Pentenediol-Type Compounds Specifically Bind to Voltage

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Pentenediol-type compounds specifically bind to voltage-dependent anion channel 1 in Saccharomyces cerevisiae mitochondria Yufu Unten, Masatoshi Murai, Takenori Yamamoto, Akira Watanabe, Naoya Ichimaru, Shunsuke Aburaya, Wataru Aoki, Yasuo Shinohara, and Hideto Miyoshi Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b01209 • Publication Date (Web): 18 Jan 2019 Downloaded from http://pubs.acs.org on January 21, 2019

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Biochemistry

Pentenediol-type compounds specifically bind to voltage-dependent anion channel 1 in Saccharomyces cerevisiae mitochondria

Yufu Unten1, Masatoshi Murai1, Takenori Yamamoto2, Akira Watanabe2, Naoya Ichimaru1, Shunsuke Aburaya1, Wataru Aoki1, Yasuo Shinohara2, and Hideto Miyoshi1* From 1Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502; 2Institute for Genome Research, University of Tokushima, Kuramotocho-3, Tokushima 770-8503, JAPAN

*To whom correspondence should be addressed: Hideto Miyoshi, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan, Tel: (+81)-75-7536119; E-mail: [email protected]

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ABBREVIATIONS ADP/ATP carrier, AAC; carboxyatractyloside, CATR; cytochrome c, cyt. c: fluorescein-5maleimide, 5-FM; 4,4’-diisothiocyano-2,2’-stilbenedisulfonic acid, DIDS; ligand-directed tosyl, LDT; N-ethylmaleimide, NEM; pentenediol, PTD; outer mitochondrial membrane, OMM; phosphate carrier, PiC; 6-carboxy-N,N,N’,N’-tetramethylrhodamine, TAMRA; type-2 NADH dehydrogenase, NDH-2; voltage-dependent anion channel 1, VDAC1.


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ABSTRACT Voltage-dependent anion channel 1 (VDAC1) situated in the outer mitochondrial membrane regulates the transfer of various metabolites and is a key player in mitochondria-mediated apoptosis. Although many small chemicals that modulate the functions of VDAC1 have been reported to date, most, if not all, of them cannot be regarded as specific reagents due to their interactions with other transporters or enzymes. By screening our chemical libraries using isolated Saccharomyces cerevisiae mitochondria, we found pentenediol (PTD)-type compounds (e.g. PTD-023) as new specific inhibitors of VDAC1. PTD-023 inhibited overall ADPuptake/ATP-release reactions in isolated mitochondria at a single digit µM level. To identify the binding position of PTDs in VDAC1 by visualizing PTD-bound peptides, we conducted liganddirected tosyl (LDT) chemistry using the synthetic LDT reagent t-PTD-023 derived from the parent PTD-023 in combination with mutagenesis experiments. t-PTD-023 made a covalent bond predominantly and subsidiarily with nucleophilic Cys210 and Cys130, respectively, indicating that PTDs bind to the region interactive with both residues. Site-directed mutations of hydrogen bond-acceptable Asp139 and Glu152 to Ala, which were selected as potential interactive partners of the critical pentenediol moiety based on the presumed binding model of PTDs in VDAC1, resulted in a decrease in susceptibility against PTD-023. This result strongly suggests that PTDs bind to VDAC1 through a specific hydrogen bond with the two residues. The present study is the first to demonstrate the binding position of specific inhibitors of VDAC1 at the amino acid level.


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INTRODUCTION Mitochondria are critical organelles within eukaryotic cells, which perform diverse cellular functions predominantly by producing ATP via oxidative phosphorylation. Mitochondrial enzymes and transporters presiding over ATP production are promising targets for the development of agrochemicals, anthelmintic reagents, and anticancer drugs. Many specific inhibitors of the mitochondrial respiratory complexes (complexes I−III) of harmful insects and fungi have been used in the agricultural field on a worldwide level (1-3). Ohmura and coworkers identified nafuredin from Aspergillus niger as a potent and specific inhibitor of helminth (Ascaris suum) respiratory complex I (4, 5). Voltage-dependent anion channel 1 (VDAC1) situated in the outer mitochondrial membrane (OMM) has been anticipated to be a druggable target for anticancer therapeutics (6, 7, 8). McLellan et al. recently identified a thiohydantoin-type compound (ML316) as the first specific inhibitor of fungal mitochondrial phosphate carrier, which may provide a new therapeutic strategy to address drug-resistant Candida species (9). Hartuti et al. demonstrated that the natural product ferulenol inhibits the growth of Plasmodium falciparum by inhibiting mitochondrial L-malate-quinone oxidoreductase, which is essential for parasite survival (such as Plasmodium apicomplexan parasites), but absent in mammalian hosts (10). Thus, the discovery of new inhibitors of the mitochondrial machineries presiding over ATP production may contribute greatly not only to the acquisition of seed compounds for the development of new reagents (drugs), but also to promising advances in basic biochemical research on these machineries. By employing assay that monitors overall ADP-uptake/ATP-release reactions using isolated Saccharomyces cerevisiae mitochondria respiring with α-ketoglutarate (11), we previously initiated the screening our own chemical libraries with the aim of identifying new low-molecularweight chemicals that specifically inhibit mitochondrial enzymes or transporters. In this assay, we detected ATP outside mitochondria, which was synthesized in the mitochondrial matrix from externally added ADP under energy-generating conditions and sequentially released outside of mitochondria, by coupling with the ATP-dependent formation of NADPH by hexokinase/glucose6-phosphate dehydrogenase sequential reactions. This assay system principally enables us to screen potential inhibitors of any of the mitochondrial machineries presiding over ATP production via oxidative phosphorylation, including respiratory enzymes (type-2 NADH dehydrogenase (NDH-2) and complexes II–IV), FoF1-ATP synthase (complex V), VDAC1, ADP/ATP carrier (AAC), and phosphate carrier (PiC). During the screening (though we have not yet disclosed the results of screening), we identified pentenediol (PTD)-type compounds (e.g. PTD-023 in Figure 1) as specific inhibitors of yeast mitochondrial VDAC1, as described hereafter in this manuscript. Since PTDs are not similar in structure to any small-molecule regulators/inhibitors of VDAC1 hitherto known, they may become potential seed compounds for the development of new reagents and, through exquisite chemical functionalization, useful molecular tools for biochemical research on VDAC1. VDAC1 situated in OMM serves as a mitochondrial gatekeeper, which allows the transfer of various substances across OMM (ranging from small Ca2+ to large cytochrome c) and is a key player in mitochondria4 ACS Paragon Plus Environment

