Design and synthesis of ATP-based nucleotide analogues and profiling of nucleotide-binding proteins

Article abstract of DOI:10.1021/bc100592q

Two nucleotide-based probes were designed and synthesized in order to enrich samples for specific classes of proteins by affinity-based protein profiling. We focused on the profiling of adenine nucleotide-binding proteins. Two properties were considered in the design of the probes: the bait needs to bind adenine nucleotide-binding proteins with high affinity and carry a second functional group suitable and easily accessible for coupling to a chromatography resin. For this purpose, we synthesized p-biotinyl amidobenzoic acid-ATP (p-BABA-ATP) and p-biotinyl aminomethylbenzoic acid-ATP (p-BAMBA-ATP). p-BABA-ATP and p-BAMBA-ATP both bind to ATP-binding cassette (ABC) proteins with at least 10-fold higher affinity than ATP. Several ABC transporters could be enriched using p-BABA-ATP or p-BAMBA-ATP.

Full text of DOI:10.1021/bc100592q

ARTICLE
pubs.acs.org/bc
Design and Synthesis of ATP-Based Nucleotide Analogues and
Profiling of Nucleotide-Binding Proteins
Justina. C. Wolters,† Gerard Roelfes,‡ and Bert Poolman*,†
†Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute,
Netherlands Proteomics Centre & Zernike Institute for Advanced Materials
‡Department of Biomolecular Chemistry, Stratingh Institute for Chemistry
University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands
S Supporting Information
b
ABSTRACT: Two nucleotide-based probes were designed and
synthesized in order to enrich samples for specific classes of
proteins by affinity-based protein profiling. We focused on the
profiling of adenine nucleotide-binding proteins. Two proper-
ties were considered in the design of the probes: the bait needs
to bind adenine nucleotide-binding proteins with high affinity
and carry a second functional group suitable and easily acces-
sible for coupling to a chromatography resin. For this purpose,
we synthesized p-biotinyl amidobenzoic acid-ATP (p-BABA-
ATP) and p-biotinyl aminomethylbenzoic acid-ATP (p-BAM-
BA-ATP). p-BABA-ATP and p-BAMBA-ATP both bind to
ATP-binding cassette (ABC) proteins with at least 10-fold
higher affinity than ATP. Several ABC transporters could be
enriched using p-BABA-ATP or p-BAMBA-ATP.
’ INTRODUCTION
groups) have been prepared, yielding nucleotide analogues with
different functionalities. The reporter analogues can be divided in
molecules with spectroscopic or radioactive moieties and probes
that can bind to antibodies or column based materials (like a
biotin moiety).12ꢀ15 The features of ATP analogues in studies of
nucleotide-binding proteins, in combination with their struc-
tures, have been exploited by us to find patterns for modifica-
tions. ATP analogues with small modifications at the nucleobase
(like azido-ATP, εATP) and phosphate (AMP-PNP, AMP-PCP,
and ATPγS) of ATP are tolerated by most adenine nucleotide-
binding proteins. Bulkier groups are generally restricted to either
the phosphate or ribose ring. The extents to which these modifica-
tions are tolerated vary for the different classes (adenine) nucleotide-
binding proteins.
Adenine nucleotide-binding proteins cover a large range of
protein classes. They can use ATP as source of metabolic energy in
living cells, fueling of transport (via transport ATPases) or move-
ment (via molecular motors), and phosphorylation of enzymes
and small metabolites via kinases. Most often, ATP (binding and/
or hydrolysis) is used in coupling reactions and drives the ender-
gonic steps. Moreover, it is a substrate and/or cofactor involved
in DNA/RNA modifying processes like transcription, DNA
In activity-based protein profiling, protein classes are enriched
via a reactive probe that requires the protein or protein domain to
be functional. The probe (bait) contains a reactive group and a
reporter tag, usually connected via a linker moiety. The binding
moiety is designed to react with a specific class of proteins, and
the reporter tag of the probe enables their detection and/or
enrichment (via fluorescent, affinity, antigen, or isotope-labeled
reporter tags). The use of different binding moieties and reactive
groups can help in the analysis of the active site of the proteins.
