholar, 2Tonks N.K. Neel B.G. Curr. Opin. Cell Biol. 2001; 13: 182-195Crossref PubMed Scopus (459) Google Scholar). The family of classical PTPs can be divided into two broad categories as intracellular and receptor-like PTPs covering a total of 17 subtypes (3Andersen J.N. Mortensen O.H. Peters G.H. Drake P.G. Iversen L.F. Olsen O.H. Jansen P.G. Andersen H.S. Tonks N.K. Moller N.P. Mol. Cell. Biol. 2001; 21: 7117-7136Crossref PubMed Scopus (588) Google Scholar). Receptor-like PTPs contain an extracellular domain, a single transmembrane domain, and one or two cytoplasmic PTP domains. Intracellular PTPs generally contain one PTP domain and an N- or C-terminal domain that targets the enzymes to specific subcellular localizations, as exemplified by the targeting of PTP1B to the endoplasmic reticulum (4Frangioni J.V. Beahm P.H. Shifrin V. Jost C.A. Neel B.J. Cell. 1992; 68: 545-560Abstract Full Text PDF PubMed Scopus (500) Google Scholar). protein-tyrosine phosphatase T cell protein-tyrosine phosphatase T cell PTP1B and TC-PTP are two closely related intracellular enzymes. PTP1B was the first protein-tyrosine phosphatase to be identified and characterized (5Tonks N.K. Diltz C.D. Fischer E.H. J. Biol. Chem. 1988; 263: 6731-6737Abstract Full Text PDF PubMed Google Scholar, 6Tonks N.K. Diltz C.D. Fischer E.H. J. Biol. Chem. 1988; 263: 6722-6730Abstract Full Text PDF PubMed Google Scholar). Shortly after this landmark event, PTP1B was cloned from a placenta cDNA library (7Chernoff J. Schievella A.R. Jost C.A. Erikson R.L. Neel B.G. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2735-2739Crossref PubMed Scopus (165) Google Scholar), and TC-PTP was cloned from a peripheral human T cell cDNA library (8Cool D.E. Tonks N.K. Charbonneau H. Walsh K.A. Fischer E.H. Krebs E.G. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5257-5261Crossref PubMed Scopus (225) Google Scholar). Despite its name, TC-PTP is ubiquitously expressed (9Ibarra-Sanchez M.D. Simoncic P.D. Nestel F.R. Duplay P. Lapp W.S. Tremblay M.L. Semin. Immunol. 2000; 12: 379-386Crossref PubMed Scopus (84) Google Scholar). Alternative splicing gives rise to two forms of TC-PTP that differ in the C termini, a 45-kDa form that is targeted to the nucleus and a 48-kDa form that localizes to the endoplasmic reticulum via a hydrophobic C-terminal region (10Lorenzen J.A. Dadabay C.Y. Fischer E.H. J. Cell Biol. 1995; 131: 631-643Crossref PubMed Scopus (112) Google Scholar). TC-PTP is tightly regulated during the cell cycle and seems to play an important role in mitogenesis (9Ibarra-Sanchez M.D. Simoncic P.D. Nestel F.R. Duplay P. Lapp W.S. Tremblay M.L. Semin. Immunol. 2000; 12: 379-386Crossref PubMed Scopus (84) Google Scholar). In a recent study, it was shown that cellular stress causes reversible cytoplasmic accumulation of the 45-kDa form of TC-PTP (i.e. the nuclear form) (11Lam M.H.C. Michell B.J. Fodero-Tavoletti M.T. Kemp B.E. Tonks N.K. Tiganis T. J. Biol. Chem. 2001; 276: 37700-37707Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Although they have a sequence identity of about 74% in the catalytic domains (see Fig. 1), TC-PTP and PTP1B clearly fulfill different biological functions, as has been demonstrated in knock-out mice. Thus, although PTP1B knock-out mice show increased insulin sensitivity and resistance to diet-induced obesity and are viable with a normal life span (12Elchebly M. Payette P. Michaliszyn E. Cromlish W. Collins S. Loy A.L. Normandin D. Cheng A. Himms-Hagen J. Chan C.-C. Ramachandaran C. Gresser M.J. Tremblay M.L. Kennedy B.P. Science. 