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Biochemistry

mediated apoptosis (7, 12, 13). VDAC1 also functions as a hub protein interacting with various O O proteins from the cytosol and endoplasmic OH HO 3 reticulum, which together regulate metabolic PTD-023 pathways (7, 14 −16). Although the structural and functional features of VDAC1 have been OH extensively studied by means of different O O S techniques, the mechanisms underlying the HO 3 O O O various functions of VDAC1 remain elusive. t-PTD-023 In the present study, we characterized the N inhibitory action of PTDs in detail by taking PTD023 as the test compound, which was the most OOC O TAMRA-N3 potent inhibitor among the PTDs identified. H N Through ligand-directed tosyl (LDT) chemistry N3 N O (17, 18), which allows for the detection and capture of PTD-bound peptides, in combination Figure 1. Structures of test compounds and reagents used in this study: PTD-023, t-PTD-023, with proteomic and mutagenesis experiments, we and TAMRA-N3. found that PTDs bind in the proximity of Cys210 while maintaining an interactive distance to Cys130, both of which may be exposed to the cytosolic side. Based on this finding, we forecasted a binding model of PTDs in VDAC1 and selected hydrogen bond-acceptable Asp139 and Glu152 as potential interactive partners of the critical pentenediol moiety of PTDs. Using mutants of Asp139 and/or Glu152 to Ala, it was suggested that the pentenediol moiety binds to VDAC1 through a specific hydrogen bond with the two residues. To the best of our knowledge, the present study is the first to demonstrate the binding position of specific inhibitors of VDAC1 at the amino acid level. OH


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EXPERIMENTAL PROCEDURES Materials Carboxyatractoside (CATR) and fluorescein-5-maleimide (5-FM) were purchased from Sigma-Aldrich (St. Louis, MO) and Thermo Fisher Scientific (Waltham, MA), respectively. [14C]ADP was purchased from PerkinElmer (Waltham, MA). Protein standards (Precision Plus Protein Standards and Precision Plus Protein Dual Xtra Standards) for SDS-PAGE were from BioRad (Hercules, CA). The Click-iT reaction buffer kit and TAMRA-N3 (Figure 1), were purchased from Life Technologies (Carlsbad, CA). A monoclonal antibody for S. cerevisiae VDAC1 was purchased from MitoSciences (Eugene, OR). Other reagents were of analytical grade. Yeast culture and isolation of mitochondria The haploid strains of S. cerevisiae used in the present study were W303-1B (MATα ade2-1 leu2-3,112 his3- 22,15 trp1-1 ura3-1 can1-100) and Δpor1 (MATα ade2-1 leu2- 3,112 his3-22,15 trp1-1 ura3-1 can1-100 por1::HIS3) (19). Yeast cells were grown in 10 mL (5 mL × 2 test tubes) of semisynthetic lactate medium (2% lactic acid, 0.3% yeast extract, 0.05% glucose, 0.05% CaCl2, 0.05% NaCl, 0.06% MgCl2, 0.1% KH2PO4, and 0.1% NH4Cl) or YPGal medium (1% yeast extract, 2% peptone, and 2% galactose) at 28 °C for 36 h with rotation at 250 rpm. The preculture was used to inoculate 800 mL (400 mL × 2 flasks) of the same medium, and cells were grown at 120 rpm at 28 °C until A600 reached 1−3. Yeast mitochondria were isolated by digesting the cell wall with Zymolyase followed by homogenization and differential centrifugation, as described previously (19, 20). The final mitochondrial pellet was resuspended in buffer containing 0.60 M mannitol, 10 mM Tris/HCl (pH 7.4), 0.1 mM EDTA, 0.1% BSA, and protease inhibitor cocktail (Sigma-Aldrich). A total of 1−2 mg of mitochondria was typically obtained from 800 mL culture. Protein concentrations were determined using the BCA Protein Assay Kit (Thermo Fisher Scientic, Maltham, MA) with BSA as the standard. Preparation of expression plasmids for POR1 mutants pRS314 P1P (YCp type containing TRP1 and the promoter region of the POR1 gene), which was prepared in our previous work (19), was used as a vector for the transformation of POR1 mutants. The DNA fragment encoding POR1 was prepared by PCR using the primers GE2266 and GE2267. The nucleotide sequence of POR1 in the database was NM_001182894. The DNA fragments encoding POR1 single mutants (C130A, C210A, D139A, and E152A) were prepared using the overlap-extension PCR method with the mutagenic primers listed in Supplemental Table S1. The DNA fragments encoding the POR1 double mutants (C130A/C210A and D139A/E152A) were prepared using the DNA fragments of single mutants (C130A and D139A) as templates of overlap-extension PCR. Prepared DNA fragments were subcloned into the NdeI and BamHI sites of pRS314-P1P, and the sequences of DNAs encoding these mutants were confirmed. For the expression of these POR1 mutants, the expression plasmids prepared were introduced into the por1-disrupted yeast strain (MATα ade2-1 leu2-3,112 his3-22,15 trp1-1 ura3-1 can1-100 por1::HIS3) (21). All recombinant DNA experiments were 6 ACS Paragon Plus Environment

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Biochemistry

performed according to the guidelines of the University of Tokushima (approval number: 26-121, http://www.tokushima-u.ac.jp/legal/reiki_honbun/x383RG00000178.html). Measurement of ADP-uptake/ATP-release in isolated mitochondria The measurement of ADP-uptake/ATP-release in isolated mitochondria was conducted based on the previously described protocol (11). Freshly isolated mitochondria (50 µg of proteins/mL) were suspended in 2.5 mL of reaction buffer (0.60 M mannitol, 0.10 mM EGTA, 2.0 mM MgCl2, 10 mM KPi, 5.0 mM α-ketoglutarate, and 10 mM Tris-HCl, pH 7.4) at 30 ˚C in the presence of an ATP-detecting system (2.5 mM glucose, hexokinase (1.7 E.U.), glucose-6-phosphate dehydrogenase (0.85 E.U.), 0.20 mM NADP+, and 10 µM Ap5A (a specific inhibitor of mitochondrial adenylate kinase)). Externally added ADP (25 mM) started the exchange reaction with ATP synthesized in the mitochondrial matrix. The formation of NADPH, which is proportional to ATP efflux, was continuously monitored spectrophotometrically for 5−10 min with a Shimadzu UV3000 (340 nm; ɛ = 6.2 mM-1 cm-1). The rate of absorbance increase was obtained from the linear part of the curve and used to calculate the amount of exchanged ADP (nmol/min/mg proteins). Measurement of [14C]ADP uptake by yeast mitochondria [14C]ADP uptake by yeast mitochondria was determined as reported previously (22) with some modifications. Briefly, in a 1.5 mL Eppendorf tube, freshly isolated mitochondria (100 µg of proteins/mL, 500 µL) were incubated with a test compound on ice for 5 min in buffer containing 250 mM sucrose, 10 mM Tris/HCl (pH 7.2), and oligomycin (5.0 µg/mL). The reaction was initiated by adding [14C]ADP (100 µM, 70 MBq/mmol) to the suspension. The mixture was further incubated on ice for 3 min, then terminated by the addition of CATR (10 µM), followed by immediate centrifugation (15,000 rpm at 4 ˚C for 5 min). The pellets were washed in the same buffer, centrifuged again, solubilized in 2.0% (w/v) SDS (200 µL), and then mixed with liquid scintillation cocktail (2 mL, Insta-Gel Plus, PerkinElmer). Radioactivity was assessed using a liquid scintillation counter (AccuFLEX LSC-8000, Hitachi, Tokyo). The radioactivity of the sample, which was conducted in the absence of mitochondria, was used to subtract the background. Measurement of respiratory enzyme activities For the assays of each respiratory complex, yeast mitochondria were permeabilized by repeated freeze-thawing in 50 mM KPi buffer (pH 7.4) to improve the accessibility of substrates, such as NADH, ATP, and cyt. c (23). NADH-Q1 oxidoreductase activity (NDH-2) was followed by the oxidation of NADH (340 nm; ε = 6.2 mM−1 cm−1) in buffer (2.5 mL) containing 50 mM KPi (pH 7.4), 4.0 mM KCN, and 50 µM Q1 at 30 °C. The protein concentration was set to 12 µg/mL, and the reaction was initiated by the addition of NADH (final 50 µM). Succinate-cyt. c oxidoreductase activity (complexes II−III) was followed by the reduction of cyt. c (550-540 nm; ε = 21 mM−1 cm−1) in buffer (2.5 mL) containing 50 mM KPi (pH 7.4), 4.0 mM KCN, and 50 µM cyt. c (from horse heart, Sigma-Aldrich) at 30˚C. The protein 7 ACS Paragon Plus Environment

