Isotopically Labeled Desthiobiotin Azide (isoDTB) Tags Enable Global Profiling of the Bacterial Cysteinome

Article abstract of DOI:10.1002/anie.201912075

Rapid development of bacterial resistance has led to an urgent need to find new druggable targets for antibiotics. In this context, residue-specific chemoproteomic approaches enable proteome-wide identification of binding sites for covalent inhibitors. Described here are easily synthesized isotopically labeled desthiobiotin azide (isoDTB) tags that shortened the chemoproteomic workflow and allowed an increased coverage of cysteines in bacterial systems. They were used to quantify 59 % of all cysteines in essential proteins in Staphylococcus aureus and enabled the discovery of 88 cysteines that showed high reactivity, which correlates with functional importance. Furthermore, 268 cysteines that are engaged by covalent ligands were identified. Inhibition of HMG-CoA synthase was verified and will allow addressing the bacterial mevalonate pathway through a new target. Overall, a broad map of the bacterial cysteinome was obtained, which will facilitate the development of antibiotics with novel modes-of-action.

Full text of DOI:10.1002/anie.201912075

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Accepted Article
Title: Isotopically Labeled Desthiobiotin Azide (isoDTB) Tags Enable
Global Profiling of the Bacterial Cysteinome
Authors: Patrick R. A. Zanon, Lisa Lewald, and Stephan M. Hacker
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201912075
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10.1002/anie.201912075
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RESEARCH ARTICLE
Isotopically Labeled Desthiobiotin Azide (isoDTB) Tags Enable
Global Profiling of the Bacterial Cysteinome
Patrick R. A. Zanon, Lisa Lewald, and Stephan M. Hacker*
Abstract: Rapid development of bacterial resistance has led to an
urgent need to find new druggable targets for antibiotics. In this
context, residue-specific chemoproteomic approaches enable
proteome-wide identification of binding sites for covalent inhibitors.
Here, we describe isotopically labeled desthiobiotin azide (isoDTB)
tags that were easily synthesized, shortened the chemoproteomic
workflow and allowed an increased coverage of cysteines in bacterial
systems. We quantified 59% of all cysteines in essential proteins in
Staphylococcus aureus and discovered 88 cysteines with high
reactivity, which correlates with functional importance. Furthermore,
we identified 268 cysteines that are engaged by covalent ligands. We
verified inhibition of HMG-CoA synthase, which will allow addressing
the bacterial mevalonate pathway through a new target. Overall, a
broad map of the bacterial cysteinome was obtained, which will
facilitate the development of antibiotics with novel modes-of-action.
reactive molecules. This technology is especially well established
for profiling cysteine residues using methods based on the
isoTOP-ABPP (isotopic tandem orthogonal proteolysis activity-
based protein profiling) platform (Figure 1a).[2a] In this technology,
a proteome of interest is split into two samples. One of these
samples is treated with a covalent inhibitor and the other one with
DMSO as control. In the next step, both samples are treated with
iodoacetamide alkyne (IA-alkyne).[9] This probe will modify many
cysteines in both samples with alkynes and this reactivity will be
blocked by the covalent inhibitor at its specific binding sites. The
samples are next modified with isotopically labeled affinity tags
using copper-catalyzed azide-alkyne cycloaddition (CuAAC).[10]
The samples are combined, enriched on streptavidin beads,
proteolytically digested and the modified peptides eluted for mass
spectrometry (MS)-based quantification. Most quantified
cysteines will have ratios R ≈ 1 between the heavy and light
channel indicating no interaction with the covalent compound
(Figure 1a). In contrast, cysteines at the specific binding sites will
show ratios of R >> 1. In this way, quantitative and site-specific
interaction studies in the whole proteome are possible with
unmodified covalent inhibitors that do not need to be equipped e.g.
with an affinity handle.
Introduction
Infections with multidrug-resistant bacteria like methicillin-
resistant Staphylococcus aureus (MRSA) are emerging as major
threats to human health.[1] Nevertheless, very few novel classes
of antibiotics have been introduced to the clinic over the last
decades.[1] Furthermore, almost all approved antibiotics
exclusively interfere with a very limited set of bacterial targets,
involved in protein, nucleic acid and cell wall biosynthesis.[1]
Therefore, developing innovative methods to discover novel
druggable targets for antibiotics is a pivotal task to guarantee
efficient treatment of bacterial infections in the future.