1999; 283: 1544-1548Crossref PubMed Scopus (1882) Google Scholar, 13Klaman L.D. Boss O. Peroni O.D. Kim J.K. Martino J.L. Zabolotny J.M. Moghal N. Lubkin M. Kim Y.B. Sharpe A.H. Stricker-Krongrad A. Shulman G.I. Neel B.G. Kahn B.B. Mol. Cell. Biol. 2000; 20: 5479-5489Crossref PubMed Scopus (1111) Google Scholar), TC-PTP knock-out mice die at 3–5 weeks of age (14Youten K.E. Muise E.S. Itie A. Michaliszyn E. Wagner J. Jothy S. Lapp W.S. Tremblay M.L. J. Exp. Med. 1997; 186: 683-693Crossref PubMed Scopus (309) Google Scholar). In accordance with these in vivo observations, substrate trapping experiments have further shown that PTP1B and TC-PTP recognize different cellular targets (15Flint A.J. Tiganis T. Barford D. Tonks N.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1680-1685Crossref PubMed Scopus (673) Google Scholar, 16Tiganis T. Bennett A.M. Ravichandran K.S. Tonks N.K. Mol. Cell. Biol. 1998; 18: 1622-1634Crossref PubMed Google Scholar). At present it is not known to which degree this is due to different inherent substrate specificity that resides within the catalytic domains or to other regulatory mechanisms. For example, the activity and function of PTP1B can be regulated at different levels, including transcription (17Fukada T. Tonks N.K. J. Biol. Chem. 2001; 276: 25512-25519Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar), alternative splicing (18Shifrin V.I. Neel B.G. J. Biol. Chem. 1993; 268: 25376-25384Abstract Full Text PDF PubMed Google Scholar), proteolytic processing (19Frangione J.V. Oda A. Smith M. Salzman E.W. Neel B.G. EMBO J. 1993; 12: 4843-4856Crossref PubMed Scopus (282) Google Scholar), and covalent modification (i.e. phosphorylation of specific residues such as Ser-50) (20Ravichandran L.V. Chen H. Li Y.H. Quon M.J. Mol. Endocrinol. 2001; 15: 1768-1780Crossref PubMed Scopus (100) Google Scholar). Likewise, as indicated above, alternative splicing may determine which substrates are recognized by TC-PTP (i.e. nuclear substrates by the 45-kDa form and cytoplasmic substrates by the 48-kDa form). However, by comparing substrates trapped with PTP1B and those trapped by targeting a TC-PTP/PTP1B chimera to the endoplasmic reticulum, Tonks and coworkers (16Tiganis T. Bennett A.M. Ravichandran K.S. Tonks N.K. Mol. Cell. Biol. 1998; 18: 1622-1634Crossref PubMed Google Scholar) provide convincing evidence that at least part of the observed differences in substrate recognition capacity between the two enzymes is due to differences in intrinsic substrate specificity. Thus, fine structural differences not readily identifiable by primary sequence analyses may account for the observed differences in substrate recognition by PTP1B and TC-PTP. Similarly, elegant catalytic domain-swapping experiments of two other homologous PTPs, SHP-1 and SHP-2, clearly indicate that substantial substrate specificity may reside in the PTP domains (21Tenev T. Keilhack H. Tomic S. Stoyanov B. Stein-Gerlach M. Lammers R. Krivtsov A.V. Ullrich A. Böhmer F.D. J. Biol. Chem. 1997; 272: 5966-5973Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 22O'Reilly A.M. Neel B.G. Mol. Cell. Biol. 1998; 18: 161-177Crossref PubMed Scopus (96) Google Scholar). In addition, areas outside the highly conserved regions surrounding the active sites may contribute to substrate binding (3Andersen J.N. Mortensen O.H. Peters G.H. Drake P.G. Iversen L.F. Olsen O.H. Jansen P.G. Andersen H.S. Tonks N.K. Moller N.P. Mol. Cell. Biol. 2001; 21: 7117-7136Crossref PubMed Scopus (588) Google Scholar). Although the exact molecular mechanism(s) underlying the above phenotype of PTP1B knock-out mice still remains to be identified, these studies indicate that PTP1B could be an attractive drug target for treatment of type 2 diabetes. As a consequence, PTPs have caught the attention of the pharmaceutical industry (23Møller N.P.H. Iversen L.F. Andersen H.S. McCormack J.G. Curr. Opin. Drug Discov. Devel. 2000; 3: 527-540PubMed Google Scholar). In particular, there is a rush for developing selective PTP1B inhibitors. We have recently used two different structure-based design approaches (attraction/repulsion and steric fit/steric hindrance) to develop selective PTP1B inhibitors (24Iversen L.F. Andersen H.S. Møller K.B. Olsen O.H. Peters G.H. Branner S. Mortensen S.B. Hansen T.K. Lau J., Ge, Y. Holsworth D.D. Newman M.J. Møller N.P.H. Biochemistry. 