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concentration was set to 12 µg/mL, and the reaction was initiated by the addition of sodium succinate (final 10 mM). Cyt. c oxidase activity (complex IV) was followed by the oxidation of reduced cyt. c (550-540 nm; ε = 21 mM−1 cm−1) in 50 mM KPi buffer (pH 7.4, 2.5 mL) at 30 ˚C. The protein concentration was set to 12 µg/mL, and the reaction was initiated by the addition of dithionite-reduced cyt. c (final 50 µM). Hydrolysis of ATP (complex V) was measured in 2.5 mL of reaction buffer containing 50 mM Tris/HCl (pH 8.0), 0.50 µM antimycin A, and 6.0 mM MgCl2 at 30 ˚C in the presence of ATPgenerating system (2.0 mM 2-phosphoenolpyrvate, 50 µL of a pyruvate kinase/lactate dehydrogenase mixture (Sigma-Aldrich), and 100 µM NADH). The protein concentration was set to 30 µg/mL, and the reaction was initiated by the addition of ATP (final 100 µM). The oxidation of NADH, which is proportional to ATP hydrolysis, was monitored spectrophotometrically at 340 nm (ɛ = 6.2 mM-1 cm-1). Labeling of yeast mitochondria by t-PTD-023, NEM, or 5-FM Yeast mitochondria (1.0−2.0 mg of proteins/mL, 100−500 µL) were suspended in buffer containing 0.60 M mannitol, 0.10 mM EGTA, 2.0 mM MgCl2, 10 mM KPi, 10 mM Tris-HCl (pH 7.4), and protease inhibitor cocktail, and were then incubated with t-PTD-023 (0.1−10 µM) at 25˚C for 1−6 h. Mitochondria treated with t-PTD-023 were collected by centrifugation (15,000 rpm, 5 min) and solubilized in buffer containing 50 mM Tris/HCl (pH 8.0) and 1.0% (w/v) SDS. The proteins alkynylated by t-PTD-023 were conjugated with fluorescent TAMRA-N3 via click chemistry using the Click-iT protein reaction buffer kit according to the manufacturer’s protocol. Proteins were recovered by methanol/chloroform precipitation and subjected to further analyses. For the chemical labeling of mitochondria by NEM, yeast mitochondria (2.0 mg/mL) were incubated with NEM (0.2 mM, 100 mmol/mg of proteins) in buffer containing 0.60 M mannitol, 0.10 mM EGTA, 2.0 mM MgCl2, 10 mM KPi, and 10 mM Tris-HCl (pH 7.4) on ice for 30 min. Non-reacted NEM was quenched with DTT (20 mM), centrifugated (15,000 rpm, 4˚C, 5 min), and the pellets were then resuspended in the same buffer and subjected to further analyses. Labeling by 5-FM was conducted according to the same method, except that the concentrations of 5-FM and DTT were set to 150 µM and 5.0 mM, respectively. For the isolation of VDAC1, mitochondria labeled by t-PTD-023, NEM, or 5-FM were solubilized with 2.0% (w/v) Triton X-100 and subjected to purification using hydroxyapatite chromatography as described elsewhere (24). The flow-through fraction containing VDAC1 was collected and treated with methanol/chloroform, and the resulting precipitate was subjected to conjugation with fluorescent TAMRA-N3. Photoaffinity labeling of yeast mitochondria by the photoreactive quinone PUQ-1 Yeast mitochondria (1.0 mg/mL, 200 µL) were resuspended in the same buffer as that used for labeling by t-PTD-023 and incubated with 1.0 µM PUQ-1 in the presence of NADH (50 µM) and PTD-023 (50 or 100 µM) at room temperature for 10 min (19). Samples were irradiated with a 8 ACS Paragon Plus Environment

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Biochemistry

long wavelength UV lamp (Black Ray model B-100A, Upland, CA) on ice for 10 min, positioned 10 cm from the light source. Labeled mitochondria were collected by centrifugation and solubilized in buffer containing 50 mM Tris/HCl (pH 8.0) and 1.0% (w/v) SDS. Proteins labeled by PUQ-1 were conjugated with fluorescent TAMRA-N3 via click chemistry as described above, and subjected to further analyses. Electrophoresis and Western analysis Proteins conjugated with fluorescent TAMRA-N3 were resolved on a 15% Laemmli-type SDS gel (25). The migration pattern of fluorescent proteins was visualized by the model FLA-5100 Bioimaging analyzer (Fuji Film, Tokyo, Japan) or Typhoon FLA9500 (GE Healthcare, Buckingham-shire, UK) using a 532 nm light source and LPG (575 nm) filter. Data processing was conducted using Multi Gauge software (Fuji Film) or Image Quant, respectively. For the detection of VDAC1, the resolved proteins were transferred onto an PVDF membrane (19), blocked with 1.0% gelatin, and incubated with an antibody against yeast VDAC1 (1:3000 dilution), and then incubated further with an alkaline phosphatase (AP)-conjugated secondary antibody. The membrane was developed with NBT/BCIP colorimetric reagents (Bio-Rad). Proteomic analysis To localize the position at which t-PTD-023 covalently modified, modified VDAC1 was roughly purified by hydroxyapatite chromatography, conjugated with fluorescent TAMRA-N3, separated on a 12.5% Laemmli-type SDS gel, and stained by CBB. TAMRA-conjugated VDAC1 was digested with Asp-N (Roche, Penzberg, Germany), Lys-C (Wako Pure Chemicals, Osaka, Japan), and CNBr in 50 mM KPi buffer (pH 8.0, containing 0.01% SDS), 50 mM Tris/HCl buffer (pH 8.5, containing 0.1% SDS), and 70% formic acid. The digests were resolved on a 16% tricin gel (16% T, 6% C, ref. 26) For the MALDI-TOF MS analysis of proteins, CBB-stained protein bands were in-gel digested with trypsin in buffer containing 25 mM NH4HCO3 at 37 °C overnight. The digests were desalted with ZipTip (Millipore) and spotted onto the target plate using CHCA as a matrix. A mass spectrometric analysis was conducted using a Bruker Autoflex III Smartbeam instrument (Bruker Daltonics). The MS spectra obtained were analyzed according to previous procedures (19). For the LC-MS/MS analysis, tryptic digests were analyzed on an LTQ Velos Orbitrap mass spectrometer equipped with Ultimate 3000 nano-LC (LC-MS, Thermo Fisher Scientific) under the same experimental conditions reported previously (27, 28). Data were analyzed using Proteome Discoverer 2.1 (Thermo Scientific) with Mascot 2.3 (Matrix Science, London, U.K.). Regarding tryptic digestion, calbamidomethylation (Cys), oxidation (Met), and modifications by NEM (against Cys) were set as dynamic modifications. MS and MS/MS spectra were compared against the SwissProt database, and were filtered at a q value of ≤0.01 corresponding to a 1% false discovery rate.