The probes can be used to screen for inhibitors via competitive
binding studies, with the clear advantage that experiments can be
performed in complex mixtures without the requirement for
prior enrichment steps (e.g., as used for the identification of drug
targets.1 This protein profiling is routinely done in cell homo-
genates rather than in vivo, because the size of the probes limits
their uptake and distribution in the cells. This limitation can be
overcome by splitting the label into two parts: a cross-linking
group and a reporter tag (fluorescent probe or biotin tag). Both
parts of the probe contain an extra reactive group to attach the
two halves, using so-called click chemistry (see refs 2,3,4 for click
chemistry implemented in activity-based probes). Numerous
reviews about activity-based proteomics and their applications
have been published in recent years.5ꢀ11
Received: January 4, 2011
For the use of ATP analogues, modifications to all three
moieties of ATP (the nucleobase, ribose ring, or phosphate
Revised:
Published: June 21, 2011
April 21, 2011
r
2011 American Chemical Society
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replication, recombination, and restriction. Besides its function
as energy storage unit or substrate for the biosynthesis of DNA/
RNA, ATP acts as a signaling molecule for proteins belonging
to the class of purinergic receptors. The probes described in
this paper were designed for screening of proteins from the
ATP-binding cassette (ABC) superfamily but are likely to have
broader uses.
p-Biotinyl-amidobenzoic Acid (p-BABA). 1H NMR (300 MHz,
CD3SOCD3): δ = 1.34ꢀ1.62 (m, 6H), 2.33 (t, J = 7.3 Hz, 2H),
2.56 (d, J = 12.5, 1H), 2.80 (dd, J = 12.5, 5.1 Hz, 1H), 3.07ꢀ3.13
(m, 1H), 4.12 (t, J = 5.1 Hz, 1H), 4.29 (t, J = 6.2 Hz, 1H), 6.39 (d,
J = 23.4 Hz, 2H), 7.68 (d, J = 8.8 Hz, 2H), 7.85 (d, J = 8.8, 2H).
13C NMR (200 MHz, CD3SOCD3): δ = 25.1, 28.3, 36.5, 55.5,
59.4, 61.3, 118.5, 125.1, 130.5, 143.4, 163.0, 167.2, 172.1. ESI-MS:
calculated mass C17H21N3O4S 363.44, found m/z [M]ꢀ 362.6.
p-Biotinyl-amidobenzoic Acid (p-BAMBA). 1H NMR (300 MHz,
CD3SOCD3): δ 1.31ꢀ1.57 (m, 6H), 2.14 (t, J = 7.1 Hz, 2H), 2.56
(d, J = 12.5 Hz, 1H), 2.81 (dd, J = 12.5, 5.1 1H), 3.06ꢀ3.09 (m,
1H), 4.11 (t, J = 5.1 Hz, 1H), 4.27ꢀ4.31 (m, 3H), 6.39 (d, J =
21.2 Hz, 2H), 7.33 (d, J = 8.4 Hz, 2H), 7.87 (d, J = 8.4 Hz, 2H), 8.41
(t, J = 5.5 Hz, 1H). ESI-MS: calculated mass C18H23N3O4S 377.47,
found m/z [M]ꢀ 376.2.
Synthesis Biotinyl-linker-ATP. The synthesis of the biotinyl-
linker-ATP complex is adapted from the synthesis of 30-O-(40-
benzoyl)benzoyl-ATP as described by Williams et al.27 Glassware
was dried at 160 °C prior to use. Biotinylated linker (0.5 mmol)
was dissolved in dry DMF (3 mL) together with carbonyldiimi-
dazole (CDI) (1.2 mmol), keeping the setup with the dried glass-
ware under a flow of nitrogen. The reaction was monitored by the
evaporation of CO2 gas, which stopped after 1 h after addition of
CDI. Then, ATP (0.5 mmol) in H2O (12 mL) was added to the
mixture, and the reaction was incubated for 6 h under constant
stirring at room temperature.