2001; 40: 14812-14820Crossref PubMed Scopus (85) Google Scholar, 25Iversen L.F. Andersen H.S. Branner S. Mortensen S.B. Peters G.H. Norris K. Olsen O.H. Jeppesen C.B. Lundt B.F. Ripka W. Møller K.B. Møller N.P.H. J. Biol. Chem. 2000; 275: 10300-10307Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Because PTP1B and TC-PTP are about 74% identical in the catalytic domains, it is anticipated that active site-directed PTP1B inhibitors might also bind to TC-PTP with equal potency. Indeed, we recently showed that two active site PTP inhibitors inhibited the two enzymes with almost identical potency. It is unclear if such potential cross-reactivity would be beneficial or perhaps cause adverse effects. Surprisingly, although PTP1B has been the focus of numerous structural studies (for an updated list of x-ray structures, see science.novonordisk.com/ptp), no x-ray structures have been reported for TC-PTP. Therefore, we decided to compare PTP1B and TC-PTP both at the functional and structural levels. Similar constructs of PTP1B and TC-PTP corresponding to the first 321 amino acid residues of the former were compared in (i) enzyme kinetic studies, (ii) inhibitor studies using a set of active site-directed PTP inhibitors, and (iii) by x-ray protein crystallography. Although as expected these studies showed very similar function and structure of the two enzymes, our analysis also indicates that they are sufficiently different to allow a structure-based design of inhibitors that are selective for either of them. PTP1B 1–321 and TC-PTP 1–314 were cloned, expressed, and purified essentially as described previously (24Iversen L.F. Andersen H.S. Møller K.B. Olsen O.H. Peters G.H. Branner S. Mortensen S.B. Hansen T.K. Lau J., Ge, Y. Holsworth D.D. Newman M.J. Møller N.P.H. Biochemistry. 2001; 40: 14812-14820Crossref PubMed Scopus (85) Google Scholar, 25Iversen L.F. Andersen H.S. Branner S. Mortensen S.B. Peters G.H. Norris K. Olsen O.H. Jeppesen C.B. Lundt B.F. Ripka W. Møller K.B. Møller N.P.H. J. Biol. Chem. 2000; 275: 10300-10307Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 26Andersen H.S. Iversen L.F. Jeppesen C.B. Branner S. Norris K. Møller K.B. Møller N.P.H. J. Biol. Chem. 2000; 275: 7101-7108Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). The cDNAs encoding these PTPs were obtained by polymerase chain reaction using primers with convenient cloning sites and appropriate cDNA templates. The coding sequences were confirmed by DNA sequencing. The constructs were inserted into the pET11a expression vector, and PTP1B and TC-PTP were expressed essentially as described previously (26Andersen H.S. Iversen L.F. Jeppesen C.B. Branner S. Norris K. Møller K.B. Møller N.P.H. J. Biol. Chem. 2000; 275: 7101-7108Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). PTP1B and TC-PTP were purified in a two-step procedure. In brief, compound 4, which is a selective PTP1B/TC-PTP inhibitor (see Fig. 2), was coupled to epoxy-activated Sepharose 6B (Amersham Biosciences) according to the manufacturer's instructions (100 mg of compound 4/g of drained column material). Lysates from Escherichia coli producing PTP1B and TC-PTP were cleared by centrifugation and applied to the column. The enzymes were eluted by a combined pH (6.2–9.0) and salt gradient (0.1–1.0 m NaCl), resulting in ∼90% pure preparations. The final polishing consists of an anion exchange purification step (Mono-Q, Amersham Biosciences). Before crystallization, buffer exchange (see below under “Crystallization”) was performed using a Superdex 200 column (Amersham Biosciences). Further experimental details will be published elsewhere. The phosphatase activity was determined using p- nitrophenyl phosphate as substrate essentially as described (24Iversen L.F. Andersen H.S. Møller K.B. Olsen O.H. Peters G.H. Branner S. Mortensen S.B. Hansen T.K. Lau J., Ge, Y. Holsworth D.D. Newman M.J. Møller N.P.H. Biochemistry. 