Purification and Crystallization of the Catalytic Domain of Human Protein Tyrosine Phosphatase 1B Expressed in Escherichia coli

doi:10.1006/jmbi.1994.1409

Journal of Molecular Biology

Volume 239, Issue 5, 23 June 1994, Pages 726-730

https://doi.org/10.1006/jmbi.1994.1409 Get rights and content

Abstract

The amino-terminal 321 residues encoding the catalytic domain of human protein tyrosine phosphatase 1B (molecular mass 37 kDa) has been expressed in Escherichia coli , purified to homogeneity and crystallized. The crystals diffract to 2·4 Å resolution when exposed to synchrotron radiation and belong to space group P3₁21 (or its enantiomorph) P 3₂21) with a = 88·4 Å, b = 88·4 Å, c = 104·0 Å, α = β = 90·0°, γ = 120·0°. There is one molecule of protein tyrosine phosphatase 1B per asymmetric unit and the crystal form is suitable for the determination of the atomic structure of the enzyme.

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Cited by (48)

Emerging role of tyrosine phosphatase, TCPTP, In the organelles of the early secretory pathway
2013, Biochimica et Biophysica Acta - Molecular Cell Research
Citation Excerpt :
PTP1B shows ER localization like TC48 and it shares many substrates with TC45. When the crystal structures of catalytic domains of TC45 and PTP1B were compared, it was found that they have virtually identical catalytic sites [29,30]. Though PTP1B shows similarity to TCPTP, there are extensive differences in their substrate selectivity and functions.
T-cell protein tyrosine phosphatase, TCPTP, is a ubiquitously expressed non-receptor type tyrosine phosphatase. There are two splice variants of TCPTP, TC48 and TC45, which differ in their sub-cellular localizations and functions. TC45 is a nuclear protein, which has both nuclear and cytoplasmic substrates, and is involved in many signaling events including endocytic recycling of platelet-derived growth factor β-receptor. TC48 is a predominantly endoplasmic reticulum (ER)-localizing protein, which dephosphorylates some of the substrates of TC45 at the ER. However, recently few specific substrates for TC48 have been identified. These include C3G (RapGEF1), syntaxin 17 and BCR-Abl. TC48 moves from the ER to post-ER compartments, the ER–Golgi intermediate compartment (ERGIC) and Golgi, and it is retrieved back to the ER. The retrieval of ER proteins from post-ER compartments is generally believed as a mechanism of targeting these proteins to the ER. However, it is possible that this shuttling of TC48 serves to regulate signaling in the early secretory pathway. For example, TC48 dephosphorylates phosphorylated C3G at the Golgi and inhibits neurite outgrowth. TC48 interacts with and dephosphorylates syntaxin 17, which is an ER and ERGIC-localizing protein involved in vesicle transport. A yeast two-hybrid screen identified several unique interacting partners of TC48 belonging to two groups — proteins involved in vesicle trafficking and proteins involved in cell adhesion. These interacting proteins could be substrates or regulators of TC48 function and localization. Thus, the role of TC48 seems to be more diverse, which is still to be explored.
The molecular details of WPD-loop movement differ in the protein-tyrosine phosphatases YopH and PTP1B
2012, Archives of Biochemistry and Biophysics
Citation Excerpt :
Unexpectedly, the conservative Trp to Phe mutation has significantly different effects in the two enzymes. The plasmid pEt-19b encoding the 37 kDa form of the human wildtype PTP1B (amino acid residues 1–321) was provided by Dr. Nicholas K. Tonks and has been previously reported [26]. Labeled and natural abundance p-nitrophenyl phosphate (pNPP, dicyclohexylammonium salt) were synthesized according to previous methods [27].
The movement of a conserved protein loop (the WPD-loop) is important in catalysis by protein tyrosine phosphatases (PTPs). Using kinetics, isotope effects, and X-ray crystallography, the different effects arising from mutation of the conserved tryptophan in the WPD-loop were compared in two PTPs, the human PTP1B, and the bacterial YopH from Yersinia. Mutation of the conserved tryptophan in the WPD-loop to phenylalanine has a negligible effect on k_cat in PTP1B and full loop movement is maintained. In contrast, the corresponding mutation in YopH reduces k_cat by two orders of magnitude and the WPD loop locks in an intermediate position, disabling general acid catalysis. During loop movement the indole moiety of the WPD-loop tryptophan moves in opposite directions in the two enzymes. Comparisons of mammalian and bacterial PTPs reveal differences in the residues forming the hydrophobic pocket surrounding the conserved tryptophan. Thus, although WPD-loop movement is a conserved feature in PTPs, differences exist in the molecular details, and in the tolerance to mutation, in PTP1B compared to YopH. Despite high structural similarity of the active sites in both WPD-loop open and closed conformations, differences are identified in the molecular details associated with loop movement in PTPs from different organisms.