Within the class of the adenine nucleotide-binding proteins,
ABC proteins play an important role in a wide range of physio-
logical functions, mostly involving translocation of compounds
across the membrane (so-called ABC transporters).16 At least
eighteen of the human ABC genes are associated to diseases,
including cystic fibrosis (ABCC7), Tangier disease (ABCA1),
immune deficiency (ABCB2/3), and many others (listed in
ref 17 and on http://nutrigene.4t.com/humanabc.htm).
ABC transporters share a conserved overall architecture with
two conserved nucleotide-binding domains (NBDs) and two
transmembrane domains (TMDs). The transmembrane domains
form a pathway through the membrane for the translocation of
the substrates. The energy needed for the translocation is
provided by ATP that binds to the NBDs and is hydrolyzed at
these sites. A limited number of ATP analogues have been ex-
ploited to probe functional and structural aspects of ABC pro-
teins. The fluorescent ATP analogues trinitrophenyl-ATP
(TNP-ATP), but also the cross-linking azido-derivatives,18 have
been used to determine binding constants for nucleotides.19,20
The non- (or slowly) hydrolyzable analogues AMP-PNP21ꢀ23
and ATPγS 24,25 have been used to dissect the effects of adenine
nucleotide binding and hydrolysis. Profiling of ABC proteins has,
to the best of our knowledge, not been reported.
The crude reaction mixture was purified in batches (5 mL)
by RP-HPLC, using a preparative C18 column (prepared from
Vydac218TPB_1015RPC column material; column dimensions
€
25/132) connected to an Akta-purifier system. The column was
Here, we describe the synthesis of two adenine nucleotide
analogues, p-biotinyl amidobenzoic acid-ATP (p-BABA-ATP)
and p-biotinyl aminomethylbenzoic acid-ATP (p-BAMBA-
ATP), their structural characterization, and their binding to the
ABC transporter OpuA. As a proof of principle for the profiling of
ABC transporters, several ABC and non-ABC transporters were
used for the characterization of the ATP analogues.
run at a flow rate of 3 mL/min, using buffer A, triethylammo-
niumacetate (TEAA, 10 mM) pH 7, and buffer B, acetonitrile
plus TEAA pH 7 (10 mM), and the following gradient: 35 min
5% buffer B, 75 min gradient 5ꢀ35% buffer B, 15 min 90% buffer
B, 15 min 0% buffer B. Fifteen millliliter fractions were collected
and analyzed by ESI-MS to determine the fractions containing
the ATP analogue. ATP analogue peak fractions of multiple runs
were pooled and freezeꢀdried, and the material was then re-
suspended in TEAA pH 7 (200 mM) and loaded onto a 1 mL
monoQ sepharose column, pre-equilibrated with 20 column
volumes TEAA pH 7 (200 mM). The column was washed with
2 ꢁ 10 column volumes TEAA pH 7 (200 mM) and eluted with
4 ꢁ 5 column volumes TEAA pH 7.0 (1M). Multiple rounds of
freezeꢀdrying (the solids were resuspended in Milli-Q) were
done to remove the (volatile) TEAA pH 7 completely. The yield
of pure product was about 20% based on the amount of the pure
ATP analogues (dry weight). The final products were analyzed
with ESI-MS:
p-Biotinyl-amidobenzoic Acid-ATP (p-BABA-ATP). (ESI-MS),
calculated mass C27H35N8O16P3S 852,61, found m/z [M]ꢀ
851.71 (ACN (50%) plus formic acid (0.1%))/ 851.3 (methanol).
p-Biotinyl-amidobenzoic acid-ATP (p-BAMBA-ATP). (ESI-
MS), calculated mass C28H37N8O16P3S 866.64, found m/z
[M]ꢀ 865.6 (ACN (50%) plus formic acid (0.1%)).