2001; 40: 14812-14820Crossref PubMed Scopus (85) Google Scholar, 27Peters G.H. Iversen L.F. Branner S. Andersen H.S. Mortensen S.B. Olsen O.H. Møller K.B. Møller N.P.H. J. Biol. Chem. 2000; 275: 18201-18209Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar) using a constant ionic strength three-component buffer at pH 6.5 (28Lohse D.L. Denu J.M. Santoro N. Dixon J.E. Biochemistry. 1997; 36: 4568-4575Crossref PubMed Scopus (156) Google Scholar, 29Ellis, K. J., and Morrison, J. F. (1982) Methods Enzymol. 405–426Google Scholar, 30Ekman P. Jager O. Anal. Biochem. 1993; 214: 138-141Crossref PubMed Scopus (88) Google Scholar). The data were analyzed using nonlinear regression hyperbolic fit to classical Michaelis-Menten enzyme kinetic models. Inhibition is expressed as Ki values in μm. The reported S.D. are calculated from at least three independent experiments. An ∼10 mg/ml TC-PTP in 10 mmTris, pH 7.5, 25 mm NaCl, 0.2 mm EDTA, and 3 mm dithiothreitol was used for crystallization. Crystals were grown by the hanging drop vapor diffusion method. Two μl of TC-PTP solution were mixed with 2 μl of reservoir solution consisting of 0.05–0.25 m Hepes buffer, pH 8.0, 0.2 mmagnesium acetate, 20% polyethylene glycol 8000, and 0.1% β-mercaptoethanol. The reservoir volume was 1 ml. Crystals grew to the size of 0.5 × 0.3 × 0.1 mm over approximately 1 week, and three or more weeks in total were used for crystal growth. Data were collected using a Mar345 image plate detector on a rotating anode (RU300, CuKα 50 kV/100 mA) equipped with Osmic multilayer mirror system. The data collection was performed on a single crystal at room temperature. A data set to 2.53-Å resolution was obtained. Data processing was performed using Denzo, Scalepack, and the CCP4 program suite (31Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref Scopus (38253) Google Scholar, 32Collaborative Computational Project, N. 4., Acta Crystallogr. Sect. D Biol. Crystallogr. 50, 1994, 760–763Google Scholar). From autoindexing and the systematic absent reflections, the space group was determined to be P41212 or P43212 with cell dimensions a =b = 60.5 Å and c = 187.6 Å. The Vm was calculated to be 2.3 Å3/dalton with a TC-PTP monomer in asymmetric unit (Vm = 2.4 Å3/dalton for the average protein crystal). A molecular replacement solution was found using Amore (33Navaza J. Acta Crystallogr. Sect. A. 1994; 50: 157-163Crossref Scopus (5026) Google Scholar) and PTP1B (Protein Data Bank code 1C88) as a search model (ligand and water molecules were omitted from the structure). The search was performed in both P41212 and P43212, with the correct solution identified in P43212. All refinements were performed using CNX version 2000 (Accelrys). Interchanging cycles of model building using X-build (Accelrys) and refinement were performed. To avoid phase bias from the molecular replacement search model, 5% of the amplitudes were omitted from the refinements and used for R-free calculations, and the lowering of R-free was monitored during all refinements. Water molecules were inserted using the X-solvate program (Accelrys). The graphical interface used was Quanta (Accelrys). For further details see Table I.Table IStatistics of x-ray and refinementsStatistics of x-ray data and structure refinements Space groupP43212 Unit cell parametersa = b = 60.5 c = 187.6 Å Completeness (20–2.56Å)97.5% Completeness (2.6–2.56Å)82.0% Multiplicity (20–2.56 Å)3.3 Rmerge (20–2.56 Å)6.7% Rmerge (2.6–2.56Å)38.1% <I/ς(I)< (20–2.56Å)11.7 <I/ς(I)> (2.6–2.56 Å)2 Unique reflections116.5 Atoms in structure2293 R-factor1-aR-factors