Báo cáo khoa học: Bacitracin inhibits the reductive activity of protein disulfide isomerase by disulfide bond formation with free cysteines in the substrate-binding domain pptx

Bacitracin inhibits the reductive activity of protein disulfide isomerase by disulfide bond formation with free cysteines in the substrate-binding domain Nina Dickerhof 1 , Torsten Kleffmann 2 , Ralph Jack 3 and Sally McCormick 1 1 Department of Biochemistry, University of Otago, Dunedin, New Zealand 2 Centre for Protein Research, University of Otago, Dunedin, New Zealand 3 Seperex Nutritionals, The Centre for Innovation, Dunedin, New Zealand Introduction Protein disulfide isomerase (PDI, EC 5.3.4.1)isan endoplasmic reticulum-resident enzyme in eukaryotic cells that catalyzes both the oxidation of cysteines to form disulfide bonds and the reduction and rearrange- ment of disulfide bonds in proteins, depending on the redox potential of the cell [1–3]. PDI comprises four structural thioredoxin-like domains, a, b, b¢ and a¢, and an x-linker region between the b¢-domain and a¢-domain [4–6]. The different activities of PDI are carried out by different redox states of the catalytic Cys-Gly-His-Cys motif present in each of the two a-domains of PDI, which can exist in either the reduced dithiol or the oxidized disulfide state [7]. The two b-domains are noncatalytic; however, the b¢-domain displays a large hydrophobic surface, and has been identified as the principal substrate-binding domain of PDI [8,9]. The peptide antibiotic bacitracin was reported to be an inhibitor of PDI in 1981 [10], and since then has been widely used to demonstrate the role of PDI in cellular processes, including glioma cell invasion [11], melanoma cell death [12], virus entry [13,14], and platelet function [15]. In vitro evidence for the specific- ity of bacitracin as a PDI inhibitor, however, is limited, and its mechanism of action remains elusive. Karala and Ruddock [16] recently questioned the use of bacitracin as a specific PDI inhibitor, after they demonstrated a partial effect on the reductive activity Keywords bacitracin; cyclic peptide; protein disulfide isomerase; protein disulfide isomerase (PDI) inhibition; substrate-binding domain; thiol–disulfide exchange Correspondence S. McCormick, Department of Biochemistry, University of Otago, PO Box 56, Dunedin, New Zealand Fax: +64 3 479 7866 Tel: +64 3 479 7840 E-mail: sally.mccormick@otago.ac.nz (Received 13 January 2011, revised 15 March 2011, accepted 6 April 2011) doi:10.1111/j.1742-4658.2011.08119.x The peptide antibiotic bacitracin is widely used as an inhibitor of protein disulfide isomerase (PDI) to demonstrate the role of the protein-folding catalyst in a variety of molecular pathways. Commercial bacitracin is a mixture of at least 22 structurally related peptides. The inhibitory activity of individual bacitracin analogs on PDI is unknown. For the present study, we purified the major bacitracin analogs, A, B, H, and F, and tested their ability to inhibit the reductive activity of PDI by use of an insulin aggrega- tion assay. All analogs inhibited PDI, but the activity (IC 50 ) ranged from 20 l M for bacitracin F to 1050 lM for bacitracin B. The mechanism of PDI inhibition by bacitracin is unknown. Here, we show, by MALDI- TOF ⁄ TOF MS, a direct interaction of bacitracin with PDI, involving disulfide bond formation between an open thiol form of the bacitracin thiazoline ring and cysteines in the substrate-binding domain of PDI. Abbreviations ACN, acetonitrile; CID, collision-induced dissociation; PDI, protein disulfide isomerase. 2034 FEBS Journal 278 (2011) 2034–2043 ª 2011 The Authors Journal compilation ª 2011 FEBS of PDI, but no effect of bacitracin on PDI-catalyzed disulfide formation or isomerization. Bacitracin, a dodecapeptide antibiotic produced by certain strains of Bacillus licheniformis and Bacillus subtilis, is a mixture of at least 22 structurally related peptides, which can be separated by RP-HPLC and characterized by MS [17]. The basic structure of bacitra- cin consists of a cyclic peptide of seven amino acids with a linear peptide side chain of five amino acids (Fig. 1A). A thiazoline ring is present at the N-terminus of the pep- tide formed either by l-cysteine and l-isoleucine or by l-cysteine and l-valine. The various analogs result from substitutions of hydrophobic amino acids within the peptide sequence and from oxidative transformation of the thiazoline ring. For example, bacitracin A, B1, B2 and B3 are transformed into bacitracin F, H1, H2 and H3, respectively, by oxidation of the amino-thiazoline ring to the keto-thiazole ring [18,19]. Bacitracin A makes up approximately 70% of the total mass of com- mercial bacitracin and, together with bacitracin B1, B2 and B3, accounts for more than 96% of total antimicro- bial activity in commercial bacitracin products [20]. The analog responsible for the reported inhibitory activity on PDI is unknown. Here, we have purified bacitra- cin A, B1–3, F and H1–3, and tested their ability to inhi- bit the reductive activity of PDI. Furthermore, we have investigated the mechanism of action of bacitracin on PDI, and demonstrated its ability to form disulfide bonds with cysteines in the substrate-binding domain of PDI. Results The separation of commercial bacitracin into individ- ual analogs was achieved by RP-HPLC (Fig. 1B). Major peaks were collected, pooled and analyzed by MALDI-TOF ⁄ TOF MS to identify isolated bacitracin analogs (Fig. 1C and Fig. S1). Bacitracin H and F, containing the keto-thiazole ring, eluted at a later retention time than bacitracin A and B, owing to their increased hydrophobicity. The homogeneity of the iso- lated fractions was subsequently assessed by analytical RP-HPLC (Fig. 1D). The bacitracin B fraction showed traces of early-eluting compounds, which are likely to be products formed by breakdown after purification. The ability of each analog to inhibit the reductive activity of PDI was tested in a turbidometric assay based on the reduction of insulin by dithiothreitol in the pres- ence of PDI. Upon reduction, insulin forms aggregates, and the rate of aggregation was followed by turbidity measurement at 562 nm (Fig. 2A–E). The kinetics of this reaction were biphasic, with an initial lag phase fol- lowed by an exponential increase in turbidity. In each case, the presence of the bacitracin analog resulted in a longer lag phase and an attenuated increase in turbidity, in a dose-dependent manner. Figure 2F shows a compar- ison of the absorbance reached at 100 min when each analog was present at a concentration of 100 lm. Bacitracin F and H were the most effective analogs. Dose–response curves were generated to obtain IC 50 values for each analog by expressing activity as absor- bance at 100 min as a percentage of the control absor- bance obtained with no inhibitor present (Fig. 3). Bacitracin F and H were found to be approximately 25-fold more active as PDI inhibitors than bacitracin A and B (IC 50 of 20 and 40 lm versus 590 and 1050 lm, respectively). The IC 50 of the commercial mix was 70 lm. The mechanism of action of bacitracin on PDI is unclear. In order to determine a direct interaction of bacitracin with PDI, we separated incubations contain- ing bacitracin analogs and PDI by SDS ⁄ PAGE. Western blot analysis with an anti-PDI serum and an antibody against bacitracin showed that each bacitra- cin analog comigrated with PDI in SDS ⁄ PAGE under nonreducing conditions (Fig. 4A). This suggests that bacitracin undergoes a robust interaction with PDI that is resistant to the denaturing conditions in SDS ⁄ PAGE. However, the PDI–bacitracin complex could not be detected under reducing conditions, indi- cating that reducible bond formation is involved in the PDI–bacitracin interaction. Furthermore, we showed that the PDI–bacitracin complex could be immunopre- cipitated with an anti-PDI serum (Fig. 4B), using pro- tein G beads. Several washing steps of the beads with NaCl ⁄ P i containing 0.1% Tween-20 did not diminish the immunoblot signals for bacitracin at the site of PDI (Fig. 4C). These data support the view of a robust covalent PDI–bacitracin interaction. We hypothesized that the thiazoline ring of bacitra- cin reacts with cysteines on PDI to form disulfide bonds. On the basis of reported disulfide formation resulting from opening of a thiazole ring to a thiol form [21], we proposed a mechanism of ring opening for bacitracin with subsequent disulfide bond forma- tion with free cysteines on PDI ( Fig. 5A). In order to test this proposed mechanism, we performed in-gel tryptic digestions of the PDI–bacitracin complex, and analyzed the generated peptides by MALDI-TOF ⁄ TOF MS. Mass spectra were investigated for peaks with a predicted [M + H] + potentially comprising cys- teine-containing PDI peptides crosslinked to the open thiol form of bacitracin. Figure 5B shows an example for a predicted crosslink between the Cys345-contain- ing PDI peptide Ile341–Arg347 ([M + H] + : 905.43) and the open thiol form of bacitracin A ([M + H] + : 1440.77). A peak at m⁄ z 2343.09 was detected in the N. Dickerhof et al. Bacitracin forms a disulfide bond with free PDI cysteines FEBS Journal 278 (2011) 2034–2043 ª 2011 The Authors Journal compilation ª 2011 