In the last step of this protocol, the modified peptides need to be
eluted from the streptavidin beads for MS-based analysis. As
previous studies have utilized biotin, which binds almost
irreversibly to streptavidin, as an affinity handle, various cleavable
linkers have been applied in order to elute the peptides from the
beads.[11] These include linkers cleaved by proteases (Figure
1b),[2a,9] acidic[11b,12] or reductive conditions.[11b,13] Due to the high
requirements on the orthogonality of these linkers, they need to
be designed very carefully, which usually requires laborious multi-
step synthesis of the tags. Furthermore, the cleavage of the linker
adds another step to the chemoproteomic protocol.
Chemoproteomic approaches are extremely powerful to globally
understand which proteins are able to bind small molecules as
ligands[2] and are particularly straightforward for covalently
reactive molecules.[2a,2c,2d] Strikingly, covalent inhibitors have
seen a resurgence of interest for the development of novel drugs
as they can increase compound selectivity, reduce resistance
formation and target shallow protein pockets.[3] This has led to the
recent clinical approval of several covalent kinase inhibitors.[4]
Especially in the field of antibiotics, covalent inhibitors are
prevalent as exemplified by β-lactams,[3] fosfomycin,[5]
showdomycin[6] and optimized arylomycins[7].
Therefore, we set out to develop isotopically labeled desthiobiotin
azide (isoDTB) tags (Figure 1c) for residue-specific proteomics.
As desthiobiotin still binds very strongly to streptavidin, all steps
up to the proteolytic digestion can be kept the same.[14] Due to the
reversibility of binding of desthiobiotin to streptavidin, in the last
step, the peptides are then easily eluted using acidic conditions
with acetonitrile as the co-solvent.[14] Because a complex
cleavable linker is not needed, we designed these tags with
exclusively two isotopically differentiated glycine residues as the
linker moiety.
Recently, methods have emerged that globally identify the exact
interaction site of covalent inhibitors in
a
competitive
fashion.[2a,2b,8] In this way, many pockets that can bind covalent
ligands are identified in parallel using a small library of covalently
After establishing the utility of the isoDTB tags for residue-specific
proteomics, we used them to globally investigate cysteines in the
proteome of S. aureus for their reactivity and their potential to bind
covalent ligands. In this way, we identified 88 highly reactive
cysteines and more than 250 cysteines that can be addressed
with covalent ligands. These residues are starting points for the
development of antibiotics with novel modes-of-action.
M. Sc. Patrick R. A. Zanon, M. Sc. Lisa Lewald, Dr. Stephan M.
Hacker
Department of Chemistry
Technical University of Munich
Lichtenbergstrasse 4, 85748 Garching, Germany
E-mail: stephan.m.hacker@tum.de
Supporting information for this article is given via a link at the end of
the document.
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10.1002/anie.201912075
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Figure 1. a) Workflow for competitive, residue-specific chemoproteomic experiments.[2a] RG = reactive group; AH = affinity handle. b) Structure of the TEV protease-
cleavable tags (TEV tags) originally used for residue-specific proteomics.[2a,9] c) Structure of the isoDTB tags developed in this study.
Results and Discussion
We synthesized the isoDTB tags using solid-phase peptide
synthesis. For this purpose, a Rink amide resin and an Fmoc
strategy were utilized. We sequentially coupled ε-azido-lysine,
two glycine residues and desthiobiotin. We used glycine with the
natural isotope distribution for the light isoDTB tag and glycine
with two 13C atoms and one 15N atom for the heavy tag. In this
way, a total mass difference between the tags of 6 Da was
obtained. Purification by RP-HPLC resulted in a yield of
approximately 70% for both isoDTB tags.
To establish that the tags are applicable to broadly investigate
cysteines in a proteomic context, we treated two identical samples
of the lysate of the methicillin-sensitive S. aureus (MSSA) strain
SH1000[15] with 1 mM IA-alkyne and modified the two samples
with the light and heavy isoDTB tag, respectively, using CuAAC.