Spectral Properties. UV spectra of p-BABA-ATP and
p-BAMBA-ATP were measured on a Cary100 Bio UVꢀvisible
spectrophotometer (Varian) with samples of 400 μL (20 μM in a
quartz cuvette, with maxima for p-BABA-ATP (264 nm),
p-BABA (260 nm), ATP (258 nm), p-BAMBA-ATP (245 nm),
and p-BAMBA (230 nm). The molar extinction coefficient was
not altered upon modification of ATP, and under the experi-
mental conditions, ATP, p-BABA-ATP, and p-BAMBA-ATP all
’ EXPERIMENTAL SECTION
Synthesis of Biotinylated Linkers. The synthesis of biotiny-
lated linkers is based on a description of biotinylated amino acid
precursors by Skander et al.26 Briefly, biotin (2.1 mmol) was dis-
solved in DMF (40 mL) with tributylamine (0.64 mL, 2.7 mmol).
isobutyl chloroformate (0.32 mL, 2.5 mmol) was added to the
solution to activate the biotin, and the mixture was incubated
at room temperature for about 15 min after which it was cooled
on ice. Meanwhile, linker (4.1 mmol) was dissolved in DMF
(40 mL) and cooled on ice. After cooling, the activated biotin
mixture was slowly added to the linker dissolved in DMF, and the
mixture was incubated for two hours at 5 °C. The solvent was
removed under vacuum, the crude precipitate was recrystallized
from 3:1 ethanol/H2O, and the obtained crystals were washed
extensively with water and ethanol. The reaction was performed
with two different linkers: p-aminobenzoic acid (p-ABA) and
p-aminomethylbenzoic acid (p-AMBA). Both biotinylated lin-
kers recrystallized as a white solid, but the recrystallized solid of
the biotinylated p-aminomethylbenzoic acid was more hygro-
scopic compared to the solid of the biotinylated p-aminobenzoic
acid. The yield of pure biotinylated linker was about 40% and the
purity of the products was analyzed by NMR:
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Figure 1. X-ray crystallography structure of the ATPase dimer of the ABC transporter MJ0796 (1L2T).49 The structure was solved in the presence of
ATP (panel A, ATP green/orange), showing the position of the nucleotides. One of the ATP molecules was overlaid with the ATP analogue p-BABA-
ATP (in blue/orange). Panel B depicts an enlargement of a space-filling model of MJ0796 with the ATP analogue, showing the accessibility of the
biotin group.
had molar extinction coefficients of around 15 000 Mꢀ1 cmꢀ1 at
their respective absorption maxima.
TNP-ATP were added via a Hamilton syringe pump (Harvard
apparatus) in steps of 4.5 μL ATP (2 mM) and for the ATP
analogues (0.14 mM) using stock solutions of ATP (357 mM)
and for the ATP analogues (25 mM), both dissolved in KPi
(50 mM) pH 7) and a mixing time of 15 s. Titration of protein
solution with KPi (50 mM) pH 7 were performed to correct for
dilution and bleaching during the measurements.
Protein and DNA Constructs. The following ABC transpor-
ters were used: OpuA,28 OpuAΔSBD,29 GlnPQ,30 Sav1866,31 and
the soluble nucleotide-binding domain (GlcV) of the ABC trans-
porter GlcSTUV.32 The following membrane proteins were used
as controls (non-ABC transporters): LacS (C320A/A635C),33
LacY,34 IIC,35 and RibU.36 The Sav1866 gene construct was
cloned into the N-Lic vector pNZnLIC as described by
Geertsema et al.37 The genes for GlcV and LacY were expressed
in E. coli, and all other proteins were produced in L. lactis as
described in the listed references. GlcV was obtained from M.
Pretz and C. van der Does (University Groningen, The Nether-
lands). Actin (from bovine muscle, A3653), myosin (from rabbit
muscle, M1636), and protein kinase A (from bovine heart,
P5511) were purchased from Sigma Aldrich.