FEBS 2035 mass spectrum, which matched the predicted [M + H] + of 2343.17 for this crosslinked peptide (Fig. 5C). The precursor m ⁄ z 2343.09 was selected for collision-induced dissociation (CID)-TOF ⁄ TOF MS analysis. The MS ⁄ MS spectrum acquired in positive ion mode showed a cluster of three peaks at m ⁄ z 871.36, 905.36 and 937.33 (Fig. 5D), which are 34 and 32 mass units apart, respectively. This peak cluster is indicative of the Ile341–Arg347 peptide being involved in a disulfide bond. The three peaks represent CID-based cleavage events at the cysteine b carbon– sulfur bond, with double bond formation between the Fig. 1. Separation of bacitracin analogs. (A) Structures of the most abundant bacitracin analogs of commercial bacitracin mixtures including the amino-thiazoline ring (a) and (b) or keto-thiazole ring (c) and (d). (B) Representative chromatogram for the separation of bacitracin by RP- HPLC, with a gradient of 10–90% ACN over 40 min and detection at 252 nm. Bacitracin A and F and bacitracin B and H were purified as pooled fractions of B 1–3 and H 1–3. (C) MALDI-TOF ⁄ TOF MS spectrum of bacitracin F: The most significant fragment ions, their structures and m ⁄ z values that were used to discriminate different bacitracin analogs by MALDI-TOF ⁄ TOF MS are shown. (D) The purity of each frac- tion was assessed by analytical RP-HPLC, with similar conditions as used for the preparative RP-HPLC. Bacitracin forms a disulfide bond with free PDI cysteines N. Dickerhof et al. 2036 FEBS Journal 278 (2011) 2034–2043 ª 2011 The Authors Journal compilation ª 2011 FEBS a and b carbons (871.36), the sulfur–sulfur bond (905.36) and the sulfur–carbon bond of the open thiaz- oline ring of bacitracin A (937.33). Negative ion mode CID-TOF ⁄ TOF MS analysis of the same precursor yielded the corresponding signature peaks (m ⁄ z 1404.88, 1438.82 and 1470.80) of the other part of the disulfide bond, i.e. the negatively charged open-ring bacitracin A ion without sulfur, free thiol and two sulfurs, respectively (Fig. 5E). All three sulfur bonds are prone to cleavage upon ionization by increased laser energy, which generates a small proportion of fragments in the ion source. The combination of in-source decay of the precursor m ⁄ z 2343.09 with CID on the in-source fragment m ⁄ z 937.22 in positive ion mode confirmed the location of the disulfide (Fig. 5F). The in-source decay ⁄ CID fragment spectrum yielded unambiguous sequence information for the Cys345-containing PDI peptide, with an additional 32 mass units at position 345 (Fig. 5F). In addition to Cys345, Cys314 was also found to be crosslinked to bacitracin (Fig. S2). Discussion The peptide antibiotic bacitracin is widely used experi- mentally in vivo as a specific PDI inhibitor, although evidence for its specificity is scarce, and the activities of its different analogs are unknown. Our study dis- sected the activities of the various major bacitracin analogs on the reductive activity of PDI, and showed that the H and F analogs are 25-fold more active than the A and B analogs. As Karala and Ruddock [16] could not demonstrate a significant effect of 1 mm commercial bacitracin on the oxidase and isomerase activities of PDI, we con- centrated on the reductive activity of PDI in this study. When Roth [10] originally identified bacitracin as an inhibitor of PDI, they studied the reduction of insulin by rat liver lysates, and found that 250 lm Fig. 2. Insulin reduction by PDI in the presence or absence of bacitracin analogs. (A–E) Insulin (1 mgÆmL )1 ) was incubated in 100 mM potas- sium phosphate and 1 m M EDTA (pH 7.4) in the absence (uncatalyzed) or presence of 10 lgÆmL )1 PDI and increasing amounts of commer- cial bacitracin or individual bacitracin analogs. The reaction was initiated with 0.1 m M dithiothreitol (at time 0). (F) Comparison of A at 100 min after incubation with 100 l M of each analog. Data are presented as mean ± standard error of independent triplicate experiments. *P < 0.05 and ***P < 0.001 for comparison with the control. N. Dickerhof et al. Bacitracin forms a disulfide bond with free PDI cysteines FEBS Journal 278 (2011) 2034–2043 ª 2011 The Authors Journal compilation ª 2011 FEBS 2037 inhibited 90% of the activity. Smith et al. [22] reported an IC 50 value of 152 lm for bacitracin for the inhibi- tion of PDI reductive activity. The IC 50 value deter- mined in our study for the inhibition of PDI reductive activity by bacitracin was 70 lm. The experimental conditions in all studies varied with respect to dith- iothreitol, PDI and insulin concentrations, which may in part account for differences in the IC 