The samples were combined either in a ratio of 1:1 or 1:4.
Subsequently, we enriched the samples on streptavidin beads,
Figure 2. a) Ratios R of all quantified cysteines in the S. aureus SH1000
proteome in experiments, in which the light and heavy labeled samples were
digested the proteins with trypsin and eluted the modified peptides
using our straightforward approach. Analysis using liquid
chromatography coupled to tandem MS (LC-MS/MS) using a Q
Exactive Plus (Thermo Fisher) mass spectrometer and evaluation
using freely available MaxQuant software[16] identified 1155
cysteines that were quantified for both conditions (Figure 2a,
Supporting Information, Table S1). This analysis revealed a
narrow distribution of the detected ratios for both samples around
the expected values. The isoDTB tags therefore reliably allowed
quantification of cysteines in the whole bacterial proteome.
both reacted with 1 mM IA-alkyne, clicked to the isoDTB tags and mixed at the
indicated ratios. Expected values of log2(R) of 0 for the 1:1 mixture and 2 for the
1:4 mixture are indicated with dashed grey lines. b) Venn diagram comparing
the number of quantified cysteines in the S. aureus SH1000 proteome using
1 mM IA-alkyne and the TEV tags or the isoDTB tags, respectively. c) Number
of quantified cysteines in a variety of Gram-positive and Gram-negative bacteria
as well as in the human cell line MDA-MB-231 using the isoDTB tags. The grey
dashed line indicates 1000 quantified cysteines. All data results from duplicates.
We next benchmarked our technology against the TEV protease-
cleavable biotin tags (TEV tags) that have been most broadly
used to residue-specifically map proteomes (Figure 2b,
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Figure 3. a) Workflow for the measurement of cysteine reactivity using the isoDTB tags.[9a] DTB = desthiobiotin. b) Plot of the reactivity ratios (R10:1) obtained by
comparing S. aureus SH1000 proteomes treated with high (100 µM) vs low concentration (10 µM) of IA-alkyne (black). Ratios (R1:1) of an experiment with high
concentration used for both samples (grey) are used as a control to ensure reliable quantification of all cysteines. c), d) Percentage of cysteines in the different
reactivity bins that are in essential proteins (c)[17] or at functional sites (d).
Supporting Information, Table S1).[2a,9] Our isoDTB tags
outperformed the TEV tags by quantifying 27% more cysteines in
the S. aureus proteome. We were able to increase the number of
cysteines quantified with the isoDTB tags even more by
additionally using chymotrypsin and AspN for the proteolytic
digest (Supporting Information, Figure S1 and Table S1). This is
not possible for the TEV tags as these proteases would cleave
the tag itself. In this way, we quantified a total of 1643 cysteines
in the S. aureus proteome using the isoDTB tags in only six
experiments. We next investigated the performance of the isoDTB
tags with IA-alkyne in different Gram-positive and Gram-negative
bacteria (Figure 2c, Supporting Information, Table S1) and
consistently quantified more than 1000 cysteines in each strain.
Moreover, we were able to quantify more than 3500 cysteines in
the human cell line MDA-MB-231, which is competitive with
previously described methods.[2a] Therefore, our isoDTB tags not
only shortened the chemoproteomic protocol but also led to
increased coverage in bacterial systems compared to the widely
used TEV tag technology.
concentration many cysteines were labeled, at the low
concentration only the most reactive cysteines were labeled
quantitatively. After CuAAC with the light (low concentration) and
heavy isoDTB tags (high concentration), respectively, the
samples were analyzed in the same way as described above.
Here, high ratios (R10:1) indicate low reactivity cysteines, whereas
the most reactive cysteines have R10:1 ≈ 1. Using this procedure,
we quantified 921 cysteines and identified 88 highly reactive
cysteines with R10:1 < 3 in 69 different proteins (Figure 3b,
Supporting Information, Table S2). Another 240 cysteines
showed medium reactivity (3 < R10:1 < 5), whereas the remaining
593 cysteines were of low reactivity (R10:1 > 5). Cysteines of all
three bins of reactivity were evenly distributed throughout the
different functional classes of proteins (Supporting Information,
Figure S2).[18] Interestingly, highly reactive cysteines were
depleted in essential proteins[17] in comparison to their
counterparts of lower reactivity (Figure 3c). It can be speculated
that evolutionary pressure has selected against highly reactive
cysteines in essential proteins as these would interact with many
reactive small molecule electrophiles that occur in nature.