Profiling with ATP Analogues. Isolation of membrane
vesicles and purification of the proteins was done as described.38
Membrane vesicles were prepared in KPi (50 mM) pH 7 at a
concentration of ca. 20 mg/mL, unless indicated otherwise; for
protein purifications, the membrane vesicles were diluted to
5 mg/mL in KPi (50 mM) pH 7, KCl (200 mM) plus glycerol
(20%) and, subsequently, solubilized with DDM (0.5%); purified
protein was obtained in KPi (50 mM) pH 7, KCl (200 mM),
glycerol (20%), and DDM (0.05%) plus imidazole (200 mM).
GlcV was purified in Na-MES (20 mM) pH 6.5 plus NaCl
(100 mM) as described by Pretz and collegues39 and stored in
aliquots at ꢀ20 °C. For profiling with the ATP analogues, pro-
tein mixtures were incubated with streptactin column material
(Streptactin Sepharose fromIBA, binding capacity300nmol/mL)
in the presence of the ATP analogue, using Biospin Poly-Prep
columns (volume 1.2 mL, Biorad). The mixture was incubated
for at least 1 h at 4 °C (under continuous mixing), before washing
and elution of the proteins bound to the column material. The
column material was washed in general, with 4 column volumes,
prior to elution of the bounds proteins with 1 column volume
of 2ꢁ SDS loading buffer, which is composed of Tris-HCl
(100 mM) pH 6 plus glycerol (20%), bromophenol blue
Partial Hydrolysis of p-BABA-ATP. A few milligrams of
p-BABA-ATP was dissolved in methanol (250 μL). Sodium
carbonate was added until the total volume was filled about half-
way with powder, to ensure saturating conditions. The mixture
was incubated for 1 h at 40 °C, under mixing (800 rpm). The
mixture was shortly spun down and the supernatant was mixed
1:1 with ACN (50%) plus formic acid (0.1%). Formic acid was
added for the ESI-MS analysis. For ESI-MS/MSMS analysis, the
samples were diluted 10ꢁ in ACN (50%) plus formic acid
(0.1%).
Analytical Methods. Nuclear magnetic resonance spectra
were recorded with a 200/300 MHz (1H NMR) or 50/75 MHz
(13C NMR) spectrometer. Chemical shifts are reported in delta
(δ) units, parts per million (ppm), and as an average shift for split
peaks. Coupling constants (J) are reported in Hz.
ESI-MS spectra were measured with a LCQ fleet mass
spectrometer (Thermo scientific) via direct injection at a flow
rate of 10 μL/min. Settings of the spectrometer were tuned with
a standard solution of ATP both in positive and negative ion
mode. Samples were diluted in ACN (50%) plus formic acid
(0.1%) prior to the measurements. ESI-MS/MSMS mass spectra
were recorded on an API3000 triple quadrupole mass spectro-
meter equipped with a TurboIonSpray source (MDS-Sciex,
Concord, Ontario, Canada) in positive ion mode with nitrogen
as collision gas, at a collision energy of 40 eV.
Analytical RP-HPLC was performed using a polystyrene/
divenyl benzene column (Source 5RPC ST 4.6/150) connected
€
to an Akta-purifier system. Two buffers were used for the
separation: Buffer A, TEAA (100 mM) pH 7.0, and Buffer B,
acetonitrile plus TEAA (100 mM) pH 7.0. The settings for the
separations were as follows: 200 μL loop, flow 1 mL/min,
gradient: 5 min 17% buffer B, 10 min gradient 17ꢀ20% buffer
B, 5 min 90% buffer B, 5 min 0% B.