50 values for bacitracin-mediated inhibition of PDI reductive activ- ity. Furthermore, on the basis of our results, the inhib- itory activity of the bacitracin mixture will depend on the concentrations of individual bacitracin analogs. Indeed, we found that bacitracin sourced from differ- ent suppliers varies in analog composition. The commercial bacitracin that we used here consisted of 65% bacitracin A and B as analyzed by analytical RP-HPLC. We therefore expected the IC 50 of the commercial bacitracin to be close to the IC 50 values for bacitracin A and B (590 and 1050 lm, respectively). However, the IC 50 of commercial bacitra- cin (70 lm) was much lower, and closer to the IC 50 of the less abundant bacitracin H and F (40 and 20 lm, respectively). We believe that, along with bacitracin H and F, there are other more active but low-abundance analogs within the commercial bacitracin mix that con- tribute to the lower than expected IC 50 . This is evident from the many minor peaks observed in the HPLC separation of commercial bacitracin (Fig. 1B), and supported by reports identifying up to 22 different bac- itracin analogs [17]. We have purified and tested only the four major analogs here, as purification of the low- abundance analogs would yield insufficient quantities for activity studies. The mechanism of the inhibitory action of bacitracin on PDI is unclear. Bacitracin acts as an antibiotic by forming a complex between divalent cations and the bacterial C 55 -isoprenyl lipid carrier, ultimately resulting in the inhibition of cell wall biosynthesis. A free amino group adjacent to a thiazoline ring has been shown to be essential for bacitracin to form a complex with divalent cations [23]. Bacitracin A and B fulfill this requirement, making them the active antimicrobial compounds of the bacitracin mix. The oxidation of the amino-thiazoline ring to the keto-thiazole ring to form bacitracin H and F results in a loss of metal binding and subsequent antimicrobial activity [20]. However, we show here that the ability to inhibit PDI is higher for bacitracin H and F, which excludes the coordina- tion of metal ions as a potential mechanism of action on PDI. Fig. 3. Dose–response curves of bacitracin analogs. PDI activity was expressed as A 562 nm at 100 min as a percentage of the A 562 nm of the control reaction containing no inhibitor after subtrac- tion of the A 562 nm of the uncatalyzed reaction. Data are presented as mean ± standard error of independent triplicate experiments. Nonlinear regression analysis revealed IC 50 values, which are given along with the 95% confidence level range in parentheses. Fig. 4. Bacitracin binding to PDI. (A) Incubations of 10 lgÆmL )1 PDI and 250 l M bacitracin analog were subjected to separation by SDS ⁄ PAGE, under either reducing or nonreducing conditions, and western blot analysis. Blots were probed with both rabbit poly- clonal anti-PDI serum and peroxidase-conjugated anti-rabbit IgG, or with a peroxidase-conjugated sheep anti-bacitracin IgG. (B) Incuba- tions of 10 lgÆmL )1 PDI and 250 lM commercial bacitracin were also subjected to immunoprecipitation with a polyclonal anti-PDI serum, using protein G beads. Immunoprecipitates were eluted from the beads after three washes with NaCl ⁄ P i containing 0.1% Tween-20, separated by SDS ⁄ PAGE, and subjected to western blot analysis under nonreducing conditions. (C) Immunoprecipitates were eluted from the beads after three, four and five washes with NaCl ⁄ P i containing 0.1% Tween-20. Bacitracin forms a disulfide bond with free PDI cysteines N. Dickerhof et al. 2038 FEBS Journal 278 (2011) 2034–2043 ª 2011 The Authors Journal compilation ª 2011 FEBS To further investigate the mechanism of inhibition of PDI by bacitracin, we tested for a direct interaction between PDI and bacitracin. We were able to demon- strate a direct and covalent interaction of each bacitra- cin analog with PDI by colocalization in immunoblot analyses after SDS ⁄ PAGE, as well as by coimmuno- Fig. 5. Disulfide bond formation between bacitracin and Cys345 on PDI. (A) Scheme of thiol formation of the thiazoline ring of bacitracin A with subsequent formation of a mixed disulfide with PDI. (B) Proposed crosslink between the PDI peptide Ile341–Arg347 and bacitracin A through a disulfide bond between Cys345 and the thiol form of bacitracin A. The thiol form of the thiazoline ring of bacitracin A and Cys345 are shown as chemical structures, the rest of bacitracin A as ‘Bacitracin A’, and all other amino acids not involved in the crosslink by the sin- gle-letter code. The PDI peptide side of the disulfide bond is named