Growth inhibitory factor/metallothionein-3 is a sulfane sulfur-binding protein
- Environmental Biology Laboratory, School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Japan
- Environmental Biology Laboratory, Faculty of Medicine, University of Tsukuba, Japan
- Doctoral Program in Biomedical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Japan
- Department of Chemistry, Graduate School of Pure and Applied Sciences, University of Tsukuba, Japan
- Centre for Biological Sciences, University of Southampton, Highfield Campus, United Kingdom
- Department of Plant Pathology and Microbiology, National Taiwan University, Taiwan
- Graduate School of Pharmaceutical Sciences, Kyushu University, Japan
- National Institute for Physiological Sciences & Exploratory Research Center on Life and Living Systems, National Institutes of Natural Sciences, Japan
- Department of Environmental Medicine and Molecular Toxicology, Tohoku University Graduate School of Medicine, Japan
- School of Chemistry and the Institute for Life Sciences, University of Southampton, Highfield Campus, United Kingdom
- Department of Chemistry, Johns Hopkins University, United States
- Center for Computational Sciences, University of Tsukuba, Japan
Figures
Figure 1
Detection of sulfane sulfur in GIF/MT-3 by MALDI–TOF/MS.
(A) Preparation of recombinant Zn7GIF/MT-3 and oxidized apo-GIF/MT-3 proteins. Recombinant human Zn7GIF/MT-3 (10 μM) was incubated in HCl (0.1 N) at 37°C for 30 min and then replaced with 20 mM Tris–HCl (pH 7.5) buffer and incubated for 36 hr at 37°C. After removal of low-molecular-weight molecules using 3 kDa centrifugal filtration, GIF/MT-3-bound zinc content was measured using ICP-MS. Each value represents the mean ± SD of three independent experiments. (B) FT–ICR–MALDI–TOF/MS spectrum (positive-ion mode) of Zn7GIF/MT-3. (C) FT–ICR–MALDI–TOF/MS spectrum (positive-ion mode) of oxidized apo-GIF/MT-3. (D) Putative oxidation reaction scheme in apo-GIF/MT-3 protein. FT-ICR, Fourier transform ion cyclotron resonance.
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Figure 1—source data 1
Source data for panel A: Zinc content measured in GIF/MT-3 samples.
- https://cdn.elifesciences.org/articles/92120/elife-92120-fig1-data1-v1.xlsx
Figure 2 with 1 supplement
Detection of sulfane sulfur in GIF/MT-3 by Raman spectroscopy.
(A) Raman spectra of Zn7GIF/MT-3, oxidized apo-GIF/MT-3, and HPE-IAM-treated GIF/MT-3 in the 250–800 cm−1 region. Optimized geometries for (B) α-domain and (C) β-domain models of apo-GIF/MT-3 (assuming some cysteines with persulfide and tetrasulfide bonds as shown). Optimized geometries for (D) α-domain and (E) β-domain models of apo-GIF/MT-3 (assuming some cysteines with disulfide bonds). (F) Calculated Raman spectra of apo-GIF/MT-3 models with/without sulfane sulfurs.
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Figure 2—source data 1
Peak assignments for apo-GIF/MT-3 model structures.
- https://cdn.elifesciences.org/articles/92120/elife-92120-fig2-data1-v1.docx
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Figure 2—source data 2
Peak assignments for Zn7S20GIF/MT-3 and Zn7GIF/MT-3 model structures.
- https://cdn.elifesciences.org/articles/92120/elife-92120-fig2-data2-v1.docx
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Figure 2—source data 3
Raw Raman spectroscopy data for panel A , and calculated Raman shifts and peak intensities for panel F .
- https://cdn.elifesciences.org/articles/92120/elife-92120-fig2-data3-v1.xlsx
Figure 2—figure supplement 1
Zn-binding GIF/MT-3 models and calculated Raman spectra.
(A) α-domain and (B) β-domain models of Zn7S20GIF/MT3 (assuming all cysteines are persulfides). (C) α-domain and (D) β-domain models of Zn7GIF/MT3 (assuming all cysteines are thiols). (E) Calculated Raman spectra for Zn7GIF/MT3 with or without sulfane sulfurs.
