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

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

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.

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 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

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

)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]
+

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

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-

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

⁄
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

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