R1, and the bacitracin side R2. (C) MALDI-TOF MS spectrum of pep- tides generated by tryptic digestion of the PDI–bacitracin complex containing the crosslinked peptide Ile341–Arg347 ⁄ bacitracin A (arrow). (D) Area of the CID-TOF ⁄ TOF MS spectrum of the precursor ion m ⁄ z 2343.09 acquired in positive ion mode, showing signature peaks for the peptide [M + H] + 905.36 involved in a disulfide bond. (E) Area of the CID-TOF ⁄ TOF MS spectrum of the precursor ion m ⁄ z 2341.09 acquired in negative ion mode, showing signature peaks for the open thiol form of bacitracin A [M – H] ) 1438.82 involved in a disulfide bond. (F) In- source decay of the precursor ion m ⁄ z 2343.09 combined with CID on the in-source fragment m ⁄ z 937.33 reveals an additional 32 mass units at Cys345 (arrow), confirming the disulfide bond at this position. N. Dickerhof et al. Bacitracin forms a disulfide bond with free PDI cysteines FEBS Journal 278 (2011) 2034–2043 ª 2011 The Authors Journal compilation ª 2011 FEBS 2039 precipitation of bacitracin and PDI with an anti-PDI serum. We showed that the interaction was disrupted under reducing conditions, indicating the involvement of disulfide bond formation. We hypothesized that bacitracin may react with cysteines on PDI to form a disulfide bond. Using MALDI-TOF ⁄ TOF MS analy- sis, we were able to show that disulfide bond forma- tion occurs between an open thiol form of bacitracin and Cys314 and Cys345 of PDI. PDI comprises four domains, a, b, b¢ and a¢, and an x-linker region between the b¢-domain and a¢-domain. The a-domains are catalytically active, the b¢-domain functions as a substrate-binding domain displaying a large hydrophobic surface area, and the x-linker func- tions to gate access to the b¢-domain [8,9,24]. The cysteines involved in disulfide bond formation with bacitracin, Cys314 and Cys345, are present in the b¢-domain and x-linker, respectively. The crystal struc- ture of this region of PDI shows that these cysteines are free [9]. We propose that bacitracin binds to the hydrophobic surface of the substrate-binding region of PDI, allowing subsequent disulfide bond formation with the free cysteines Cys314 and Cys345. The pres- ence of the covalently bound bacitracin in this region would impair the binding of the substrate insulin, ulti- mately inhibiting its reduction. Although all bacitracin analogs seem to interact covalently with PDI (Fig. 4), there is a 25-fold increase in inhibitory activity between bacitracin A and B and bacitracin F and H, respectively. If we assume that bacitracin interacts with the hydrophobic surface of the substrate-binding site prior to the disulfide bond formation with Cys314 or Cys345, bacitracin H and F, which are more hydro- phobic than bacitracin A and B, might be more potent binding partners. Karala and Ruddock [16] tested the effect of bacitra- cin on the reductive activity of a truncated PDI con- taining the catalytic a-domain, but lacking the independent substrate-binding b¢-domain. Bacitracin did not seem to affect the rate of catalysis of the trun- cated PDI, whereas full-length PDI showed a signifi- cantly lower rate of catalysis in the presence of bacitracin. These findings can be explained by our results showing that bacitracin targets the substrate- binding domain of PDI, but not the catalytic domain. Furthermore, Karala and Ruddock [16] showed no effect of bacitracin on the oxidase activity of PDI, which can be carried out by either of the catalytic domains, a or a¢, with no requirement for the sub- strate-binding domain [25]. Although bacitracin has been commonly used as a specific PDI inhibitor, its ability to react with cysteines is unlikely to be limited to PDI. Indeed, we have tested the binding of bacitracin to various proteins, and found it to bind BSA and IgG (Fig. S3). Interestingly, it did not bind apolipoprotein A1 or pepsin (Fig. S3), neither of which has any free cysteines. We assume that the same mechanism of bacitracin binding as shown here for PDI applies to other proteins contain- ing free cysteines. In conclusion, we show a mechanism of action of bacitracin on PDI that involves covalent binding of an open thiol form of bacitracin to free cysteines in the substrate-binding domain of PDI. However, the inter- action between bacitracin and PDI is nonspecific, and applies to other proteins containing free cysteines. Experimental procedures Materials As commercial bacitracin has been reported to contain pro- teases [26], we incubated 5 mm bacitracin from different sources with 1 mg of BSA, and tested for albumin degrada- tion by SDS ⁄ PAGE. No evidence of degradation was found