were calculated using all data from 20 to 2.56 Å. Crystallographic R-factor=||Σ(hkl)Fo|-|Fc||/Σ(hkl)|Fo21.5 R-free1-bR-free=Σ(hkl)εT||Fo|-|Fc||/Σ(hkl)εT|Fo| where T is a test set containing a random 5% of the observations omitted from the refinement process.28.8Root mean square deviations from idealized geometry Bond lengths (Å)0.007 Bond angles (°)2.41-a R-factors were calculated using all data from 20 to 2.56 Å.Crystallographic R-factor=||Σ(hkl)Fo|-|Fc||/Σ(hkl)|Fo1-b R-free=Σ(hkl)εT||Fo|-|Fc||/Σ(hkl)εT|Fo| where T is a test set containing a random 5% of the observations omitted from the refinement process. Open table in a new tab Compounds used here (Fig. 2) were synthesized as described previously (25Iversen L.F. Andersen H.S. Branner S. Mortensen S.B. Peters G.H. Norris K. Olsen O.H. Jeppesen C.B. Lundt B.F. Ripka W. Møller K.B. Møller N.P.H. J. Biol. Chem. 2000; 275: 10300-10307Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 26Andersen H.S. Iversen L.F. Jeppesen C.B. Branner S. Norris K. Møller K.B. Møller N.P.H. J. Biol. Chem. 2000; 275: 7101-7108Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). The 1–321 PTP1B construct is the best characterized enzyme domain within the PTP family, and its activity and structure have been studied in numerous reports since its original identification and cloning. Therefore, to compare directly with this gold standard in the PTP field we decided to clone and express the similar construct of TC-PTP,i.e. residues 1–314. The sequence identity between the catalytic domains of TC-PTP and PTP1B is 74% (Fig. 1) with the major differences clustered in four stretches of amino acid residues, (i) 1–35, (ii) 129–148, (iii) 158–174, (iv) 235–246 (TC-PTP numbering is used). To compare PTP1B and TC-PTP at the functional level, we first determined the steady state kinetic parameters,kcat and Km for each enzyme using p- nitrophenyl phosphate as substrate. As shown in Table II, the catalytic efficiencies of these two enzymes are almost identical, thus providing an initial indication that the high degree of primary sequence identity also translates into a high degree of similarity at the structural and functional levels.Table IIKinetic constants for the hydrolysis of p-nitrophenyl phosphate at pH 6.5 and 25 °CEnzymekcatKmkcat/Km× 10−3s−1mms−1 m−1PTP1B51.2 ± 2.00.47 ± 0.02109.5TC PTP53.9 ± 1.80.48 ± 0.03113.2 Open table in a new tab We next probed the active site cavities of the two enzymes with a set of PTP inhibitors. We have previously shown that 2-(oxalylamino)-benzoic acid (Fig. 2, compound 1) is a general PTP inhibitor with the two carboxyl groups interacting with conserved residues in the active sites of these enzymes (26Andersen H.S. Iversen L.F. Jeppesen C.B. Branner S. Norris K. Møller K.B. Møller N.P.H. J. Biol. Chem. 2000; 275: 7101-7108Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). Of note in the present context, we also found that an additional ring system could dramatically change the inhibitory profiles of these compounds. Thus, although the naphthyl-based compound 2 is a relatively potent inhibitor of PTP-LAR at pH 5.5, the indole-based compound3 is a poor inhibitor of this enzyme. Conversely, compound3 is a relatively potent inhibitor of SHP-1, whereas compound 2 is about 3-fold less potent. Accordingly, this shows that even very close to the active site, structural differences among PTPs can be detected with these low molecular weight inhibitors. As shown in Table III, only minor differences could be demonstrated when compounds 1–4 were analyzed for inhibitory activity against PTP1B and TC-PTP. This is in agreement with our recent findings where two pyran-based inhibitors were found to inhibit these two enzymes with similar efficiency (24Iversen L.F. Andersen H.S. Møller K.B. Olsen O.H. Peters G.H. Branner S. Mortensen S.B. Hansen T.K. Lau J., Ge, Y. Holsworth D.D. Newman M.J. Møller N.P.H. Biochemistry. 