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RESULTS

Figure 2. Inhibition of overall ADP-uptake/ATPrelease reactions and [14C]ADP uptake by PTDs. (A) Isolated yeast mitochondria (50 µg of proteins/mL) were incubated with PTD-023, tPTD-023, or CATR (10 µM each) in reaction buffer supplemented with an ATP-detecting system (see the Experimental Procedures). Overall ADPuptake/ATP-release reactions were monitored by detecting ATP efflux, which is proportional to the formation of NADPH. The average ATP efflux in the absence of inhibitor was 0.9 ± 0.05 µmol ATP/min/mg of proteins. Values show means ± SEM (n = 3−6). (B) Mitochondria (100 µg of proteins/mL, 500 µL) were incubated with PTD023 or CATR and then [14C]ADP (100 µM, 70 MBq/mmol) was added to the mitochondrial suspension, followed by incubation on ice for 3 min, as described in the Experimental Procedures. [14C]ADP uptake was terminated by the addition of 10 µM CATR, followed by the quantification of radioactivity. The average uptake of [14C] ADP in the absence of inhibitor was 4.8 ± 0.18 nmol/mg of proteins. Values show means ± SEM (n = 3−9).

PTD-023 inhibits overall ADP-uptake/ATPrelease in isolated yeast mitochondria We previously initiated the screening of our own chemical libraries with the aim of discovering new small chemicals that specifically inhibit the mitochondrial enzymes or transporters, as mentioned above. During this screening, we found that some PTDs (e.g. PTD-023) efficiently inhibit overall ADP-uptake/ATP-release reactions in isolated mitochondria respiring with αketoglutarate: 10 µM PTD-023 markedly inhibited the reaction (Figure 2A). The IC50 value, which is the molar concentration required for 50% inhibition, was 5.4 (± 0.3) µM (the final mitochondrial protein concentration was 50 µg/mL). We confirmed, as a control, that 10 µM carboxyatractyloside (CATR, an inhibitor of AAC), antimycin A (an inhibitor of complex III), and SF6847 (a protonophoric uncoupler) almost completely inhibits the reactions, as shown in Figure 2 taking CATR as an example. We also examined the effects of PTD-023 on 14 [ C]ADP uptake by non-respiring yeast mitochondria. We used a two-fold higher mitochondrial protein concentration in this assay (100 µg of proteins/mL) than the above ADPuptake/ATP-release assay to secure the accuracy of measurements. PTD-023 inhibited [14C]ADP uptake; incorporated radioactivity decreased to ~40 and ~50% of the control in the presence of 20 and 40 µM PTD-023, respectively (Figure 2B). The extent of the inhibition of [14C]ADP uptake was considerably less than that of the ADPuptake/ATP-release reactions. CATR (10 µM) inhibited [14C]ADP uptake by ~90% under the same experimental conditions. Effects of PTD-023 on the respiratory enzymes Since the mitochondrial respiratory system 10

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Biochemistry

provides the driving force for ADP uptake and ATP synthesis by forming proton electrochemical potential, PTD-023-induced impairments in the respiratory system results in the inhibition of overall ADP-uptake/ATPrelease reactions. Therefore, we examined the inhibitory effects of PTD-023 on each respiratory complex using appropriate substrate pairs (note that permeability barrier of OMM to NADH, ADP/ATP, and cytochrome c was previously lowered by repeated freeze-thawing under N2 atmosphere (23)). PTD-023 hardly exhibited the inhibition of each respiratory complex including ATP hydrolysis by ATP synthase (Figure 3). In these assays, the concentration of PTD-023 was set to 200 nmol/mg of proteins, which is equivalent to that used in the measurement of overall ADP-uptake/ATPrelease reactions above (10 µM PTD-023 at 50 µg of proteins/mL). Thus, the respiratory enzyme complexes can be excluded as a potential target of PTD-023, suggesting that the target is one of the transporters of substrates that are essential for ATP synthesis: VDAC1, AAC, or PiC (or more than one).

Figure 3 Effects of PTD-023 on the respiratory enzyme activities. The effects of PTD-023 on the activity of each respiratory complex. Isolated yeast mitochondria were permeabilized by repeated freeze-thawing (23). NADH-UQ1 oxidoreductase (NDH-2, A), succinate-cyt. c oxidoreductase (complexes II−III, B), NADH-cyt. c oxidoreductase (NDH2 − complex III, C), and cyt. c oxidase (complex IV, D) activities in addition to ATP hydrolysis (complex V, E) were measured in the presence of PTD-023. In these assays, the concentration of PTD-023 was set to 200 nmol/mg of proteins, which was equivalent to 10 µM PTD-023 (at 50 µg of proteins/mL) used in the measurement of overall ADP-uptake/ATP-release assay. Values show means ± SEM (n = 3−5).

Synthesis of the LDT chemistry reagent t-PTD023 To identify the target protein of PTD-023, we conducted LDT chemistry that allows for the detection and capture of PTD-023-bound protein in the mitochondrial environment. LDT chemistry is based on the principle of affinity labeling and uses a proper labeling reagent, in which a high-affinity ligand of the target protein and a tag of choice (a first tag) are connected by an electrophilic tosylate (phenylsulfonate) group (17, 18) (Figure S1). The LDT reagent specifically binds to a target