with bacitracin sourced from Calbiochem (Gibbs- town, NJ, USA), and this was used for subsequent experi- ments. PDI from bovine liver was purchased from Sigma (St Louis, MO, USA), porcine monocomponent insulin from Nova Research (Copenhagen, Denmark), dithiothrei- tol from Roche (Mannheim, Germany), peroxidase-conju- gated goat anti-rabbit IgG from Thermo Fisher Scientific (Rockford, IL, USA), and Pure Proteome Protein G Mag- netic Beads from Millipore (Billerica, MA, USA). The anti- bacitracin IgG was purchased from GenWay Biotech (San Diego, CA, USA), and conjugated with peroxidase by use of a Lightning Link labelling kit (Innova Biosciences, Cam- bridge, UK). Polyclonal rabbit anti-rat PDI serum, which crossreacts with bovine PDI [27], was a generous gift from M. Hubbard (University of Melbourne). Purification of bacitracin analogs and MALDI-TOF ⁄ TOF MS analysis The nomenclature of Ikai et al. [20] for the different baci- tracin analogs was used in this study. Bacitracin A, B1–3, H1–3 and F (referred to as bacitracin A, B, H and F, respectively) were isolated by semipreparative RP-HPLC on C-18 resin, using a Jasco (Great Dunmow, UK) HPLC sys- tem with an LG 2080-02 Ternary Gradient pump, a DG 2080 Degasser, and an MD 2010-plus detector. Baci- tracin was dissolved at a concentration of 50 mg ÆmL )1 in 10% acetonitrile (ACN) containing 0.1% trifluoroacetic acid, and filtered through a 0.2 lm filter; 10 mg was then injected for separation on a preparative XTerra MS C-18 column (5 lm, 10 · 150 mm; Waters, Milford, MA, USA). A gradient was run from 10% to 90% ACN containing Bacitracin forms a disulfide bond with free PDI cysteines N. Dickerhof et al. 2040 FEBS Journal 278 (2011) 2034–2043 ª 2011 The Authors Journal compilation ª 2011 FEBS 0.1% trifluoroacetic acid over 40 min at a flow rate of 7mLÆmin )1 to elute the various analogs, which were moni- tored by absorbance at 252 nm. Fractions were subse- quently analyzed for purity by analytical RP-HPLC, with the same gradient, at a flow rate of 0.6 mLÆmin )1 ,onan analytical XTerra MS C-18 column (3.5 lm, 3 · 10 mm). The structural identity of the isolated analogs was con- firmed by MALDI-TOF ⁄ TOF MS, with a 4800 MALDI- TOF ⁄ TOF Analyzer (AB SCIEX, Framingham, MA, USA). MALDI-TOF and TOF ⁄ TOF MS spectra were acquired with 800–1000 and 2000–2800 laser shots per sam- ple spot, respectively. For based MS ⁄ MS, the 2-kV opera- tion mode was used, with air as collision gas at a pressure of 1 · 10 6 Torr. Purified fractions were dried by rotary evaporation under vacuum, dissolved in 80% 2-methyl-propan-2-ol containing 0.05% HCl, and subsequently lyophilized. The weight of the dry bacitracin analog was determined, and it was dissolved in NaCl ⁄ P i for activity assays. Testing the effect of bacitracin analogs on PDI reductive activity PDI activity was measured with an assay that measures the catalytic reduction of insulin, as described by Holmgren [28]. Insulin (1 mgÆmL )1 ) was incubated in 100 mm potas- sium phosphate and 1 mm EDTA (pH 7.4), in the presence of 10 lgÆmL )1 PDI and varying amounts of bacitracin ana- log at room temperature. The reaction was initiated after 10 min by the addition of 0.1 mm dithiothreitol, and the increase in turbidity was monitored at 562 nm on an Elx808 Ultra Microplate Reader (Bio-Tex Instruments, Winooski, VT, USA) over 100 min. Dose–response curves were generated, with expression of PDI activity as absor- bance at 100 min as a percentage of the absorbance of the control reaction containing no inhibitor, after subtraction of the absorbance of the uncatalyzed reaction containing no PDI. IC 50 values were determined by applying a nonlinear least squares fit of the equation Y = bot- tom + (top – bottom) ⁄ {1 + 10^[(log IC 50 – X)*Hill slope]} to activity versus the log of inhibitor concentration, using graph pad prism Version 5.0 for MacOSX (San Diego, CA, USA). Although the Hill slope was variable, the bottom and top values were constrained to 0 and 100, respectively. Analysis of PDI–bacitracin interaction by SDS ⁄ PAGE and immunoprecipitation PDI at 10 lgÆmL )1 was incubated with 250 lm bacitracin analog in a total volume of 100 lL for 30 min at room temperature. The reaction mixtures were separated by 10% SDS ⁄ PAGE under either reducing or nonreducing condi- tions, and subsequently transferred to nitrocellulose membrane. Blots were probed with both rabbit polyclonal anti-PDI serum and peroxidase-conjugated anti-rabbit IgG or with peroxidase-conjugated sheep antibody against bacitracin. For immunoprecipitation studies, 5 lL of polyclonal anti-PDI serum was added to the PDI–bacitracin