Solution oxygen-17 NMR application for observing a peroxidized cysteine residue in oxidized human SOD1

Transcription

1 Hyperfine Interact (2016) 237:114 DOI /s Solution oxygen-17 NMR application for observing a peroxidized cysteine residue in oxidized human SOD1 Noriko Fujiwara 1 Daisaku Yoshihara 1 Haruhiko Sakiyama 1 Hironobu Eguchi 1 Keiichiro Suzuki 1 Springer International Publishing Switzerland 2016 Abstract NMR active nuclei, 1 H, 13 Cand 15 N, are usually used for determination of protein structure. However, solution 17 O-NMR application to proteins is extremely limited although oxygen is an essential element in biomolecules. Proteins are oxidized through cysteine residues by two types of oxidation. One is reversible oxidation such as disulphide bonding (Cys-S-S-Cys) and the other is irreversible oxidation to cysteine sulfinic acid (Cys- SO 2 H) and cysteine sulfonic acid (Cys-SO 3 H). Copper,Zinc-superoxide dismutase (SOD1) is a key enzyme in the protection of cells from the superoxide anion radical. The SH group at Cys 111 residue in human SOD1 is selectively oxidized to -SO 2 Hand-SO 3 H with atmospheric oxygen, and this oxidized human SOD1 is also suggested to play an important role in the pathophysiology of various neurodegenerative diseases, probably mainly via protein aggregation. Therefore, information on the structural and the dynamics of the oxidized cysteine residue would be crucial for the understanding of protein aggregation mechanism. Although the -SO 3 H group on proteins cannot be directly detected by conventional NMR techniques, we successfully performed the site-specific 17 O-labeling of Cys 111 in SOD1 using 17 O 2 gas and the 17 O-NMR analysis for the first time. We observed clear 17 O signal derived from a protein molecule and show that 17 O-NMR is a sensitive probe for studying the structure and dynamics of the 17 O-labeled protein molecule. This novel and unique strategy can have great impact on many research fields in biology and chemistry. Keywords 17 O-NMR oxygen-17 SOD1 Superoxide dismutase Oxo-acid This article is part of the Topical Collection on Proceedings of the International Conference on Hyperfine Interactions and their Applications (HYPERFINE 2016), Leuven, Belgium, 3-8 July 2016 Noriko Fujiwara noriko-f@hyo-med.ac.jp 1 Department of Biochemistry, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo , Japan

2 114 Page 2 of 6 Hyperfine Interact (2016) 237:114 1 Introduction The thiol group of cysteine (Cys) residues in proteins is susceptible to reactive oxygen species (ROS). Upon oxidation by ROS, the thiol group can form a disulfide (S-S) with a surrounding thiol group or thiolate anion. Formation of oxo-acid derivatives involving sulfenic acid (-SOH), sulfinic acid (-SO 2 H) and sulfonic acid (-SO 3 H) is another important oxidation reaction. These reactions often trigger a conformational change in the protein, with resultant loss of function, degradation and aggregation. Cu,Zn-superoxide dismutase (SOD1) is a key enzyme for protecting cells against the highly reactive superoxide anion radical by converting it to hydrogen peroxide. SOD1 is a homodimer containing one copper ion and one zinc ion in each 16-kDa subunit (Fig. 1). Mice lacking SOD1 exhibit various disorders, such as fatty liver [1], liver fibrosis [2], hemolytic anaemia [3] and renal iron accumulation [4]. On the other hand, mutations in the SOD1 gene have been found from patients of familial amyotrophic lateral sclerosis (FALS). We previously reported that the free -SH group on Cys 111 of human SOD1 (a) is selectively oxidized to -SO 2 H(b) and-so 3 H(c) under atmospheric oxygen (Fig. 1) [5]. Interestingly, only a Cys 111 at one of the subunits is asymmetrically air oxidized [5 7]. The oxidation of Cys 111 enhances aggregation and resulting cytotoxicity [8, 9]. Immunohistochemical analysis using a specific antibody against Cys 111 -SO 3 H of human SOD1 indicated clear staining of Lewy-body-like hyaline inclusions and vacuole rims in the spinal cord of FALS model mice [5]. In addition, the survival rate and the disease duration of the double mutant mice harbouring His46Arg and Cys111Ser, but not the other double mutant mice harbouring His46Arg and overexpression of thioredoxin, were significantly longer than the original ALS mutant mice harbouring His46Arg [10]. These results indicate that oxo-acid derivatives involving sulfenic acid (-SOH), sulfinic acid (-SO 2 H) and sulfonic acid (-SO 3 H) at Cys 111 are involved in the pathophysiology of ALS. These produced oxo-acids at Cys residues could change the local hydrogen-bond environment and electrostatic polarities and might induce structural and dynamic alterations in the proteins. Therefore, a method for analysing the chemical environment of R-SO 2 H/R- SO 3 H forms of Cys residues will help understand the role of oxidized proteins in such conformational changes and associated disease processes. For the investigation of oxidized Cys residues, 13 C-NMR has been used in combination with mass spectrometry [11]. C β chemical shifts of Cys residues reflect the oxidative state of the thiol group. On the other hand, 17 O-NMR is a direct method for detecting not only sulfoxide (S=O) groups but also other oxygen-containing functionalities like hydroxyl (-OH), ether (-O-), carbonyl (C = O) and phosphate (P = O) [12]. Therefore, wide application of 17 O-NMR in organic chemistry for identification of structure and reaction mechanism has been developed based on the wide chemical-shift range and distinguishable oxidation states [12]. However, its use in biological applications for protein structural analysis has been hampered in spite that the oxygen is an important conponent of proteins. Since the natural abundance of NMR active 17 O is only %, stable-isotope labeling using a 17 O-donatable species is needed to get a clear signal. Furthermore, because 17 O has a quadrupole moment (I = 5 / 2 ), 17 O- NMR signal is broader than those obtained by 1 H- and 13 C-NMR. Therefore, 17 O-NMR has seldomly been applied in the studies of biological samples compared with other nuclei NMR. In particular, solution 17 O-NMR measurements have only been performed to study the protein-ligand interactions with 17 O-labeled low-molecular-weight ligands. The examples are limited to 17 O-labeled carbon monoxide binding to heme proteins and 17 O-labeled