TNP-ATP Binding. Purified OpuA (0.7 μM) was preincubated
for 10 min with TNP-ATP (2 μM) in KPi (50 mM) pH 7 plus
glycerol (20%), KCl (200 mM), and DDM (0.04%). Fluores-
cence was measured in a total volume of 800 μL under constant
stirring at 25 °C on a Fluorolog-3 spectrophotometer (Jobin
Yvon, settings: excitation wavelength of 409 nm (slit 1 nm),
emission wavelength of 540 nm (slit 5 nm), and data points were
averaged from measurements of 20 s). Nucleotides to chase the
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Figure 2. Schematic representation of activity based profiling using an ATP analogue as a probe. Panel A: The ATP (reactive group) moiety of the probe
binds specifically to nucleotide-binding proteins within a complex sample. Panel B: The biotin (reporter group) moiety of the probe is used for
enrichment of the proteins on streptactin column material. Panel C: The proteins enriched via this method can be analyzed after elution.
(0.002%), and SDS (4%) plus β-mercaptoethanol (1%). Analysis
of the protein samples was done on SDS-PAGE gels (12.5%,
stained with Coomassie brilliant blue) or subsequent immuno-
blotting on polyvinylidene difluoride (PVDF) membrane, using
the semidry electroblotting technique (blotting for 35 min at
80 mA per blot). Immunodetection was done with an antibody
(Amersham Pharmacia Biotech) against the His-tag of the vari-
ous proteins. The signals were imaged with a CCD camera
(Fujifilm, Las-3000 imaging system), using the Western light kit
(Tropix Inc.), and quantification of the signals from the SDS-
PAGE gels and immunoblots was done with the AIDA image
analyzer software (version 4.15).
buffer B2, 10 min gradient 17ꢀ20% buffer B2, 5 min 90% buffer
B2, 5 min 0% B.
’ RESULTS AND DISCUSSION
Design of the ATP Analogues. (+)-Biotin-hex-acyl-ATP
(BHacATP) has been used for activity-based profiling of protein
kinases.40 Protein kinases have a conserved lysine in the binding
pocket, i.e., near the phosphates of the nucleotide.41,42 The elec-
trophilic acyl-phosphate bond of the analogue couples with the
ε-amine from this lysine, connecting the biotinylated linker to the
protein and freeing it from the nucleotide. Using this analogue,
136 proteins, mostly ATP phosphohydrolases, (protein) kinases,
and several heat shock proteins, were identified in lysates of
breast cancer cells.40 However, this probe is not suitable for ABC
proteins, because the space for the hexyl-linker with the biotin
moiety would be too limited. On the basis of the structures of
ABC transporters, relatively large modifications are likely to be
tolerated at the 20- and 30-OH of the ribose (Figure 1). The
Analytical RP-HPLC. Analytical RP-HPLC was performed
using a polystyrene/divenyl benzene column (Source 5RPC
€
ST 4.6/150) connected to an Akta-purifier system. Two buffers
were used for the separation: Buffer A2, triethyl ammonium
acetate (TEAA, 100 mM) pH 7.0, and Buffer B2, acetonitrile plus
TEAA (100 mM) pH 7.0. The settings for the separations were as
follows: 200 μL loop, flow rate 1 mL/min, gradient: 5 min 17%
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Figure 3. Chemical structures and reaction schemes for the synthesis of the two ATP analogues. Panel A: Synthesis of the biotinylated linkers p-biotinyl-
amidobenzoic acid (p-BABA) and p-biotinyl-amidomethylbenzoic acid (p-BAMBA). Panel B: Synthesis of p-biotinyl-amidobenzoic acid-ATP, shown as
two isoforms, from the biotinylated linker and ATP.
Figure 4. Nucleotide-binding properties of the ATP analogues p-BABA-ATP and p-BAMBA-ATP. Binding of ATP or the ATP analogues was measured
by competition with the TNP-ATP bound to the purified ABC transporter OpuA. Panels A, B, and C show the fluorescence decay upon addition of ATP,
p-BABA-ATP, and p-BAMBA-ATP, respectively, and fitting of the results with single (dashed lines) and double (solid lines) exponential decay fits. Panel
D compiles the results collectively on a logarithmic scale.