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Figure 2—figure supplement 1—source data 1
Calculated Raman spectroscopy data showing Raman shifts and peak intensities.
- https://cdn.elifesciences.org/articles/92120/elife-92120-fig2-figsupp1-data1-v1.xlsx
Figure 3 with 2 supplements
Sulfane sulfur assay optimization and quantification of MT sulfane sulfur content.
(A) Schematic showing the detection of sulfane sulfur in Zn7GIF/MT-3. (B) Sulfane sulfur detected in Zn7GIF/MT-3 after incubation with the indicated concentrations of HPE-IAM at 60°C for 36 hr in 20 mM Tris-HCl (pH 7.5). (C) Sulfane sulfur detected in Zn7GIF/MT-3 after incubation with 5 mM HPE-IAM at 60°C for 36 hr in 20 mM Tris-HCl (pH 7.5). (D) Sulfane sulfur detected in Zn7GIF/MT-3 after incubation with 5 mM HPE-IAM at 37°C or 60°C for the indicated times in 20 mM Tris-HCl (pH 7.5). (E) Sulfane sulfur detected in human Zn7MT-1, Zn7MT-2, Zn7GIF/MT-3, wild-type (WT) Zn7GIF/MT-3, and apo-GIF/MT-3 with all Cys residues mutated to Ala (all C/A), each incubated with 5 mM HPE-IAM at 60°C for 36 hr in 20 mM Tris-HCl (pH 7.5). Sulfane sulfur content was measured using LC-MS/MS. HPE-IAM, β-(4-hydroxyphenyl)ethyl iodoacetamide. Each value represents the mean ± SD of three independent experiments.
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Figure 3—source data 1
Source data for panels B-F: Protein-bound sulfane sulfur content measured in GIF/MT-3 samples.
- https://cdn.elifesciences.org/articles/92120/elife-92120-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
Amino acid sequences of human MTs and GIF/MT-3 mutant.
Figure 3—figure supplement 2
Determination of sulfane sulfur and zinc content in wild-type and several mutant proteins.
(A) Sulfane sulfur detected in chemically synthesized α- and β-domains of GIF/MT-3. (B) Sulfane sulfur (left) and zinc (right) detected in recombinant GIF/MT-3 wild-type and mutant proteins. Sulfane sulfur was quantified using LC–MS after incubation with HPE-IAM (5 mM) at 60°C for 36 h. GIF/MT3-bound zinc content was determined using ICP-MS. Each value represents the mean ± SD of three independent experiments. The amino acid sequences of GIF/MT-3 mutant proteins are shown in Figure 3—figure supplement 1.
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Figure 3—figure supplement 2—source data 1
Protein-bound sulfane sulfur and zinc content measured in GIF/MT-3 samples.
- https://cdn.elifesciences.org/articles/92120/elife-92120-fig3-figsupp2-data1-v1.xlsx
Figure 4 with 1 supplement
Sulfane sulfur stability in apo-GIF/MT-3 and its restoration by a reducing agent.
(A) Stability of sulfane sulfur in apo-GIF/MT-3 incubated with or without (Cont) zinc. To prepare apo-GIF/MT-3, Zn7GIF/MT-3 was incubated in 0.1 M HCl for 30 min, then the buffer was replaced with 20 mM Tris–HCl (pH 7.5). To examine the stability of sulfane sulfur in apo-GIF/MT-3, freshly prepared apo-GIF/MT-3 (2 µM) with or without added zinc ions was incubated at 37°C for up to 24 hr. (B) Effect of tris(2-carboxyethyl)phosphine (TCEP) on sulfane sulfur binding and free SH/SSH groups in oxidized apo-GIF/MT-3. To prepare oxidized apo-GIF/MT, Zn7GIF/MT-3 was incubated in HCl (0.1 N) at 37°C for 30 min and then replaced with 20 mM Tris–HCl (pH 7.5) buffer and incubated for 36 hr at 37°C. The resulting oxidized apo-GIF/MT-3 protein (10 µM) was incubated with 0, 1, 10, 50, or 100 mM TCEP in 20 mM Tris–HCl (pH 7.5) at 37°C for 1 hr, then low-molecular-weight molecules were removed by 3 kDa ultrafiltration for six times. Sulfane sulfur content was determined using LC–ESI–MS/MS, and the concentrations of free SH/SSH groups were measured using Ellman’s reagent. Each value represents the mean ± SD of three independent experiments.