We next applied our method to analyze the reactivity of cysteines
in the bacterial proteome (Figure 3a).[9a] As the reactivity of
cysteines is linked to their functional relevance in human cells,[9a]
we reasoned that this feature might also be conserved in bacteria
and in this way lead to the identification of functionally important
cysteine residues. In order to study cysteine reactivity, two
identical samples of the proteome of the S. aureus strain SH1000
were treated with either a high (100 µM) or a low (10 µM)
concentration of IA-alkyne. In this way, while at the high
There is a strong enrichment of the highly and medium reactive
cysteines at functional sites (Figure 3d). These cysteines include
many residues that are directly involved in the catalytic
mechanism (e.g. C178 in the GTP cyclohydrolase FolE2 (UniProt
code Q2G0L1),[19] C112 in FabH (UniProt code Q2FZS0) and C88
in the probable acetyl-CoA acyltransferase (UniProt code
Q2G124), which are all essential proteins). Furthermore, several
highly reactive cysteines are close to cofactor-binding sites (e.g.
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C239 of the CTP synthase PyrG (UniProt code Q2FWD1) and
could be observed (Supporting Information, Figure S8b)
C45 in MnmG (UniProt code Q2FUQ3)) or metal-binding sites (e.g. indicating that it is possible to identify highly active and still
C145 of alcohol dehydrogenase Adh (UniProt code Q2G0G1) and
C65 of biotin synthase BioB (UniProt code Q2FVJ7)). Therefore,
residue-specific proteomics using our isoDTB tags allowed global
profiling of the reactivity of cysteines in the bacterial proteome and
enabled the identification of functionally relevant residues.
selective electrophiles. As we cannot rule out unspecific effects
for the highly promiscuous compounds, we excluded these from
all further analysis. While the low promiscuity compounds are
most interesting for further compound development, the medium
promiscuity compounds are most useful for the global profiling
approach performed here.
We next set out to study which cysteines in the S. aureus
proteome can be targeted with covalent ligands. For this purpose,
we obtained a library of 211 electrophilic cysteine-directed
compounds (EN001 - EN211, Supporting Information, Table S3),
mainly α-chloroacetamides. These compounds were initially
screened for antibacterial activity by performing minimum
inhibitory concentration (MIC) experiments. While we did not
expect these small compounds to be completely specific, we used
this phenotypic pre-filter in order to prioritize compounds, whose
target spectrum includes essential proteins that can be addressed
in intact cells. Based on an initial screen in three MSSA strains,
we selected 24 compounds (Supporting Information, Figure S3)
based on their MIC values and structural diversity for further
studies. Interestingly, many of these compounds contain a
2-aminothiazole moiety, which seems to be beneficial for activity.
23 compounds had MICs of ≤ 100 µM in all three strains with six
compounds having MICs of ≤ 12.5 µM in all three strains
(Supporting Information, Figure S4). Furthermore, 14 compounds
showed activity (MIC ≤ 100 µM) in two tested MRSA strains with
two compounds (EN085 and EN177) having an MIC ≤ 10 µM in all
five tested strains. This shows that electrophilic compounds with
desired biological activity could efficiently be identified from a
small compound library.
Taking all 25 investigated conditions together (19 compounds at
200 µM, five compounds at 20 µM and a DMSO control) we
compiled a competitive data table (Supporting Information, Table
S4), which includes all cysteines that were quantified for at least
three of the conditions. In this way, we obtained information on
1756 cysteines in 905 different proteins, which corresponds to a
coverage of 33% of all the cysteines encoded in the S. aureus
genome. As cysteines in essential proteins[17] are enriched in our
data over the genomic background (Supporting Information,
Figure S9a), this equates to the quantification of 59% of all
cysteines in essential proteins. Each cysteine was quantified on
average for 21 of the 25 conditions (Supporting Information,
Figure S10). Therefore, our method allowed obtaining information
on many cysteine residues in S. aureus in a reproducible manner.