3 Hyperfine Interact (2016) 237:114 Page 3 of Fig. 1 Structure of the human SOD1 dimer (pdb: 3T5W). 17 O 2 -oxidized SOD1 contains three forms as in a, b, andc palmitic acid, oxalate and biotin binding to model proteins [13 17]. Because the chemical shifts and line-widths of the R-SO n H signal could be good reporters of the surrounding environment, the direct observation technique by solution 17 O-NMR also has the potential to analyse the structural property of the R-SO n H group. However, there had been no report to directly observe 17 O-NMR signals derived from 17 O-labeled proteins. We successfully labelled Cys 111 -SH of human SOD1 by oxidation with 17 O 2 -gas and studied the 17 O-labeled Cys 111 -SO 3 H using solution 17 O-NMR [18]. 2 Experiment Reduced wild type (Cys 111 -SH form) recombinant human SOD1 (residues 1 153) was oxidized with 17 O 2 gas (70 % isotope enrichment, ISOTEC) for 24 h at room temperature [18]. The color of SOD1 solution changed to blue (Fig. 2a). Peroxidation of Cys 111 -SH to Cys SO 2 H and Cys 111 -SO 3 H in SOD1 was confirmed by SDS-PAGE. As shown in Fig. 2b, the upper-shifted bands were observed in SOD1 oxidized with 17 O 2 gasaswellasair( 16 O 2 ) oxidized SOD1. The upper-shifted bands indicate oxidized subunit of SOD1 [5]. The 17 O- labeled position and the labeling ratio at Cys 111 of SOD1 were also confirmed by using HPLC peptide mapping according to previously reported protocols [5, 19] combined with ESI-TOF-MS analysis. The oxidation state of 17 O-SOD1 was confirmed that the molar ratio of Cys 111 -SH: Cys 111 -SO 2 H: Cys 111 -SO 3 H = 63:16:21 [18] based on the HPLC profile of the peptides was identical with the previously reported pattern [5]. The standard 17 O-chemical shifts of R-SO 3 HandR-SO 2 H were confirmed by using N-acetylated cysteine sulfonic acid (AcNH-Cys-SO 3 H) and N-acetylated cysteine sulfinic acid (AcNH-Cys-SO 2 H) before performing solution 17 O-NMR on the 17 O-SOD1. As shown in Fig. 3a, 17 O-NMR signal of AcNH-Cys-SO 3 H was observed at the position of 171 ppm (line-widths; 720 Hz). In contrast, AcNH-Cys-SO 2 H provided a broader signal at 141 ppm (line-widths; 1,890 Hz) (Fig. 3b), which would be due to the unsymmetrical oxygen nucleus. These results indicate that the solution 17 O-NMR technique clearly distinguishes between Cys-SO 2 HandCys-SO 3 H in terms of chemical shifts and line-widths.