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Figure 4—source data 1
Source data for panels A and B: Protein-bound sulfane sulfur and free SH/SSH content measured in GIF/MT-3 samples.
- https://cdn.elifesciences.org/articles/92120/elife-92120-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
A putative reaction scheme for DTNB with RSSH.
Figure 5
Reactivity of HPE-IAM with tetrasulfide derivatives as models of tetrasulfide bridges in apo-GIF/MT-3.
(A) Reactivity of HPE-IAM with N-acetylcysteine (NAC) derivatives. Oxidized NAC (oxiNAC), NAC-trisulfide (NAC-S1), and NAC-tetrasulfide (NAC-S2) (each 10 µM) were incubated with HPE-IAM (5 mM) at 60°C for 1 or 36 hr with or without TCEP (1 mM) in 100 mM Tris-HCl (pH 7.5). (B) Reactivity of HPE-IAM with diallyl polysulfide derivatives. Diallyl disulfide (DADS), diallyl trisulfide (DATS), or diallyl tetrasulfide (DATetraS) (each 10 µM) was incubated with HPE-IAM (5 mM) at 60°C for 1 or 36 hr with or without TCEP (1 mM) in 100 mM Tris-HCl (pH 7.5). (C) Scheme showing possible reactions of tetrasulfide derivatives with HPE-IAM and TCEP. Bis-S-HPE-AM, bis-S-β-(4-hydroxyphenyl)ethyl acetamide. Each value represents the mean ± SD of three independent experiments.
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Figure 5—source data 1
Source data for panels A and B: Measured Bis-S-HPE-AM concentrations in polysulfide derivatives after HPE-AM incubation.
- https://cdn.elifesciences.org/articles/92120/elife-92120-fig5-data1-v1.xlsx
Figure 6
Redox-dependent release of zinc ions and recycling of sulfane sulfur in GIF/MT-3.
(A) Quantitation of zinc ions released from Zn7GIF/MT-3 by H2O2 and S-nitroso-N-acetylpenicillamine (SNAP). To examine the release of zinc ions by H2O2 and SNAP, Zn7GIF/MT-3 (10 µM) was treated with H2O2 (1 or 5 mM) or SNAP (1 or 5 mM) in 100 mM Tris–HCl (pH 7.5) at 25°C for 30 min. After removing H2O2/SNAP using 3 kDa ultrafiltration four times, free SH/SSH groups and sulfane sulfur content in GIF/MT-3 were determined. (B) Free SH/SSH content in Zn7GIF/MT-3, determined by H2O2 or SNAP treatment after incubation with TCEP. To examine the interaction of Zn7GIF/MT-3 with H2O2 or NO, Zn7GIF/MT-3 (10 µM) was incubated with H2O2 (1 or 5 mM) or SNAP (1 or 5 mM) in 100 mM Tris–HCl (pH 7.5) at 25°C for 30 min. After removing H2O2/SNAP using 3 kDa ultrafiltration four times, the resulting proteins (5 µM) were incubated with TCEP (50 mM) in 100 mM Tris–HCl (pH 7.5) at 37°C for 1 hr. After removing TCEP using 3 kDa ultrafiltration for five times, sulfane sulfur content was determined using LC–ESI–MS/MS and the concentrations of free SH/SSH groups were measured using Ellman’s reagent. (C) Proposed reactions between a zinc/persulfide cluster in GIF/MT-3 and H2O2 or NO. Each value represents the mean ± SD of three independent experiments.
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Figure 6—source data 1
Measured protein-bound zinc, sulfane sulfur, and free SH/SSH in GIF/MT-3 samples.