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protein due to a specific ligand-protein interaction. Once a ligand-protein binding complex is formed, nucleophilic amino acid residues (such as Asp, Cys, Glu, His, and Tyr) near the ligandbinding position attack an electrophilic tosylate group to make a covalent bond with the first tag via nucleophilic substitution (Figure S1). As such, LDT chemistry provides a powerful means to covalently attach a synthetic tag to target protein with high location specificity (17, 18). In order for LDT chemistry to be successful, LDT reagent that binds to the same target protein and same position in the protein as the parent ligand needs to be designed. Based on preliminary structure-activity study on PTDs (not published yet), we found that the presence of two free OH groups in the pentenediol moiety (the left side of the molecule) is critical for the inhibition of ADPuptake/ATP-release reactions in yeast mitochondria. Therefore, we supposed that the alkyl side chain moiety (the right side of the molecule) is a suitable position for the introduction of a tosylate group. We synthesized t-PTD-023 (Figure 1), in which the side chain of PTD-023 was replaced by a tosylate group having a terminal alkyne as a first tag. This alkyne group allows the conjugation of the second tag of choice, such as fluorophores and biotin, via so-called Cu+catalyzed click chemistry (29) (Figure S1), as demonstrated in previous LDT chemistry studies (27,28,30,31). We confirmed that t-PTD-023 inhibits ADP-uptake/ATP-release reactions in isolated yeast mitochondria (Figure 2A), thought the inhibitory potency of t-PTD-023, in terms of the IC50 value, was slightly weaker than that of PTD-023 (5.4 (± 0.3) vs. 7.5 (± 1.1) µM). Chemical labeling of VDAC1 via LDT chemistry using t-PTD-023 Isolated yeast mitochondria (1.0 mg of proteins/mL) were incubated with t-PTD-023 (10 µM) at 25˚C for 6 h without a respiration substrate. Note that since we were unable to maintain mitochondria at energized state for 6 h, we conducted LDT chemistry with non-respiring mitochondria. t-PTD-023-treated mitochondria were solubilized with 1.0% (w/v) SDS and then subjected to conjugation with a fluorescent TAMRA-N3 tag (Figure 1), followed by resolution on a 15% Laemmli-type SDS gel. A fluorescent band was observed at ~30 kDa (Figure 4A, WT) and fluorescent intensity increased with an increase in the concentration of t-PTD-023 used (Figure 4B). The fluorescent intensities observed for other regions were very weak, less than 5% of the ~30 kDa band in the fluorescence imaging analysis. VDAC1 (30.4 kDa), AAC (34.1 kDa), and PiC (33.5 kDa) migrate at approximately 30 kDa on SDS gel. Since hydroxyapatite chromatography is useful for roughly purifying these membrane proteins (21, 24), we attempted to purify the labeled protein using this technique. Yeast mitochondria (1.0 mg of proteins/mL) were incubated with 10 µM t-PTD-023 at 25 ˚C for 6 h, solubilized with 2.0% Triton X-100, passed through a hydroxyapatite column, and then subjected to a conjugation reaction with fluorescent TAMRA-N3 via click chemistry. As shown in Figure 4C (WT), the flow-through fraction of hydroxyapatite chromatography contained an ~30 kDa protein corresponding to the fluorescent band in Figure 4A. We identified this CBB-stained protein as VDAC1 by LC-MS/MS (95% coverage, Figure S2A). This CBB band contained no other membrane proteins. The ∼34 kDa protein marked with an asterisk was identified as AAC by MALDI-TOF MS (36% coverage, Table S2). 12 ACS Paragon Plus Environment

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Biochemistry

Figure 4. LDT chemistry using t-PTD-023 with yeast mitochondria. (A) Yeast mitochondria (1.0 mg of proteins/mL) isolated from wild-type and Δpor1 mutant S. cerevisiae cells were incubated with 10 µM t-PTD023 at 25 ˚C for 6 h. Labeled mitochondria were solubilized with 1.0% (w/v) SDS, conjugated with a fluorescent TAMRA-N3 tag, and subjected to resolution on a 15% Laemmli-type SDS gel. The fluorescent ~30 kDa band, which was only detected in mitochondria isolated from the wild-type strain, was indicated by an arrowhead. Approximately 40 µg of mitochondrial proteins were loaded in each well. (B) The concentration dependency of labeling of the ~30 kDa protein. Mitochondria (1.0 mg of proteins/mL) isolated from wild-type cells were incubated with various concentrations of t-PTD-023 (0−20 µM), followed by conjugation with fluorescent TAMRA-N3 tag. Proteins were separated on a 15% Laemmli-type SDS gel, followed by fluorescent gel imaging. (C) Mitochondria (1.0 mg of proteins/mL) isolated from wild-type and Δpor1 mutant S. cerevisiae cells were labeled with 10 µM t-PTD-023, and partially purified by hydroxyapatite chromatography, as described in the Experimental Procedures. Purified proteins were conjugated with a fluorescent TAMRA-N3 tag, separated on a 15% Laemmli-type SDS gel, and then subjected to CBB staining and fluorescent gel imaging. An arrowhead indicates VDAC1. The ∼34 kDa protein marked with an asterisk was identified as an ADP/ATP carrier. Data are representative of three independent experiments.


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To further verify this, we performed LDT chemistry with mitochondria isolated from Δpor1 S. cerevisiae that lacks VDAC1. No fluorescent band was observed at ~30 kDa for the protein samples prepared from before and after hydroxyapatite chromatography (Figures 4A and 4C, Δpor1). Altogether, these results clearly revealed that t-PTD-023 specifically binds to VDAC1 and makes a covalent bond(s) with some nucleophilic amino acid(s) via LDT chemistry. Target nucleophilic amino acid(s) will be investigated in a later section. To examine whether t-PTD-023 binds to the same position as that of PTD-023 in VDAC1, we performed a competition test between t-PTD-023 and PTD-023. Yeast mitochondria were incubated with t-PTD023 (1.0 µM) at 25 ˚C for 1 h in the presence of PTD-023 (50 or 100 µM). PTD-023 distinctly, but not completely, suppressed Figure 5. Effects of VDAC1 ligands on the labeling of the labeling of VDAC1 by t-PTD-023; 50 VDAC1 by t-PTD-023. (A) Competition tests were and 100 µM PTD-023 suppressed the conducted between t-PTD-023 and VDAC1 ligands. Yeast mitochondria (1.0 mg of proteins/mL) were labeling by ~40 and ~60%, respectively incubated with 1.0 µM t-PTD-023 at 25 ˚C for 1 h in the (Figure 5A). This result strongly suggests presence of PTD-023 (50 and 100 µM), itraconazole (100 µM), or DIDS (50 and 100 µM). Labeled proteins were that the binding position of t-PTD-023 conjugated with a fluorescent TAMRA-N3 tag, separated overlaps with that of PTD-023. A possible on a 15% Laemmli-type SDS gel, and subjected to fluorescent gel imaging. Approximately 40 µg of cause of the incomplete suppression by an mitochondrial proteins were loaded in each well. Data excess of PTD-023 is that since LDT are representative of three independent experiments. (B) chemistry requires long reaction times, Effects of itraconazole and DIDS on overall ADPuptake/ATP-release reactions in isolated mitochondria. mitochondria and t-PTD-023 need to be Yeast mitochondria (50 µg of proteins/mL) were incubated incubated for long periods of time (1 h in with itraconazole (100 µM) or DIDS (10 µM), and ATP efflux was measured under the same experimental this case), which enhances the opportunity conditions as those described in Figure 2A. Values show for the covalent (irreversible) binding of tmeans ± SEM (n = 3−6). PTD-023 even in the presence of PTD-023, a non-covalent (reversible) ligand. We did not use higher concentrations of PTD-023 (>100 µM) because high concentrations of hydrophobic chemicals tend to non-specifically alter the structures of membrane proteins and/or the membrane environment.