4 114 Page 4 of 6 Hyperfine Interact (2016) 237:114 Fig. 2 Oxidation of human SOD1 by 17 O 2 gas. a; The color change of SOD1 solution before and after 17 O 2 -oxidation. b; SDS-PAGE of human SOD1 before and after oxidation Fig O-NMR spectra of N-acetylated cysteine sulfonic acid (a; 1.4 M) and N-acetylated cysteine sulfinic acid (b; 1.0 M). The spectra are measured in 10 mm phosphate buffer (ph 5.0) at 20 C Then, observation of Cys 111 -SO 3 H/-SO 2 H signals in 17 O 2 -oxidized SOD1 was performed by using solution 17 O-NMR. One clear 17 O-NMR spectrum of 17 O 2 -oxidized SOD1 with the Cu 2+ -form was observed at 165 ppm (line-width; 470 Hz) (Fig. 4a). To examine a paramagnetic Cu 2+ effect, when Cu 2+ at the catalytic centre of SOD1 was reduced with isoascorbic acid, a sharper 17 O-NMR signal was obtained at 164 ppm (line-width; 130 Hz) (Fig. 4b). Reduction with 2-mercaptoethanol also provided the Cu + -form, giving an identical signal (data not shown). In addition, SOD1 peptide mixture produced by treatment with lysylendopeptidase, which is including the peptide (residues ) containing Cys SO 2 H/Cys 111 -SO 3 H, gave a sharp peak at the position of 171 ppm with a line-width of 250 Hz, which is analogous to the signal obtained with AcNH-Cys-SO 3 H(Fig.4c). Since the peptide mixture does not contain metal ions, one can exclude the possibility of the Cu 2+

5 Hyperfine Interact (2016) 237:114 Page 5 of Fig O-NMR spectra of 17 O 2 -oxidized SOD1. All spectra were measured in 10 mm phosphate buffer (ph 5.0) at 20 C; a original Cu 2+ -form, b Cu + -form prepared by addition of 10 mm isoascorbic acid. c SOD1 peptide mixture produced by treatment with lysylendopeptidase paramagnetic effect. These results indicate that only Cys 111 -SO 3 H form but not Cys SO 2 H form in oxidized SOD1 is successfully detected by solution 17 O-NMR although the 17 O 2 -oxidized SOD1 included both Cys 111 -SO 2 H and Cys 111 -SO 3 H forms in a ratio of 16:21. The selective observation of Cys 111 -SO 3 H signal can likely be attributed to an inherent property associated with the symmetrical oxygen nucleus. The signal-broadening effect observedincu 2+ -form (Fig. 4a) is probably caused partly by a Cu 2+ paramagnetic effect. The slight shift to the right by 1 ppm of the Cys 111 -SO 3 H signal from Cu + -SOD1 may indicate the subtle structural or environmental difference between Cu 2+ -SOD1 and Cu + -SOD1. The distance between the Cu cation at the active site and Cys 111 is about 17.8 Å based on a previously reported SOD1 structure (PDB ID: 3T5W; Fig. 1) [7]. Although the distance of the Cu 2+ is rather far from 1 H (usually sensitive up to 11 Å) and 13 C(upto6Å) nuclei [20], the Cu cation may still affect 17 O-nuclei at Cys Conclusion and outlook We have reported the first example of solution 17 O-NMR for Cys 111 -SO 3 H/SO 2 Hon human SOD1. The distinct chemical shifts and line-widths of Cys 111 -SO 3 H on SOD1 were observed in the Cu 2+ -andcu + - forms as well as peptide mixture, which strongly suggest environmental, structural and dynamic differences between Cu 2+ -form and Cu + -form of oxidized SOD1. Although oxygen atoms are often included in the functional groups involved in the biological activity of a protein, 17 O-NMR has found limited application thus far. Our work suggests that measurements observing 17 O-labelled amino acids, such as Cys and Met, by 17 O-NMR could be very useful for investigating protein structure, dynamics, and functional modulation in combination with conventional 1 H-, 13 C- and 15 N-NMR. The progress in solution 17 O-NMR when combined with an efficient 17 O-labeling strategy will break the status quo and allow for the direct analysis of biological molecules.

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