2001; 40: 14812-14820Crossref PubMed Scopus (85) Google Scholar). Thus, we conclude that, from a functional point of view, the catalytic clefts of PTP1B and TC-PTP are very similar.Table IIIInhibition constants (K1 values in μm)EnzymesCompound 1Compound 2Compound 3Compound 4PTP1B34.0 ± 1.17.3 ± 0.619.3 ± 0.61.01 ± 0.04TC PTP39.8 ± 1.67.5 ± 0.520.2 ± 0.61.47 ± 0.21 Open table in a new tab Although the above enzyme kinetic characterization and inhibitor studies as well as molecular modeling (not shown) clearly indicate that the catalytic domains of PTP1B and TC-PTP must be very similar, protein x-ray crystallography is needed for unequivocal structural comparison of the two enzymes. We first attempted to co-crystallize TC-PTP with several of the above ligands that were found to inhibit PTP1B and TC-PTP with similar potency and for which several x-ray PTP1B-inhibitor complex structures have been reported (24Iversen L.F. Andersen H.S. Møller K.B. Olsen O.H. Peters G.H. Branner S. Mortensen S.B. Hansen T.K. Lau J., Ge, Y. Holsworth D.D. Newman M.J. Møller N.P.H. Biochemistry. 2001; 40: 14812-14820Crossref PubMed Scopus (85) Google Scholar, 25Iversen L.F. Andersen H.S. Branner S. Mortensen S.B. Peters G.H. Norris K. Olsen O.H. Jeppesen C.B. Lundt B.F. Ripka W. Møller K.B. Møller N.P.H. J. Biol. Chem. 2000; 275: 10300-10307Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 26Andersen H.S. Iversen L.F. Jeppesen C.B. Branner S. Norris K. Møller K.B. Møller N.P.H. J. Biol. Chem. 2000; 275: 7101-7108Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). However, to our surprise and for reasons that will be discussed below, we were not able to obtain any crystals of TC-PTP complexed with any of these inhibitors. Therefore, we turned our attention to the uncomplexed version and obtained the apo structure. Residues 5–277 of TC-PTP were identified and built into the electron density maps. The final structure contains two disordered loops, 117–121 and 236–242, with only backbone atoms traceable in the 2 Fo − Fc electron density maps. Cys-94 and Cys-95 both contained covalently bound β-mercaptoethanol (used as a reducing agent during purification and crystallization). Forty water molecules were inserted during refinements. The WPD loop of TC-PTP was found in an open conformation. Thus, all structural comparisons with PTP1B are based on the apo form of PTP1B (Protein Data Bank code 2HNP). Overall, no differences were identified in the secondary or tertiary structures between TC-PTP and PTP1B. Minor differences are observed in loop areas between the two structures (see Fig. 3). The root mean square deviation between the TC-PTP and PTP1B structure (for all equivalent atoms) is 1.82 Å (calculated using Quanta). Our lack of success in co-crystallizing TC-PTP with inhibitors is most likely explained by unusual crystal packing along the 21 axis (space group P43212) involving the active site pocket and surrounding residues. A loop corresponding to residues 130–132, the “DDQ loop,” from one molecule was inserted in the active site of a neighboring molecule, resulting in a continuous row of TC-PTP molecules (Fig. 4 a). The active site blockage is not limited to the DDQ loop but involves a total surface area of 1183 Å2, with 892 Å2 of hydrophilic and 291 Å2 of hydrophobic nature. Residues 129–133, 135, 145–148, 150, 155, 158, and 176 on one molecule form a surface patch (inhibiting patch) that interacts with the active site on the neighboring molecule (residues 43, 48–50, 120–121, 183–184, 216–222, 260, and 264); see Fig. 4 b. The binding of the inhibitory patch is further stabilized by several hydrogen-bonding water molecules (see Fig. 5, a and b). Generally speaking, the surface loop consisting of residues 129–135 and the outermost β-strand flanking the