PDBsum entry 3eu0

Hydrolase

PDB id

3eu0

Contents

			Protein chain

					281 a.a.

			Waters ×63

References listed in PDB file

Key reference

Title

Cysteine s-Nitrosylation protects protein-Tyrosine phosphatase 1b against oxidation-Induced permanent inactivation.

Authors

Y.Y.Chen, H.M.Chu, K.T.Pan, C.H.Teng, D.L.Wang, A.H.Wang, K.H.Khoo, T.C.Meng.

Ref.

J Biol Chem, 2008, 283, 35265-35272. [DOI no: 10.1074/jbc.M805287200]

PubMed id

18840608

Abstract

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 A 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 H(2)O(2)-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.



	Figure 1. Application of the quantitative MS method and structural analysis for identification of the most susceptible Cys residue of PTP1B to S-nitrosylation. A-D, recombinant PTP1B treated with 1 mm SNAP or 0.01 mm SNAP was subjected to differential isotope labeling for quantitative MALDI-MS analysis as described in Scheme 1. The full scan MALDI-MS profile (A) revealed three pairs of cICAT-labeled tryptic peptides with a 9-Da difference, which could be assigned to T4, T28, and T15, as shown in expanded views (B-D), corresponding to the cICAT-labeled peptide pairs containing Cys-32, Cys-215, or Cys-92, respectively. The ratio of light/heavyc ICAT-labeled peak is shown below the spectrum. E, the crystal of PTP1B was soaked with 1 mm SNAP at room temperature for 20 min and subjected tox-ray crystallography. The 2F[o] - 2F[c] electron density map showed a mixture of reduced and S-nitrosylated states of Cys-215. Other Cys residues (Cys-32, -92, -121, -226, and -231) remained in the completely reduced form. Inset, the expended view of electron density map illustrates the presence of an S-nitrosothiol form of Cys-215.		Figure 2. Protective effect of Cys S-nitrosylation on preventing PTP1B from further irreversible oxidation. A, recombinant PTP1B was pretreated with 1 mm N-acetylpencillamine or SNAP for 20 min, followed by 1 mm H[2]O[2] for 10 min, and then digested by trypsin in solution. The tryptic peptides were subjected to LC-nESI-MS analysis. T28 carrying the Cys-215 in Cys-SH, Cys-SO[2]H, Cys-SO[3]H, and Cys-SNO forms were first identified by manually examining the nESI-MS profile at the expected retention time. For a semiquantitative assessment, the extracted ion chromatograms for the respective signals are plotted in A without normalization or correcting for nESI-MS response factor. The amount of the irreversibly oxidized SO[2]H/SO[3]H form (eluting at 17.7-18.5 min) elicited by 1 mm H[2]O[2] is clearly reduced to basal level in SNAP-pretreated sample, compared with N-acetylpencillamine-pretreated sample. The corresponding nESI-MS profiles for this time point are shown in B, where the signals identified as the irreversibly oxidized T28 (m/z 736.4 and 741.7) are detectable at increasing intensity when PTP1B was treated with increasing concentration of H[2]O[2] but not when it was first S-nitrosylated by SNAP. C, recombinant PTP1B, either directly exposed to H[2]O[2], SNAP, or GSNO or pretreated with SNAP or GSNO followed by H[2]O[2] treatment, was subjected to immunoblotting with an anti-oxidized PTP active site (anti-oxi-PTP) antibody (top) or an anti-PTP1B antibody (FG6) (bottom). The effect of SNAP or GSNO on inhibiting the level of H[2]O[2]-induced irreversible oxidation of PTP1B was observed in three independent experiments.

PROCHECK

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