- https://cdn.elifesciences.org/articles/92120/elife-92120-fig6-data1-v1.xlsx
Figure 7
Contribution of sulfane sulfur in GIF/MT-3 to zinc binding.
(A) To eliminate sulfane sulfur in Zn7GIF/MT-3 by cyanolysis, Zn7GIF/MT-3 (10 µM) was reacted with KCN (75 mM) in 100 mM Tris–HCl (pH 7.5) at 37°C for 14 hr. After removal of KCN, the resulting protein was incubated with TCEP (10 mM) in 100 mM Tris–HCl (pH 7.5) at 37°C for 1 hr. After removal of TCEP, sulfane sulfur content in GIF/MT-3 was determined using LC–ESI–MS/MS. (B) Comparison of zinc-binding capacity of GIF/MT-3 before and after cyanolysis. Zn7GIF/MT-3 (10 µM) was incubated with KCN (75 mM) in 100 mM Tris–HCl (pH 7.5) at 37°C for 14 hr. After removal of KCN, the resulting protein was incubated with TCEP (10 mM) in 100 mM Tris–HCl (pH 7.5) at 37°C for 1 hr. After removal of TCEP, the resulting protein (5 µM) was incubated with zinc chloride (50 µM) in 50 mM Tris–HCl (pH 7.5) at 37°C for 1 hr. Low-molecular-weight molecules were removed using 3 kDa ultrafiltration after each step. Protein-bound zinc content was determined using ICP–MS. *p<0.05 and **p<0.01. Each value represents the mean ± SD of three independent experiments.
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Figure 7—source data 1
Protein-bound sulfane sulfur and zinc content measured in GIF/MT-3 samples.
- https://cdn.elifesciences.org/articles/92120/elife-92120-fig7-data1-v1.xlsx
Figure 8
Reduction of apo-GIF/MT-3 by thioredoxin (Trx) and subsequent regeneration of sulfane sulfur.
(A) Velocity (V) of Trx-catalyzed reduction of oxidized apo-GIF/MT-3 substrate (S). Oxidation of NADPH was followed by measuring the absorbance of NADPH at 340 nm. (B) Comparison of substrate reduction by NADPH and the Trx system. (C) NADP+ formation upon incubation of: oxidized apo-GIF/MT-3 with Trx/TR, TRP14/TR, or TRP32/TR; and Zn7GIF/MT-3 with Trx/TR. (D) Regeneration of sulfane sulfur in oxidized apo-GIF/MT-3 after incubation with the Trx/TR system. TR, Trx reductase; TRP14, Trx-related protein 14; TRP32, Trx-related protein 32. Representative data are shown. Similar results were obtained in at least two independent experiments. For panel D, each value represents the mean ± SD of three independent experiments.
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Figure 8—source data 1
NADPH oxidation over time and protein-bound sulfane sulfur at endpoint during incubation of GIF/MT-3 with the Trx/TR system.
- https://cdn.elifesciences.org/articles/92120/elife-92120-fig8-data1-v1.xlsx
Figure 9 with 4 supplements
Structural modeling of sulfane sulfur in GIF/MT-3 using MOE, and a reaction scheme for sulfane sulfur-based zinc/persulfide cluster.
(A) Comparison of three-dimensional structures of Zn7GIF/MT-3 (pink) and Zn7S20GIF/MT-3 (green). (B) Cyclohexane-like Zn3Cys9 cluster in the GIF/MT-3 homology model, and bicyclononane-like Zn4Cys11 cluster derived from PDB structure 2F5H with (lower) or without (upper) sulfane sulfur. Yellow, orange, and gray spheres indicate cysteine residues, sulfane sulfur, and zinc ions, respectively. (C) Thermostability and zinc-binding affinity scores of GIF/MT-3 with different numbers of sulfane sulfurs at each cysteine residue. (D) A proposed model for redox-dependent hold-and-release regulation of zinc ions by GIF/MT-3.
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Figure 9—source data 1
Thermostability score of sulfane sulfur-bound MT isoforms with or without Zn.