reactions and incubated overnight at 4 °C. The reaction mixture was then added to 50 lL of magnetic protein G beads and incu- bated for 90 min at room temperature. The beads were washed three times with NaCl ⁄ P i containing 0.1% Tween- 20, before elution of the complex from the beads by addi- tion of SDS buffer and heating at 90 °C for 10 min. To demonstrate a robust interaction between bacitracin and PDI, the beads were also washed four and five times with NaCl ⁄ P i containing 0.1% Tween-20 before elution. The imunoprecipitates were separated by 10% SDS ⁄ PAGE under nonreducing conditions, and subjected to western blot analysis as described above. Analysis of PDI–bacitracin interaction by MALDI-TOF ⁄ TOF MS PDI was incubated with commercial bacitracin, and reac- tion mixtures were separated by 10% SDS ⁄ PAGE under nonreducing conditions, as described above. After staining with Coomassie Blue, the protein band was excised from the SDS polyacrylamide gel and subjected to in-gel diges- tion with trypsin to generate crosslinked peptides. Proteins were digested with trypsin at a ratio of 1 lg of protease to 10 lg of protein at 37 °C for 15 h. Tryptic fragments were eluted from the gel matrix and analyzed by MALDI-TOF ⁄ TOF MS. The PDI–bacitracin was characterized by a combination of in-source decay and CID-TOF ⁄ TOF MS, based on the method described by Kleffmann et al. [29]. Briefly, for a selected precursor analysis, spectra were investigated for peaks with a predicted [M + H] + potentially containing PDI peptides with a cysteine crosslinked to bacitracin. Diagnostic crosslinked peptide ions were subjected to MALDI-TOF ⁄ TOF MS analysis. Spectra were acquired in the 2-kV positive and 1-kV negative ion mode, with 2000– 2800 laser shots per sample spot. For unambiguous deter- mination of the amino acids involved in the crosslink for- mation, peptide ions generated by in-source decay of the crosslinked peptide were selected for further fragmentation by CID. Acknowledgements We are very grateful to S. Huettenhain for the use of his HPLC equipment and for sending bovine PDI. We thank S. Wilbanks and G. Jameson for helping us to interpret data and providing insights into potential inhibitory mechanisms. We would also like to thank N. Dickerhof et al. Bacitracin forms a disulfide bond with free PDI cysteines FEBS Journal 278 (2011) 2034–2043 ª 2011 The Authors Journal compilation ª 2011 FEBS 2041 A. von Zychlinksy-Kleffmann and D. Petras for their kind assistance regarding the purification, as well as M. Hubbard for the polyclonal antibody against PDI. This work was supported in part by the National Heart Foundation, New Zealand (Grant No. 1321). References 1 Bassuk JA & Berg RA (1989) Protein disulphide isom- erase, a multifunctional endoplasmic reticulum protein. Matrix 9, 244–258. 2 Freedman RB (1989) Protein disulfide isomerase: multi- ple roles in the modification of nascent secretory proteins. Cell 57, 1069–1072. 3 Noiva R & Lennarz WJ (1992) Protein disulfide isomer- ase. A multifunctional protein resident in the lumen of the endoplasmic reticulum. J Biol Chem 267, 3553– 3556. 4 Freedman RB, Gane PJ, Hawkins HC, Hlodan R, McLaughlin SH & Parry JW (1998) Experimental and theoretical analyses of the domain architecture of mam- malian protein disulphide-isomerase. Biol Chem 379, 321–328. 5 Klappa P, Ruddock LW, Darby NJ & Freedman RB (1998) The b¢ domain provides the principal peptide- binding site of protein disulfide isomerase but all domains contribute to binding of misfolded proteins. EMBO J 17, 927–935. 6 Pirneskoski A, Klappa P, Lobell M, Williamson RA, Byrne L, Alanen HI, Salo KE, Kivirikko KI, Freedman RB & Ruddock LW (2004) Molecular characterization of the principal substrate binding site of the ubiquitous folding catalyst protein disulfide isomerase. J Biol Chem 279, 10374–10381. 7 Schwaller M, Wilkinson B & Gilbert HF (2003) Reduc- tion–reoxidation cycles contribute to catalysis of disul- fide isomerization by protein-disulfide isomerase. J Biol Chem 278, 7154–7159. 8 Byrne LJ, Sidhu A, Wallis AK, Ruddock LW, Freed- man RB, Howard MJ & Williamson RA (2009) Mapping of the ligand-binding site on the b¢ domain of human PDI: interaction with peptide ligands and the x-linker region. Biochem J 423, 209–217. 9 Denisov AY, Maattanen P, Dabrowski C, Kozlov G, Thomas DY & Gehring K (2009) Solution structure of the bb¢ domains of human protein disulfide isomerase. FEBS J 276, 1440–1449. 10 Roth RA (1981) Bacitracin: an inhibitor of the insulin degrading activity of glutathione-insulin transhydrogen- ase. Biochem Biophys Res Commun 98, 431–438. 