268 cysteines in 200 different proteins were engaged by at least
one ligand (Figure 5a). In many proteins, we detect one ligandable
cysteine that is engaged by several compounds, while the other
cysteine(s) are never engaged (Supporting Information, Figure
S11). This indicates that our method measures local target
engagement of the cysteines rather than global changes to the
protein structure. While ligandable cysteines are enriched in
enzymes, we also identify them in other functional classes of
proteins (Supporting Information, Figure S12).[18] Ligandable
cysteines are enriched at functional sites (Supporting Information,
Figure S13) and similarly abundant in essential proteins as
compared to other quantified cysteines (Supporting Information,
Figure S9b). When we compared this data to the cysteine
reactivity data (Supporting Information, Figure S14), we could see
that, while the highly reactive cysteines are clearly more likely to
be ligandable, there are also many ligandable cysteines of
medium and low reactivity indicating that specific non-covalent
interactions are important in these cases.
The selected 24 compounds were screened at 200 µM
concentration in residue-specific chemoproteomic experiments
using our isoDTB tags in duplicates (Figure 1a, Supporting
Information, Figure S5 and Figure S6). For three of the
compounds (EN007, EN085 and EN177), we performed an
additional set of biologically independent duplicates. Due to the
high reproducibility between the biologically independent
experiments (Supporting Information, Figure S5), we performed
the remaining profiling in duplicates and prioritized screening
more compounds over performing more replicates. Five of the
compounds (EN007, EN085, EN135, EN177 and EN201) that
showed MIC values ≤ 25 µM in all five tested strains were
additionally tested at 20 µM concentration (Supporting Information,
Figure S7).
Next, we looked at the binding of our covalent ligands to a
selection of ligandable cysteines in more detail (Figure 5b). While
the most ligandable cysteines tend to be engaged by the most
promiscuous compounds, there is clear evidence for more
specific interactions between less ligandable cysteines with more
selective compounds. For example, the active site residue C112
of FabH (UniProt code Q2FZS0), an essential enzyme in fatty acid
synthesis,[20] is exclusively targeted by three compounds of
tempered promiscuity (EN002, EN204 and EN208, Figure 5b).
This residue has previously been shown to be covalently modified
e.g. by the inhibitors 4,5-dichloro-1,2-dithiol-3-one and
cerulenin.[21] Furthermore, the residue C323 in the isoleucine-
tRNA ligase IleS (UniProt code Q2FZ82) is only targeted by
another set of three compounds (EN012, EN077 and EN201,
Figure 5b), which could open up the possibility of inhibiting
In all experiments, we consider cysteines that have a ratio of R > 4
(log2(R) > 2) and whose R-value is statistically significantly
different from R = 1 (p-value < 0.05 in a one-sample t-test) to be
engaged by the covalent ligand. We identified a large range of
values for the fraction of cysteines that are engaged by the
different compounds (Figure 4a‑c, Supporting Information Figure
S6 and Figure S8a). Nine compounds showed low promiscuity
(< 2% of all quantified cysteines are engaged, Figure 4a), ten
compounds showed medium promiscuity (2% to 10%, Figure 4b)
and five compounds showed excessive promiscuity (> 10%,
Figure 4c). Strikingly, no correlation between MIC and promiscuity
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Figure 4. a‑c) Volcano plots for three representative compounds of low (a), medium (b) and excessive promiscuity (c). Plots show the log2(R) of the ratio between
the heavy (DMSO-treated) and light (compound-treated) channels and the –log10(p) of the statistical significance in a one-sample t-test for all quantified cysteines.
In plot a) the data point for the ligandable active site residue C111 of the essential putative HMG-CoA synthase (UniProt code Q2FV76) is highlighted in grey. All
data results from duplicates. For EN007 an additional set of biologically independent duplicates was included.
bacterial translation through a novel target. Overall, we detect
many binding events that are strongly dependent on the
compound and on the targeted cysteine indicating that our
method can detect specific ligand-binding events.
is modified by EN085, which opens up the possibility to target
translation through a novel mechanism. The highly reproducible
results between the MSSA and MRSA strains demonstrate that
our data delivers a broadly applicable map of ligandable cysteines
in the S. aureus proteome that will guide the design of antibiotics
with novel modes-of-action.