- https://cdn.elifesciences.org/articles/92120/elife-92120-fig9-data1-v1.docx
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Figure 9—source data 2
Source data for panel C: Thermostability and zinc binding affinity scores of GIF/MT-3 variants with varying sulfane sulfur counts at each cysteine.
- https://cdn.elifesciences.org/articles/92120/elife-92120-fig9-data2-v1.xlsx
Figure 9—figure supplement 1
Homology modeling of GIF/MT-3.
(A) Sequences of GIF/MT3 and templates. (B) 3D structure of GIF/MT3 and the template structures with PDB codes 4MT2 and 2F5H.
Figure 9—figure supplement 2
3D structural models of human Zn7MT-1A and Zn7MT-2.
(A) Sequences of MT1A and MT2 and the PDB structures with accession numbers 4MT2 and 1MHU. (B) Homology model of Zn7MT1A based on template PDB 4MT2. (C, D) Model 3D structures of Zn7S20MT1A. (E) Homology model of Zn7MT2 based on templates 4MT2 and 1MHU. (F, G) Model 3D structures of Zn7S20MT2.
Figure 9—figure supplement 3
Schematic structures of Zn7GIF/MT-3 (A) and Zn7S20GIF/MT-3 (B).
Figure 9—figure supplement 4
Zn3Cys9 and Zn4Cys11 clusters containing the polysulfide form of sulfane sulfur.
(A) Zn3Cys9 cluster with 10 sulfane sulfurs (5 RSSSH), taken from the homology model of MT-3. (B) Zn4Cys11 cluster with 10 sulfane sulfurs (5 RSSSH), taken from PDB structure 2F5H. Structures were rendered for display using Molecular Operating Environment software. Yellow, orange, and gray spheres indicate cysteine residues, sulfane sulfur atoms, and zinc ions, respectively.
Figure 10
Separation of sulfane sulfur-binding proteins from mouse brain cytosol using column chromatography.
(A) Diethylaminoethyl Sepharose CL-6B column. (B) Sephacryl S-100 column. (C) Blue Sepharose column. Triangles, closed circles, and dotted lines indicate sulfane sulfur, protein, and NaCl concentrations, respectively. Portions of each fraction were incubated with 5 mM of HPE-IAM in 20 mM Tris (pH 7.5) at 37°C for 30 min and the sulfane sulfur content was determined from the bis-S-HPE-AM adduct concentration measured using LC–MS/MS. Protein concentration was determined using the bicinchoninic acid assay. Isolation of sulfane sulfur-binding protein was performed as described in the Experimental procedures.
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Figure 10—source data 1
Fragment sequences of a mouse brain sulfane sulfur-binding protein, determined using nano-UPLC–MS.
- https://cdn.elifesciences.org/articles/92120/elife-92120-fig10-data1-v1.docx
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Figure 10—source data 2
Source data for panels A-C: Protein-bound sulfane sulfur and protein concentrations measured in each fraction.
- https://cdn.elifesciences.org/articles/92120/elife-92120-fig10-data2-v1.xlsx
Tables
Table 1
Thermostability and metal-binding affinity scores of growth inhibitory factor (GIF)/metallothionein-3 (MT-3) with or without sulfane sulfur.
Values were calculated using the Protein Design module of the Molecular Operating Environment (MOE) software.
| Metal | Sulfane sulfur | Affinity (kcal/mol) | Stability (kcal/mol) |
|---|---|---|---|
| Zn7 | 0 | 11 | –302 |
| 20 | –154 | –407 | |
| Cd7 | 0 | 12 | –282 |
| 20 | –82 | –344 | |
| Hg7 | 0 | 13 | –276 |
| 20 | –112 | –354 |
Additional files
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Supplementary file 1
Pdb file of Zn7S20GIF/MT-3 generated by homology modeling.
- https://cdn.elifesciences.org/articles/92120/elife-92120-supp1-v1.zip
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MDAR checklist
- https://cdn.elifesciences.org/articles/92120/elife-92120-mdarchecklist1-v1.docx
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Growth inhibitory factor/metallothionein-3 is a sulfane sulfur-binding protein
eLife 12:RP92120.
https://doi.org/10.7554/eLife.92120.4