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A second linker, that is, 4-aminomethylbenzoic acid, was used
to synthesize 4-biotinylamidomethylbenzoic acid (p-BAMBA)
(Figure 3A). The presence of an additional methylene group
compared to p-BABA resulted in a significant decrease in the
solubility of the biotintylated linker in DMF. The coupling of the
biotinylated linker to adenosine triphosphate (ATP) was based
on a method described for 30-O-(4-benzoyl)benzoyl-ATP.27 This
involved the activation of the acid group from biotin with
carbonyldiimidazole,46 which subsequently reacted with a hydro-
xyl group on the ribose ring of ATP to form an ester linkage
(Figure 3B).
The ratio between ATP and p-BABA was varied from 1:8 to
2:1. With an 8-fold excess of p-BABA, hardly any p-BABA-ATP
was formed (data not shown). The optimal yield of p-BABA-ATP
was found using equimolar amounts of ATP and p-BABA as
judged by reversed-phase HPLC analysis. p-BABA-ATP always
gave a double peak in the RP-HPLC analysis (Supporting
Information Figure S5A-B). However, ESI MS analysis showed
only a single peak at m/z 851. This can be explained by the
formation of two isomeric products, resulting from the coupling
of the biotinylated linker to the 20- and 30-OH group of the ribose
ring of ATP.
The reaction time was varied from 1 to 25 h (Supporting
Information Figure S5B) with the highest yield after 25 h. Notably,
the p-BABA-ATP isomer of the first peak was formed first and the
other isomer only after longer conversion times (>1 h).
Figure 5. SDS-PAA gels of protein binding to streptactin columns
(30 nmol binding sites) in the presence or absence of the ATP analogue
p-BABA-ATP (30 nmol), using the nucleotide-binding protein GlcV of a
glucose ABC transporter from S. solfataricus as test sample. Panel A
shows the binding of GlcV in the presence or absence of p-BABA-ATP as
a purified fraction of GlcV (3.4 nmol). Panel B shows the binding of
purified GlcV (0.6 nmol) mixed in a L. lactis crude cell lysate (0.2 mg)
after washing of the column fraction with 1 or 10 mL of washing buffer
using 50 mM Na-MES pH 7. Panel C shows the profiling of GlcV
present in L. lactis crude cell lysates (0.2 mg) at concentrations of
1.4 nmol to 140 pmol GlcV, corresponding to about 27ꢀ0.027% of GlcV
protein in the lysate using 50 mM KPi pH7 as buffer. Annotations are as
follows: start material (S) and fractions eluted from the column (C); (+)
means that proteins were incubated with the column material in the
presence of p-BABA-ATP and (ꢀ) in the absence of ATP analogue.
Equal volumes of each fraction were loaded on the gel.
Two purification steps were performed to obtain the pure ATP
derivatives: after preparative RP-HPLC (pRP-HPLC) purifica-
tion, the ATP-derivative containing fractions were pooled and
further purified on a strong anion exchange resin (monoQ
sepharose). p-BABA-ATP and p-BAMBA-ATP eluted at 1 M
TEAA pH 7 and the ESI-MS analysis of the pooled elution
fractions (elution fractions 1ꢀ3) showed a clean signal for the
ATP analogue (Supporting Information Figure S5C-G).
Structural Properties. The ESI-MS spectra of the product
proved that the coupling of p-BABA to ATP was successful but
did not establish connectivity of the product, that is, the position
of the coupling. Although literature reports suggest the present
method to result at the 20-OH or 30-OH position of the ribose
ring,27 in principle, also, coupling to the γ-phosphate or the
amine group of the adenine ring can occur. NMR analysis was
complicated as a result of the formation of the two isomers
(Supporting Information Figure S4). However, the appearance
of new signals in the 1H NMR spectra between 5 and 6 ppm with
the expected multiplicity corresponds to what was reported for
the ribose modified ATP-analogue BzATP,27 indicating that the
linker is coupled to the ribose hydroxy groups. Measurements
with 13C NMR (13C NMR and HSQC) were limited by the low
solubility of the ATP analogue. The spectra did not improve
enough at higher temperatures or longer measurement times (up
to 1 week).