11 Goplen D, Wang J, Enger PO, Tysnes BB, Terzis AJ, Laerum OD & Bjerkvig R (2006) Protein disulfide isom- erase expression is related to the invasive properties of malignant glioma. Cancer Res 66, 9895–9902. 12 Lovat PE, Corazzari M, Armstrong JL, Martin S, Pagliarini V, Hill D, Brown AM, Piacentini M, Birch-Machin MA & Redfern CP (2008) Increasing melanoma cell death using inhibitors of protein disulfide isomerases to abrogate survival responses to endoplasmic reticulum stress. Cancer Res 68, 5363– 5369. 13 Markovic I, Stantchev TS, Fields KH, Tiffany LJ, Tomic M, Weiss CD, Broder CC, Strebel K & Clouse KA (2004) Thiol ⁄ disulfide exchange is a prerequisite for CXCR4-tropic HIV-1 envelope-mediated T-cell fusion during viral entry. Blood 103, 1586–1594. 14 Ryser HJ, Levy EM, Mandel R & DiSciullo GJ (1994) Inhibition of human immunodeficiency virus infection by agents that interfere with thiol–disulfide interchange upon virus–receptor interaction. Proc Natl Acad Sci USA 91, 4559–4563. 15 Essex DW, Li M, Miller A & Feinman RD (2001) Pro- tein disulfide isomerase and sulfhydryl-dependent path- ways in platelet activation. Biochemistry 40, 6070–6075. 16 Karala AR & Ruddock LW (2010) Bacitracin is not a specific inhibitor of protein disulfide isomerase. FEBS J 277, 2454–2462. 17 Govaerts C, Li C, Orwa J, Van Schepdael A, Adams E, Roets E & Hoogmartens J (2003) Sequencing of baci- tracin A and related minor components by liquid chro- matography ⁄ electrospray ionization ion trap tandem mass spectrometry. Rapid Commun Mass Spectrom 17, 1366–1379. 18 Konigsberg W & Craig LC (1962) On bacitracin F. J Organic Chem 27, 934–938. 19 Pavli V & Kmetec V (2001) Optimization of HPLC method for stability testing of bacitracin. J Pharm Biomed Anal 24, 977–982. 20 Ikai Y, Oka H, Hayakawa J, Matsumoto M, Saito M, Harada K, Mayumi T & Suzuki M (1995) Total struc- tures and antimicrobial activity of bacitracin minor components. J Antibiot 48, 233–242. 21 Stepuro AI, Piletskaya TP & Stepuro II (2005) Role of thiamine thiol form in nitric oxide metabolism. Biochemistry (Mosc) 70, 339–349. 22 Smith AM, Chan J, Oksenberg D, Urfer R, Wexler DS, Ow A, Gao L, McAlorum A & Huang SG (2004) A high-throughput turbidometric assay for screening inhibitors of protein disulfide isomerase activity. J Biomol Screen 9, 614–620. 23 Ming L-J & Epperson JD (2002) Metal binding and structure–activity relationship of the metalloantibiotic peptide bacitracin. J Inorganic Biochem 91, 46–58. 24 Wang C, Chen S, Wang X, Wang L, Wallis AK, Freed- man RB & Wang CC (2010) Plasticity of human pro- tein disulfide isomerase: evidence for mobility around the X-linker region and its functional significance. J Biol Chem 285, 26788–26797. Bacitracin forms a disulfide bond with free PDI cysteines N. Dickerhof et al. 2042 FEBS Journal 278 (2011) 2034–2043 ª 2011 The Authors Journal compilation ª 2011 FEBS 25 Freedman RB, Klappa P & Ruddock LW (2002) Pro- tein disulfide isomerases exploit synergy between cata- lytic and specific binding domains. EMBO Rep 3, 136–140. 26 Rogelj S, Reiter KJ, Kesner L, Li M & Essex D (2000) Enzyme destruction by a protease contaminant in baci- tracin. Biochem Biophys Res Commun 273, 829–832. 27 Shnyder SD & Hubbard MJ (2002) ERp29 is a ubiqui- tous resident of the endoplasmic reticulum with a dis- tinct role in secretory protein production. J Histochem Cytochem 50, 557–566. 28 Holmgren A (1979) Thioredoxin catalyzes the reduction of insulin disulfides by dithiothreitol and dihydrolipoa- mide. J Biol Chem 254, 9627–9632. 29 Kleffmann T, Jongkees SA, Fairweather G, Wilbanks SM & Jameson GN (2009) Mass-spectrometric charac- terization of two posttranslational modifications of cys- teine dioxygenase. J Biol Inorg Chem 14 , 913–921. Supporting information The following supplementary material is available: Fig. S1. Assignment of MALDI-TOF ⁄ TOF MS signa- ture peaks for bacitracin analogs. Fig. S2. Disulfide bond formation between bacitracin and Cys314 on PDI. Fig. S3. Bacitracin binding to different proteins. This supplementary material can be found in the online version of this article. Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. N. Dickerhof et al. Bacitracin forms a disulfide bond with free PDI cysteines FEBS Journal 278 (2011) 2034–2043 ª 2011 The Authors Journal compilation ª 2011 FEBS 2043 . Bacitracin inhibits the reductive activity of protein disulfide isomerase by disulfide bond formation with free cysteines in the substrate-binding domain Nina. mechanism of action of bacitracin on PDI that involves covalent binding of an open thiol form of bacitracin to free cysteines in the substrate-binding domain of