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Biochemistry

Effects of other VDAC1 ligands on LDT chemistry by t-PTD-023 Head et al. (6) demonstrated that antifungal itraconazole specifically binds to mitochondrial VDAC1 and inhibits its functions in human endothelial cells. We conducted a competition test between t-PTD-023 (1.0 µM) and itraconazole (100 µM) under the same experimental conditions as above. An excess of itraconazole hardly suppressed the labeling by t-PTD-023 (Figure 5A), suggesting that their binding positions differ or the binding affinity of itraconazole to yeast VDAC1 is significantly lower than that of t-PTD-023. In support of the latter, itraconazole did not inhibit overall ADP-uptake/ATP-release reactions in isolated yeast mitochondria even at 100 µM (Figure 5B) (note that the final mitochondrial protein concentration for the ADP-uptake/ATPrelease assay (50 µg/mL) was 12-fold lower than that for LDT chemistry (600 µg/mL)). These results indicate that itraconazole does not bind to yeast VDAC1 presumably due to the absence of specific sequences or structural elements in yeast VDAC1 that are present in human VDAC1; yeast VDAC1 is 24% identical to human VDAC1. We next conducted a competition test between t-PTD-023 and 4,4’-diisothiocyano-2,2’stilbenedisulfonic acid (DIDS), a non-specific inhibitor of anion channels/exchangers. Several anion transport inhibitors including DIDS have been shown to inhibit the functions of rat liver mitochondrial VDAC1 (32). DIDS suppressed the labeling of VDAC1 by t-PTD-023 (1.0 µM), though the extent of suppression appeared to be apparently saturated at ~40−50%, even with increasing concentrations up to 100 µM (Figures 5A). We confirmed that DIDS inhibits ADPuptake/ATP-release reactions by ~40% at 10 µM (Figure 5B), which roughly corresponds to 100 µM in the LDT chemistry experiment. Thus, DIDS may share a binding position, at least in part, with t-PTD-023. Identification of the binding position of t-PTD-023 in VDAC1 To roughly localize the position labeled by t-PTD-023, VDAC1 labeled by t-PTD-023 was partially purified using hydroxyapatite chromatography, conjugated with fluorescent TAMRA-N3 via Cu+-catalyzed click chemistry and cleaved with cyanogen bromide (CNBr), lysylendopeptidase (Lys-C), or endoprotease Asp-N (Asp-N). The CNBr cleavage gave two major CBB-stained fragments (apparent molecular masses of ~15 and ~7 kDa) with relative fluorescent intensities of approximately 1:5 (Figure 6A). The ~15 kDa fragment contained an internal sequence corresponding to Ser109 −Arg124 (confirmed by MALDI-TOF MS, m/z = 1800.97) and the ~7 kDa fragment contained sequences corresponding to Leu212−Arg224 and Glu249−Lys267 (m/z = 1474.80 and 1958.16, respectively). Based on the theoretical cleavage sites for CNBr, the ~15 and ~7 kDa fragments were assigned as the peptides Ser2−Met142 (15.2 kDa) and Asn209 − Ala283 (8.0 kDa), respectively. These results indicate that t-PTD-023 covalently modifies at least two different positions and the predominant reactive position is in the ~7 kDa fragment. The Lys-C and Asp-N digestion of the t-PTD-023-labeled VDAC1 afforded fluorescent bands at ~4 kDa region (Figure 6A). However, the assignment of each digest was unsuccessful because we were unable to find an appropriate pair of Lys-C and Asp-N digests sharing a common region 15 ACS Paragon Plus Environment

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Figure 6. Localization of the t-PTD-023-labeled position in VDAC1. (A) Yeast mitochondria (1.0 mg of proteins/mL) were incubated with 10 µM t-PTD-023 at 25 ˚C for 4 h, partially purified by hydroxyapatite chromatography, and proteins were conjugated with fluorescent TAMRA-N3 tag, followed by digestion with CNBr, Lys-C, or Asp-N. The digests were subjected to resolution on a 16.5% Schägger-type SDS gel (16.5% T and 6% C containing 6.0 M urea). Data are representative of three independent experiments. (B) Schematic presentation of the digestion of yeast VDAC1. The theoretical cleavage patterns by CNBr, LysC, and Asp-N were generated by PeptideMass (https://web.expasy.org/peptide_mass/). Representative cleavage sites are denoted with arrowheads and indicated by their residue numbers in the sequences of S. cerevisiae VDAC1 (SwissProt entry P04840). Cys130 and Cys210 are indicated by red circles.

based on the theoretical digestion sites (Figure 6B). Since strong fluorescence was observed at the front of the gel, the major digests by the Lys-C or Asp-N treatment may have been too small to be resolved on a tricine gel. Therefore, we attempted another approach as described below. VDAC1 contains only two cysteines that are conserved from yeast to mammals: mammalian VDAC1 contains cysteines at positions 127 and 232; however, cysteines are located at positions 130 and 210 in S. cerevisiae VDAC1 (33). Each of the two regions identified above (Ser2− Met142 and Asn209 − Ala283) contains a cysteine residue, which generally elicits strong nucleophilicity. Taking this into consideration, we adopted the working hypothesis that both cysteines may bring about nucleophilic attack on the tosylate group of t-PTD-023. To assess this hypothesis, the labeling of VDAC1 by t-PTD-023 was carried out using mitochondria pretreated with N-ethylmaleimide (NEM, 100 nmol/mg of proteins) to mask cysteines using the method described in the Experimental Procedures. As shown in Figure 7, the extent of t-PTD-023 labeling was markedly reduced by the NEM treatment, indicating that t-PTD-023 makes a covalent bond with both cysteines (Cys130 and Cys210). Nevertheless, to confirm whether the two cysteines in VDAC1 were indeed modified by NEM, 16 ACS Paragon Plus Environment

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Biochemistry

we analyzed VDAC1 in NEM-treated mitochondria by MS. The SDS gel piece containing VDAC1 from NEM-treated mitochondria (equivalent to lane “+NEM” in Figure 7) was subjected to in-gel tryptic digestion, followed by extensive characterization by LC-MS/MS. As expected, among the 35 VDAC1-derived peptides identified (95% coverage, Figure S2A), we detected two peptides, G125AFDLCLK132 and 206 224 A TMNCKLPNSNVNIEFATR , that were modified by NEM at Cys130 and Cys210, respectively (MS/MS spectra were shown in Figure S2B). Although the reaction yields of the NEM modifications were not assessed by this experiment, given the remarkable suppression of Figure 7. Effects of NEM on the labeling of VDAC1 via LDT chemistry using t-PTD-023. t-PTD-023 labeling by NEM, the NEM treatment The NEM-treated mitochondria were prepared may have almost quantitatively masked the according to the method described in the Experimental Procedures. The NEM-treated cysteines. mitochondria were incubated with 10 µM t-PTDTo examine a difference in reactivities 023 at 25 ˚C for 2 h and subjected to SDS-PAGE. The position of VDAC1 was indicated by an against t-PTD-023 between Cys130 and Cys210, arrowhead. Labeling by t-PTD-023 was also we prepared two single mutants (Cys130Ala and conducted using mitochondria, which were treated with DTT, but not with NEM, and shown as a Cys210Ala) and one double mutant control. Approximately 40 µg of mitochondrial (Cys130Ala/Cys210Ala) by site-directed proteins were loaded in each well. Data are mutagenesis. Mitochondria (1.0 mg of representative of three independent experiments. proteins/mL) isolated from each mutated yeast were incubated with t-PTD-023 (10 µM) at 25 ˚C for 2 h, conjugated with fluorescent tag TAMRAN3, and subjected to resolution on a 15% Laemmli-type SDS gel, followed by fluorescent gel imaging or Western blotting using an anti-yeast VDAC1 antibody (Figure 8A). The fluorescent intensities of each VDAC1 mutant were normalized by the expression levels of VDAC1 assessed by Western blotting. As shown in Figure 8A, labeling by t-PTD-023 extensively decreased (by ~80%) due to the mutation of Cys210, while the mutation of Cys130 only suppressed labeling by ~15%. The extent of suppression in the double mutant (Cys130Ala/Cys210Ala) was significant (>90%), almost identical to that in wild-type mitochondria pretreated with NEM (Figure 7). Taken together, we conclude that t-PTD-023 reacts predominantly and subsidiarily with Cys210 and Cys130, respectively. Thus, PTDs bind to the region interactive with both cysteines, albeit potentially closer to Cys210. Saletti et al. showed by MS analysis that two cysteines in VDAC1 isolated from rat liver mitochondria were over-oxidized, though the extent of oxidation was not quantitatively determined (34). Oxidation of the cysteines in VDAC1 may affect the t-PTD-023labeling because nucleophilic reactivities of the oxidized products decrease. Since a difference 17 ACS Paragon Plus Environment