Conformation-sensing antibodies stabilize the oxidized form of PTP1B and inhibit its phosphatase activity
2011, Cell
Citation Excerpt :
X-ray data were collected in-house to a resolution of 2.8 Å using a Rigaku RU800 X-ray generator and Mar345 area detector and processed using the HKL program (Minor et al., 2006). The structure was refined using reduced wild-type PTP1B as a starting model (PDB code: 2HNQ) (Barford et al., 1994b) using CNS (Brünger et al., 1998) and analyzed using COOT (Emsley and Cowtan, 2004). Construction of scFv Phage Display Library
Protein tyrosine phosphatase 1B (PTP1B) plays important roles in downregulation of insulin and leptin signaling and is an established therapeutic target for diabetes and obesity. PTP1B is regulated by reactive oxygen species (ROS) produced in response to various stimuli, including insulin. The reversibly oxidized form of the enzyme (PTP1B-OX) is inactive and undergoes profound conformational changes at the active site. We generated conformation-sensor antibodies, in the form of single-chain variable fragments (scFvs), that stabilize PTP1B-OX and thereby inhibit its phosphatase function. Expression of conformation-sensor scFvs as intracellular antibodies (intrabodies) enhanced insulin-induced tyrosyl phosphorylation of the β subunit of the insulin receptor and its substrate IRS-1 and increased insulin-induced phosphorylation of PKB/AKT. Our data suggest that stabilization of the oxidized, inactive form of PTP1B with appropriate therapeutic molecules may offer a paradigm for phosphatase drug development.
Insights into the reaction of protein-tyrosine phosphatase 1B: Crystal structures for transition state analogs of both catalytic steps
2010, Journal of Biological Chemistry
Citation Excerpt :
This full array of structures permits a step-by-step description of the structural changes involved in binding and catalysis, particularly those related to the following protein segments: 1) the P-loop (residues 215–222), which includes the nucleophilic cysteine (Cys215) and an important serine (Ser222) that stabilizes its thiolate form, and the arginine (Arg221) involved in binding and transition state stabilization; 2) the WPD-loop (residues 179–187), bearing the general acid/base (Asp181), which is active when this loop alternates from the open to the closed conformation; 3) the Q-loop (residues 261–262), including the residue Gln262, an important player in the second catalytic step; 4) the lysine loop (residues 119–121), containing Lys120, a conserved residue involved in key interactions with the WPD-loop (31); and 5) the Tyr(P) recognition loop (residues 47–49), involved in substrate recognition and binding. The expression and purification of wild type PTP1B were slightly modified from previously reported methods (32) (see supplemental material). Protein concentrations were monitored by UV using an A1 mg/ml280 nm = 1.24.
Catalysis by protein-tyrosine phosphatase 1B (PTP1B) occurs through a two-step mechanism involving a phosphocysteine intermediate. We have solved crystal structures for the transition state analogs for both steps. Together with previously reported crystal structures of apo-PTP1B, the Michaelis complex of an inactive mutant, the phosphoenzyme intermediate, and the product complex, a full picture of all catalytic steps can now be depicted. The transition state analog for the first catalytic step comprises a ternary complex between the catalytic cysteine of PTP1B, vanadate, and the peptide DADEYL, a fragment of a physiological substrate. The equatorial vanadate oxygen atoms bind to the P-loop, and the apical positions are occupied by the peptide tyrosine oxygen and by the PTP1B cysteine sulfur atom. The vanadate assumes a trigonal bipyramidal geometry in both transition state analog structures, with very similar apical O–O distances, denoting similar transition states for both phosphoryl transfer steps. Detailed interactions between the flanking peptide and the enzyme are discussed.