Looking at the targets of the two compounds that showed the best
antibacterial activity in the initial MIC assays (EN085 and EN177,
Figure 5c,d), we saw that both compounds show engagement of
several cysteines at 20 µM (31 for EN085, 10 for EN177). Both
In order to validate the interaction of a selected compound with
an identified ligandable cysteine, we investigated C111 of the
compounds strongly target C152 of MurC (UniProt code Q2FXJ0), putative HMG-CoA synthase (UniProt code Q2FV76), which is an
which is a key enzyme essential for cell wall synthesis.[22] EN177
additionally binds to C410 of pyruvate kinase (UniProt code
Q2FXM9) and C88 of the essential probable acetyl-CoA
acyltransferase (UniProt code Q2G124). The latter cysteine forms
an acyl-thioester intermediate during catalysis.[23] EN085 binds to
C1030 in the DNA polymerase PolC (UniProt code Q2G1Z8),
C199 in the tRNA-specific methyl transferase MnmA (UniProt
code Q2FXV6), which forms a cysteine persulfide intermediate
during catalysis,[23] as well as the catalytically active nucleophile
C151 in glyceraldehyde-3-phosphate dehydrogenase (UniProt
code Q2G032).[24] Both compounds, therefore, bind to several
essential target proteins that have the potential to become novel
targets of covalent antibiotic compounds.
essential enzyme in the mevalonate pathway and in this way
might open up targeting bacteria through this so far clinically
unexplored pathway.[25] In gel-based experiments (Figure 6a,
Supporting Information, Figure S17), strong labeling by IA-alkyne
was observed for the recombinant wildtype protein, but not for the
C111A mutant. This is in good agreement with the high reactivity
of C111 (R10:1 = 0.84) in the reactivity experiments (Figure 3,
Supporting Information, Table S2). Furthermore, the low
promiscuity compound EN106 that we identified to target HMG-
CoA synthase (Figure 4a) blocked labeling at low micromolar
concentrations indicating covalent binding of this compound to
C111. Using intact protein MS (IPMS, Figure 6b, Supporting
Information, Figure S18, S19), we detected quantitative single
modification of the HMG-CoA synthase wildtype with EN106. No
modification of the C111A mutant was detectable, strongly
indicating that C111 is the site of covalent modification with
EN106. In order to study the activity of HMG-CoA synthase
(Figure 6c, Supporting Information, Figure S20) we set up a
coupled assay with acetyl-CoA-acyl transferase (ACAT).[26] ACAT
forms acetoacetyl-CoA from two molecules of acetyl-CoA.
HMG-CoA synthase then catalyzes the reaction with another
molecule of acetyl-CoA to give HMG-CoA. The free thiol group of
the CoA-SH liberated in both steps was detected using Ellman’s
reagent (Figure 6c). Addition of Wildtype HMG-CoA synthase to
the assay strongly increased the formation of free CoA-SH over
the ACAT background reaction. This activity was reduced to the
level without HMG-CoA synthase, when the C111A mutant was
used or when the wildtype was pretreated with 10 µM EN106.
EN106 had no effect on the detected ACAT activity in absence of
HMG-CoA synthase or presence of the inactive C111A mutant
In order to investigate if the results obtained in the MSSA strain
SH1000 are transferable to other S. aureus strains, compounds
EN085 and EN177 were additionally screened at 20 µM in the
MRSA strain USA300, for which they show MIC values of 6.3 µM
and 3.1 µM, respectively (Supporting Information, Figure S15 and
Table S5). We detect a very good correlation of the data obtained
in the two different strains (Supporting Information, Figure S16).