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of the t-PTD-023-labeling between Cys130Ala and Cys210Ala was comparable to a difference of fluorescence intensities between the labeled peptides Asn209−Ala283 (containing Cys210) and Ser2−Met142 (Cys130) (Figure 6A), the effects of oxidation may not be significant if any.

Figure 8. Experiments using VDAC1 mutants (Cys130Ala, Cys210Ala, and Cys130Ala/Cys201Ala). (A) LDT chemistry using t-PTD-023 with mutated VDAC1. (i) LDT chemistry using t-PTD-023 was conducted using three VDAC1 mutants. Yeast mitochondria (1.0 mg of proteins/mL) expressing wild-type or mutant VDAC1 were labeled with t-PTD-023 (10 µM) at 25 ˚C for 2 h and conjugated with fluorescent TAMRA-N3, followed by resolution on a 15% Laemmli-type SDS gel. The gel was further subjected to fluorescent gel imaging (upper) and a Western blot analysis using an anti-yeast VDAC antibody (lower). Approximately 40 µg of mitochondrial proteins were loaded in each well. (ii) Extent of t-PTD-023 labeling with wild-type and mutated VDAC1s. The fluorescent intensities of TAMRA incorporated into each VDAC1 were normalized by the expression level of VDAC1 estimated by Western blotting. Values show means ± SEM (n = 3−5). (B) Effects of cysteine mutations on overall ADP-uptake/ATP-release reactions. Overall ADP-uptake/ATP-release reactions were measured in the absence or presence of PTD-023 (10 µM) with mitochondria isolated from yeast cells expressing mutated VDAC1 under the same experimental conditions as those described in Figure 2A. Values show means ± SEM (n = 3−10). (C) Labeling of cysteine residues in VDAC1 by 5FM. Yeast mitochondria (2.0 mg of proteins/mL) were treated with 150 µM 5-FM, followed by partial purification by hydroxyapatite chromatography according to the method described in the Experimental Procedures. Samples were separated on a 15% Laemmli-type SDS gel, and the gel was subjected to fluorescence gel imaging (upper) and CBB staining (lower). Data are representative of three independent experiments.


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Biochemistry

ADP/ATP transport activities of Cys130 and Cys210 mutants We investigated the roles of Cys130 and Cys210 in the transport of ADP/ATP. While overall ADP-uptake/ATP-release activities somewhat varied among the mutants (Cys130Ala, Cys210Ala, and Cys130Ala/Cys210Ala), no drastic decreases in the activity were observed (Figure 8B). These results indicate that Cys130 and Cys210 are not essential for the native substrate transport, in agreement with the findings obtained with rat mitochondrial VDAC1 using a Cys127Ala/Cys232Ala double mutant (33). The susceptibilities of the three mutants to PTD-023 were slightly reduced compared with the wild-type VDAC1; however, 10 µM PTD-023 distinctly inhibited (by approximately 60−70%) ADP-uptake/ATP-release reactions with the mutants (Figure 8B). These results indicate that the interaction with the cysteines is not critical for the inhibitory action of PTD-023, though t-PTD-023, having a reactive tosylate group, specifically reacts with the residues in its bound state. To get insights into the topologies of the two cysteines, their reactivities against fluorescein-5maleimide (5-FM), a negatively charged membrane-impermeable SH-reagent, were examined using the three mutants (Cya130Ala, Cys210Ala, and Cys130Ala/Cys210Ala). Mitochondria (2.0 mg of proteins/mL) isolated from wild-type and mutated yeast were treated with 150 µM 5FM at 37 ˚C for 30 min and VDAC1s were partially purified by hydroxyapatite chromatography, followed by SDS-PAGE and fluorescent gel imaging. 5-FM reacted with wild-type and single mutated VDAC1s, but not with the double mutant (Figure 8C). The CBB-stained VDAC1 band was slightly shifted to the upper side by the treatment with 5-FM. The average relative labeling yields of the single mutants by 5-FM, which were estimated based on the fluorescence intensities of the bands normalized by CBB-staining intensities, were approximately 70% of the wild type; hence, the simple sum of the labeling yields against Cys130 and Cys210 was more than 100%. This overestimation may have been due to some issue associated with image scanning of the bands because their signal intensities markedly differed between the wild type and the mutants. These remarkable differences reflected the highly various recovery yields of VDAC1 from hydroxyapatite chromatography (as well as the expression levels of the proteins). We note that purification by hydroxyapatite chromatography was an essential experimental process because 5FM reacted with the cysteines of a number of proteins. Nevertheless, although quantitative comparisons of 5-FM labeling between the wild type and the mutants were difficult, the results indicate that the two cysteine residues are reactive with 5-FM. It is, therefore, likely that these residues are exposed to the cytosolic side. Effects of PTD-023 on the binding of quinone to VDAC1 By using a photoaffinity labeling technique, we previously demonstrated that the quinone head-rings of two photoreactive ubiquinones (PUQ-1 and PUQ-2, Figure S3) specifically bind to VDAC1 in S. cerevisiae mitochondria and that the labeled position is in the C-terminal region (Phe221–Lys234) connecting the 15−16th β-strand sheets (19). To elucidate relative binding positions of PTD-023 and the quinone in VDAC1, we performed photoaffinity labeling using PUQ-1 (1.0 µM) in the presence of an excess of PTD-023 (50 and 100 µM). PTD-023 did not 19 ACS Paragon Plus Environment

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suppress the binding of PUQ-1 to VDAC1 (Figure 9), indicating that the binding positions of PTD-023 and PUQ-1 do not overlap.

Figure 9. Effects of PTD-023 on the binding of PUQ-1 to VDAC1. Yeast mitochondria (1.0 mg of proteins/mL) were photoaffinity labeled with 1.0 µM PUQ-1 in the presence of 50 or 100 µM PTD-023. Samples were denatured with 1.0% (w/v) SDS, followed by conjugation with fluorescent TAMRA-N3 via Cu+-catalyzed click chemistry. Proteins were separated on a 15% Laemmli-type SDS gel and subjected to fluorescent gel imaging and CBB staining. Approximately 40 µg of mitochondrial proteins were loaded in each well. Data are representative of three independent experiments.