Cysteine S-nitrosylation protects protein-tyrosine phosphatase 1B against oxidation-induced permanent inactivation
2008, Journal of Biological Chemistry
Citation Excerpt :
The peptide samples were subjected to MALDI-MS and MS/MS analyses using an Applied Biosystems 4700 proteomics analyzer mass spectrometer (Applied Biosystems), as described previously (17, 25). Crystallization and Structural Analysis of PTP1B S-Nitrosylation—Crystallization of the 37-kDa PTP1B was carried out as described previously (27). The crystal was incubated with SNAP (1 mm) at room temperature for 20 min and then mixed with the mother liquid containing 29% glycerol as an antifreezing reagent.
Protein S-nitrosylation mediated by cellular nitric oxide (NO) plays a primary role in executing biological functions in cGMP-independent NO signaling. Although S-nitrosylation appears similar to Cys oxidation induced by reactive oxygen species, the molecular mechanism and biological consequence remain unclear. We investigated the structural process of S-nitrosylation of protein-tyrosine phosphatase 1B (PTP1B). We treated PTP1B with various NO donors, including S-nitrosothiol reagents and compound-releasing NO radicals, to produce site-specific Cys S-nitrosylation identified using advanced mass spectrometry (MS) techniques. Quantitative MS showed that the active site Cys-215 was the primary residue susceptible to S-nitrosylation. The crystal structure of NO donor-reacted PTP1B at 2.6 Å resolution revealed that the S-NO state at Cys-215 had no discernible irreversibly oxidized forms, whereas other Cys residues remained in their free thiol states. We further demonstrated that S-nitrosylation of the Cys-215 residue protected PTP1B from subsequent H₂O₂-induced irreversible oxidation. Increasing the level of cellular NO by pretreating cells with an NO donor or by activating ectopically expressed NO synthase inhibited reactive oxygen species-induced irreversible oxidation of endogenous PTP1B. These findings suggest that S-nitrosylation might prevent PTPs from permanent inactivation caused by oxidative stress.
Mass spectrometry-based analyses for identifying and characterizing S-nitrosylation of protein tyrosine phosphatases
2007, Methods
All members in the protein tyrosine phosphatase (PTP) family of enzymes contain an invariant Cys residue which is absolutely indispensable for catalysis. Due to the unique microenvironment surrounding the active center of PTPs, this Cys residue exhibits an unusually low pKa characteristic, thus being highly susceptible to oxidation or S-nitrosylation. While oxidation-dependent regulation of PTP activity has been extensively examined, the molecular details and biological consequences of PTP S-nitrosylation remain unexplored. We hypothesized that the catalytic Cys residue is targeted by proximal nitric oxide (NO) and its derivatives collectively termed reactive nitrogen species (RNS), leading to nitrosothiol formation concomitant with reversible inactivation of PTPs. To test this hypothesis, we have developed novel strategies to examine the redox status of Cys residues of purified PTP1B that was exposed to NO donor S-Nitroso-N-penicillamine (SNAP). A gel-based method in conjunction with mass spectrometry (MS) analysis revealed that the catalytic Cys215 of PTP1B was reversibly modified when PTP1B was briefly treated with SNAP. In order to further identify the exact mode of NO-induced modification, we employed an online LC–ESI-MS/MS analysis incorporating a mass difference-based, data-dependent acquisition function that effectively mapped the S-nitrosylated Cys residues. Our results demonstrated that treating PTP1B with SNAP led to S-nitrosothiol formation of the catalytic Cys215. Interestingly, SNAP-induced modifications were strictly reversible as highly oxidized Cys derivatives (Cys–SO₂H or Cys–SO₃H) were not identified by MS analyses. Thus, the methods introduced in this study provide direct evidence to prove the direct link between S-nitrosylation of the catalytic Cys residue and reversible inactivation of PTPs.

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Journal of Molecular Biology

Crystallization NotePurification and Crystallization of the Catalytic Domain of Human Protein Tyrosine Phosphatase 1B Expressed in Escherichia coli

Abstract

Crystallization Note
Purification and Crystallization of the Catalytic Domain of Human Protein Tyrosine Phosphatase 1B Expressed in Escherichia coli