central β-sheet of PTPs, including residues 145–150, define the major part of the inhibitor patch.Figure 5The DDQ loop binding motif. a, binding of Asp-131 from the DDQ loop (from one TC-PTP molecule) and two water molecules to the active site pocket P loop and Gln-260 (of a neighboring molecule). The atoms are colored according to atom type: carbons are in light green (in dark green for the Asp-131 carbon atoms), oxygens are in red, and nitrogens are in blue. The distances for the marked possible hydrogen bonds are in Å. b, stereo picture of the DDQ loop, the two water molecules, Gln-260, and P loop together with the final 2 Fo − Fc electron density map. The atoms are colored as described in a. The 2 Fo − Fc electron density map is colored at one sigma level in blue and three sigma level in red. c, stereo picture of the PTP1B(C215S)-phosphotyrosine (pTyr) structure (Protein Data Bank code 1PTV) superimposed on the TC-PTP structure for comparison of the binding mode between the Asp-131 with water molecules and the natural substrate phosphotyrosine. TC-PTP atoms are colored in light green (Asp-131 in dark green), and PTP1B(C215S)–phosphotyrosine atoms are in yellow. The critical oxygen atoms are colored in red.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In the following, the residues that belong to the patch that is on the blocking (or inhibiting) molecule are denoted “inhibitor residues” (in italics), whereas the residues that belong to the active site patch of the neighboring molecule are described as “TC-PTP residues” (in bold). Both of the carboxylic acid groups of Asp-130 and Asp-131 from the DDQ loop hydrogen bonds to the amine side chain group of Gln-260. Furthermore, the side chain of Asp-131 interacts with the P loop via two hydrogen-bonding water molecules (see details below). The amine of Gln-132 hydrogen bonds to the amine ofGln-264. As indicated, a number of van der Waals contacts stabilize the complex formation as follows. (i) the Cβ atom of Thr-129 interacts with the Cβ and Cγ side chain atoms ofAsp-50; (ii) the Glu-133 Cγ atom interacts with the Cβ atom of Phe-183; (iii) Leu-135 interacts with Phe-183; (iv) Leu-145 interacts withTyr-48; (v) Leu-146 interacts withVal-121; (vi) Val-150 interacts with the Cγ and Cδ atoms of both Arg-43 and Arg-49; and finally (vii) Leu-158 is in van der Waals contact with the Cβ ofSer-120. Furthermore, the side chain hydroxyl group of Ser-147 is in hydrogen bond contact with the backbone carbonyl group of Ser-120, and Glu-148 hydrogen bonds to the backbone nitrogen of Arg-49 as well as to a water molecule that further hydrogen bonds to the backbone nitrogens of both Arg-49 and Asp-50. Finally, the side chains of Thr-155 and His-176 (via a water molecule) hydrogen bond with the guanidinium group of Arg-49. As described, Asp-131 is positioned into the active site cleft of TC-PTP. However, the carboxylic acid group is not in direct contact with the P loop as previously seen for charged ligands in the active site pocket of PTPs (24Iversen L.F. Andersen H.S. Møller K.B. Olsen O.H. Peters G.H. Branner S. Mortensen S.B. Hansen T.K. Lau J., Ge, Y. Holsworth D.D. Newman M.J. Møller N.P.H. Biochemistry. 2001; 40: 14812-14820Crossref PubMed Scopus (85) Google Scholar, 25Iversen L.F. Andersen H.S. Branner S. Mortensen S.B. Peters G.H. Norris K. Olsen O.H. Jeppesen C.B. Lundt B.F. Ripka W. Møller K.B. Møller N.P.H. J. Biol. Chem. 2000; 275: 10300-10307Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 26Andersen H.S. Iversen L.F. Jeppesen C.B. Branner S. Norris K. Møller K.B. Møller N.P.H. J. Biol. Chem. 2000; 275: 7101-7108Abstract Full Text Full Text" @default.
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• W2026671374 title "Structure Determination of T Cell Protein-tyrosine Phosphatase" @default.
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