All cysteines in essential proteins discussed above were also
engaged by the same compound in USA300. While no new
engaged cysteines were identified for EN177, we identified five
additional engaged cysteines for EN085 in USA300 that were not
quantified at all in SH1000. Among those, two cysteines are in
essential proteins.[17] EN085 binds to the active site C119 in MurA
(UniProt code A0A0H2XGP3), which is a key enzyme in cell wall
biosynthesis[22] and also targeted by fosfomycin.[5] Additionally,
C565 in the aspartate-tRNA ligase AspS (UniProt code Q2FG97)
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Figure 5. a) Total number of ligandable and other quantified cysteines and proteins in our competitive data table. b) Heat map of the log2(R) values for a selection
of ligandable cysteines with all tested compounds at 200 µM. Cysteines discussed in the text are highlighted in boldface. Compounds are sorted from left to right by
decreasing promiscuity and cysteines are sorted from top to bottom by decreasing number of identified ligands. c,d) Volcano plots for compounds EN085 (c) and
EN177 (d) at 20 µM in S. aureus SH1000. Plots show the log2(R) of the ratio between the heavy (DMSO-treated) and light (compound-treated) channels and the –
log10(p) of the statistical significance in a one-sample t-test for all quantified cysteines. Ligandable cysteines in essential proteins that are engaged by the respective
compound are highlighted in red. 1: C152 of MurC (UniProt code Q2FXJ0), 2: C199 of MnmA (UniProt code Q2FXV6), 3: C1030 of PolC (UniProt code Q2G1Z8),
4: C151 of glyceraldehyde-3-phosphate dehydrogenase (UniProt code Q2G032), 5: C410 of pyruvate kinase (UniProt code Q2FXM9), 6: C88 of probable acetyl-
CoA acyltransferase (UniProt code Q2G124). All data results from duplicates. For compounds EN007, EN085 and EN177 in panel b) and all data in panels c) and
d) an additional biologically independent set of duplicates was also included.
(Supporting Information, Figure S21) showing that EN106 does
not inhibit the ACAT reaction or hinder detection by alkylation of
the free CoA-SH or the product of the Ellman’s reagent.
Conclusion
Furthermore, inhibition remained after gel-filtration to remove
excess free EN106 showing irreversible inhibition and further
excluding interference of EN106 with other components of the
assay (Supporting Information, Figure S22). Covalent
modification of HMG-CoA synthase at C111 with compound
EN106 therefore led to effective inhibition of its activity. HMG-CoA
synthase is therefore a promising target for the development of
novel antibiotics that interfere with the essential mevalonate
pathway.
We describe the synthesis of isotopically labeled desthiobiotin
azide (isoDTB) tags and their application in chemoproteomic
experiments. These tags were easily synthesized by solid-phase
peptide synthesis in high yields and showed excellent
physicochemical properties. By using desthiobiotin, these tags
circumvented the need to use complex cleavable linkers[11a] for
peptide elution and thus significantly shortened the
chemoproteomic protocol, while increasing the coverage of
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10.1002/anie.201912075
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proteomes. Interestingly, highly reactive cysteines were less likely
to be found in essential proteins. This points to the fact that
evolution may have selected against highly reactive cysteines in
essential proteins in order to protect bacteria from the influence of
reactive electrophiles occurring in nature during metabolism or as
environmental chemicals.
Finally, we applied the isoDTB tags to broadly understand, which
cysteines in the bacterial proteome can be engaged with covalent
ligands. For this purpose, we compiled the competitive data for 19
α-chloroacetamides and were able to profile 1756 cysteines
including 59% of all cysteines in essential proteins. We identified
268 cysteines that can bind covalent ligands in 200 different
proteins. The targeted cysteines include many functionally
relevant residues in essential proteins involved in many different
pathways. In this way, the data presented will be the starting point
for more specific covalent inhibitors to develop antibiotics with
novel modes-of-action. The presented isoDTB tags will allow
monitoring the on- and off-target effects of the compounds and in
this way streamline the development process.
We investigated inhibition of HMG-CoA synthase in more detail.
Modification at the ligandable cysteine residues was detected and
this interaction led to inhibition of the enzyme activity. HMG-CoA
synthase inhibition by modification of C111 using the human
HMG-CoA synthase inhibitor hymeglusin has been described but
this inhibitor suffers from a short half-life of the thioester in the
covalent protein adduct.[27] Therefore, permanent covalent
inhibition by the low promiscuity compound EN106 is a very
promising starting point to explore the antibiotic potential of this
protein.[25] This case study demonstrates that our map of
ligandable cysteines is an excellent resource to quickly identify
residues that can be targeted in a functionally relevant manner.