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Biochemistry

DISCUSSION VDAC1 situated in OMM serves as a mitochondrial gatekeeper that allows the transfer of various substances, such as metabolites, ions, and reactive oxygen species (12, 13). Since VDAC1 also plays a key role in mitochondrial permeability transition and mitochondria-mediated apoptosis, it has potential as a druggable target for anticancer therapeutics (7, 8, 16). By screening our chemical libraries using overall ADP-uptake/ATP-release assay with isolated S. cerevisiae mitochondria, we identified PTDs as new specific inhibitors of VDAC1, which are not similar in structure to any chemical ligands of VDAC1 hitherto known. PTD-023 inhibited native substrates (ADP/ATP) transport via VDAC1 in respiring mitochondria at a single digit µM level. Although direct comparisons of data from different experiments are difficult, the inhibitory potency of PTD-023 appears to be at the same level as those of erastin (14) and itraconazole (6), which regulate the functions of VDACs in isolated mitochondria in the range of a single digit to a few tens of µM. Since the principle structural framework of PTDs is simple, there may be ample room for further structural modifications in anticipation of significant enhancements in the inhibitory potency and/or selectivity among VDAC1s from different organisms. Thus, the identification of PTDs will provide valuable insights for the synthetic development of further unique inhibitors of VDAC1. Many studies reported the small chemicals that regulate the various functions of VDAC1 (35 −42). However, it should be realized that the modes of interactions between the chemicals and VDAC1 were not necessarily investigated by direct (or specific) binding experiments in earlier studies. For example, some studies examined the effects of chemicals on the channel conductance of lipid bilayer-reconstituted VDAC1 (oblimersen (35), avicin (36)) or various VDAC1-related phenomena, such as the opening/closing of the mitochondrial permeability transition pore (cisplatin (37)), the association/dissociation of cytoplasmic kinases from VDAC1 (endostatin (38), methyl jasmonate (39, 40)), and the induction/inhibition of VDAC1 oligomerization (VBIT-3 and VBIT-4 (41), cyathin-R (42)). Thus, the molecular mechanisms responsible for the interactions between these chemicals and VDAC1 remain largely elusive. In this context, Head et al. (6) for the first time directly demonstrated a specific ligand (itraconazole)VDAC1 interaction in human umbilical vein cells using a photoaffinity labeling technique; however, the binding position of itraconazole in VDAC1 was not investigated in that study. In the present study, we assessed the effects of PTDs on native substrates (ADP/ATP) transport mediated by VDAC1 and, for the first time, revealed the binding position of the specific inhibitors (PTDs) at the amino acid level, as discussed later. Although De Pinto et al. reported that the carboxylic group modifier [14C]DCCD binds to Glu72 in bovine mitochondrial VDAC1 (43), this reagent is not a specific ligand of VDAC1. The X-ray crystallographic and NMR structures of mammalian VDAC1 independently revealed a 19-stranded β-barrel fold with an N-terminal α-helix lining the pore interior (44−46), though discrepancies concerning its structure between structural and biochemical/biophysical studies have been debated (47−49). VDAC1 contains only two cysteines that are conserved from yeast to mammals; mammalian VDAC1 contains cysteines at positions 127 and 232, whereas they 21 ACS Paragon Plus Environment

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are located at positions 130 and 210 in S. cerevisiae VDAC1. According to the structural models and membrane topology map (44−46), Cys127 and Cys232 are located in the middle of the pore and lipid/cytosol interface, respectively. Although biochemical studies aimed at elucidating position (or topology) of the cysteine residues in mammalian VDAC1 remain controversial (33, 47, 50-52), Aram et al. (33) concluded that neither residues is essential for the VDAC1 channel activity in the bilayer-reconstituted system and the induction of apoptosis mediated by VDAC1 overexpression in T-REx-293 cells. We also confirmed the non-essential roles of Cys130 and Cys210 of yeast VDAC1 in the transport of ADP/ATP (Figure 8B). Although the positions of Cys130 and Cys210 in S. cerevisiae VDAC1 cannot be precisely identified from the mammalian models because of the different positions of the cysteines, we predicted their positions by SWISSMODEL (53) based on human VDAC1 (2JK4.pdb), as shown in Figure 10A. This structural model suggests that both cysteines are exposed to the cytosolic side, being consistent with the result showing that the two residues were modified by membrane impermeable 5-FM (Figure 8C).

Figure 10. The binding model of PTDs in VDAC1 in S. cerevisiae mitochondria. The structure of yeast VDAC1 was produced by SWISS-MODEL (53) based on human VDAC1 (2JK4.pdb). (A) The positions of Cys130 and Cys210 are shown. (B) Schematic presentation of the binding model of PTD-023 in yeast VDAC1. The polar toxophoric pentenediol moiety and hydrophobic side chain are tentatively located on the inside and outside of the pore, respectively. (C) All hydrogen bond-acceptable residues, which were searched from the region colored in light brown as potential interactive partners of the pentenediol moiety, are shown. The position of nucleophilic Tyr155 is also shown (see the text). (D) Left: The key basic residues on the αhelix (Lys12, Arg15, Lys20), the 15th barrel wall (Arg218), and conserved Lys174 at the entry point, which contribute to ATP permeation via the path 1 in mouse VDAC1 (55), are shown. Right: All potential Lys/Arg residues on the N-terminal α-helix and along the presumed path 1 including conserved Lys174 in yeast VDAC1 are shown. (E) The region labeled by the quinone head-rings of photoreactive PUQ-1 and PUQ-2 (Phe221– Lys234 connecting the 15−16th β-strand sheets, ref. 19) are shown in blue.


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Biochemistry

We confirmed that the positions of Cys130 and Cys210, which were modeled by MODELLER (54) based on human VDAC1, are similar to those obtained by SWISSMODEL (Figure S4). Based on the present results, we forecast a binding model of PTDs in yeast VDAC1. We assumed that PTDs bind to the cytosolic side between Cys210 and Cys130, albeit somewhat closer to the former. Since the pore interior of VDAC1 is predominantly hydrophilic (55), it is difficult to image that a whole PTD molecule, including the hydrophobic side chain moiety, enters the interior. Therefore, we tentatively located the polar toxophoric pentenediol moiety and Figure 11. Effects of Asp139/Glu152 mutations on the side chain on the inside and outside of the susceptibility against PTD-023. Overall ADPpore, respectively, crossing the loop uptake/ATP-release reactions were measured in the absence or presence of PTD-023 (10 µM) with connecting the 12−13th β-strand, as mitochondria isolated from yeast cells expressing schematically presented in Figure 10B. mutated VDAC1 (Asp139Ala, Glu152Ala, and Asp139Ala/Glu152Ala) under the same experimental This binding manner may allow the tosylate conditions as those described in Figure 2A. Values group of t-PTD-023 to form intensive show means ± SEM (n = 3 − 5). The statistical significance of pairwise comparisons to the wild type contact with Cys210 while maintaining an was assessed with Dunnett’s test. *P

Pentenediol-Type Compounds Specifically Bind to Voltage

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