Figure 6. a) Result of gel-based labeling experiments with HMG-CoA synthase.
1 µM recombinant wildtype (WT) HMG-CoA synthase was added into 1 mg/mL
soluble lysate of S. aureus SH1000. As controls, 1 µM of the HMG-CoA
synthase mutant (C111A) or no HMG-CoA synthase (none) were added. The
samples were treated with the indicated concentrations of EN106 or with DMSO
as control. The samples were labeled with IA-alkyne and modified with TAMRA-
azide using CuAAC. Analysis using SDS-PAGE with subsequent in-gel
fluorescence scanning and Coomassie staining is shown. b) IPMS analysis of
the modification of HMG-CoA synthase by EN106. 1 µM HMG-CoA synthase
wildtype (WT) or mutant (C111A) was treated with DMSO as control or 10 µM
EN106. Deconvoluted IPMS spectra are shown. The mass difference between
the wildtype treated with EN106 or DMSO (∆m = 258) exactly corresponds to
the modification of the protein with one molecule of EN106. c) Results of activity
assays with HMG-CoA synthase. 1 µM HMG-CoA synthase wildtype (WT) was
treated with 10 µM EN106 or DMSO as a control. Acetyl-CoA, acetyl-CoA-acyl-
transferase (ACAT) and Ellman’s reagent were added and the reaction progress
was followed by measuring the absorbance at 410 nm over time. HMG-CoA
synthase activity was calculated by a linear fit of the linear portion of this curve.
Controls with the HMG-CoA synthase mutant (C111A), no HMG-CoA synthase
or no acetyl-CoA-acyl-transferase (no ACAT) were included. The graph shows
Taken together, our isoDTB tags are important new tools for
residue-specific proteomics in bacterial systems. They allowed
studying the bacterial cysteinome globally and should be
transferable to studying other amino acids in a straightforward
manner.[8] The cysteines that were characterized to be able to
bind to covalent ligands in this study are the foundation for the
development of covalent inhibitors that could lead to antibiotics
with totally new modes-of-action.
mean
± standard deviation. All data results from triplicates. mAU: milli
absorbance units.
cysteines in the proteome of S. aureus. The isoDTB tags allowed
quantifying many cysteines across different Gram-positive and
Gram-negative bacterial proteomes and gave results comparable
to the TEV tags also in the human proteome.[2a]. Due to the easy
synthesis of the tag, the shortened workflow, the use of freely
available MaxQuant data evaluation software[16] and the excellent
performance, this technology will make residue-specific
proteomics applicable in many laboratories not specialized in
chemoproteomics.
Acknowledgements
We gratefully acknowledge Prof. Dr. Stephan A. Sieber and his
group for their generous support. We thank Dr. Markus
Lakemeyer and Dr. Franziska Mandl for critical reading of the
manuscript. SMH and PRAZ acknowledge funding by the Fonds
der Chemischen Industrie through a Liebig Fellowship and a
Ph.D. fellowship. We acknowledge financial support by the TUM
Junior Fellow Fund.
The isoDTB tags were applied to study the reactivity of cysteines
in the proteome of S. aureus. We identified 88 highly reactive
cysteine residues that are strongly enriched at functional sites of
proteins. This indicates that reactivity of cysteines is a proxy for
the functional relevance of certain residues also in bacterial
Keywords: Antibiotics • Covalent Inhibitors • Isotopic labeling•
Protein modifications • Proteomics
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10.1002/anie.201912075
Angewandte Chemie International Edition
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10.1002/anie.201912075
Angewandte Chemie International Edition
RESEARCH ARTICLE
RESEARCH ARTICLE
Patrick R. A. Zanon, Lisa Lewald, and
Stephan M. Hacker*
Page No. – Page No.
Isotopically Labeled Desthiobiotin
Azide (isoDTB) Tags Enable Global
Profiling of the Bacterial Cysteinome
Isotopically labeled desthiobiotin azide (isoDTB) tags were synthesized in a
straightforward manner and allowed residue-specific profiling of the cysteinome in
S. aureus. In this way, 268 cysteines that can be engaged by covalent ligands were
identified. These cysteines are targets for the development of antibiotics with novel
modes-of-action.
This article is protected by copyright. All rights reserved.