A variety of other methods were used to demonstrate the
coupling of the biotinylated linker to the hydroxyl groups of
the ribose ring of ATP. Measurements of p-BABA-ATP and
ATP with 31P NMR showed equal spectra (both with shifts at
39.2 ppm (t) and 51.2 ppm (dd)), which excludes coupling of the
p-BABA to the γ-phosphate of ATP. Partial hydrolysis and
detection by ESI-MS (and MSMS) of p-BABA-ATP was used
to distinguish between coupling of the biotinylated linker to
the amine group of the adenine ring or the hydroxyl groups of
the ribose ring. The ESI-MS spectrum of the ATP analogue
after hydrolysis with sodium carbonate in methanol did show
structure of the nucleotide binding protein HlyB indicates that
certain modifications may affect the interaction between the ribose
of the nucleotide and a glutamine residue in the C-loop of the ABC
proteins.43 We thus designed two ATP analogues with a linker
coupled to the ribose to keep the biotin group accessible for binding
to a chromatography resin. We introduced an aromatic group in the
linker, as previous studies had shown that an aromatic group like
trinitrophenol linked to ATP (TNP-ATP) binds to ABC proteins
with much higher affinity than genuine ATP.19,44,45 In fact, ABC
transporters have been reported to bind ATP with KD values in the
submillimolar to low millimolar range, which is too low for efficient
profiling of the proteins. Guided by these observations, the
aromatic ring-structure was used as the basis for the design of the
linkers to couple the biotin moiety to ATP. For the activity-based
protein profiling with these probes (as summarized in Figure 2), the
ATP moiety is the bait to enrich for the ABC proteins, and the
biotin moiety is the reporter tag for enrichment.
Synthesis of the ATP Analogues. The general synthetic
approach was designed and optimized for biotinyl-amidobenzoic
acid-ATP (p-BABA-ATP). The synthesis of biotinyl-amidoben-
zoic acid (Figure 3A) was achieved as described previously for
biotinylated amino acid precursors, using isobutyl chloroformate
to activate the biotin prior to reaction with the amine of the linker
moiety.26
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Figure 6. Immunoblot (panel A) and SDS-PAA gel (panels B and C) monitoring the binding of ABC transporters, non-ABC transporters, and other
nucleotide-binding proteins from mixtures of purified proteins [15ꢀ30 μg purified protein (panel A and B) and 80 μg protein (panel C)] to streptactin
[15 nmol (panel A and B) and 30 nmol (panel C) binding sites] in the presence of p-BABA-ATP [15 nmol (panel A and B) and 30 nmol (panel C)].
50 mM KPi pH 7 plus 200 mM KCl was used as buffer for these experiments, supplemented with 0.04% DDM for the samples with purified membrane
proteins. The following ABC transporter proteins: OpuA, GlnPQ, and Sav1866 were used; RibU, LacS, and the IIC domain of the mannitol-PTS (panel
A and B) served as controls, as these proteins do not contain a nucleotide-binding site; actin and myosin (myosin heavy chains (myosin HC) binds the
ATP) (panel C) were used as a second set of controls as these are non-ABC type nucleotide-binding proteins. Annotations are as follows: start material
(S), flowthrough (F), wash fraction (W), and fractions eluted from the column (C), (+) means that proteins were incubated with the column material in
the presence of p-BABA-ATP and (ꢀ) in the absence of ATP analogue. Equal volumes of each fraction were loaded on the gel.
transesterification of p-BABA-ATP (Supporting Information
Figure S6) and the methyl ester of p-BABA could be detected.
Reaction of the amide bonds under these conditions is highly
unlikely, as it is illustrated that the amide bond between the biotin
and linker moiety remained intact under these conditions. In
combination, these experiments demonstrate that p-BABA is
indeed attached to the 20/30-OH group via an ester linkage.
Nucleotide-Binding Properties. A competition assay was
used to determine the binding properties of p-BABA-ATP and
p-BAMBA-ATP relative to that of ATP. The purified ABC
transporter OpuA from Lactococcus lactis47 was used as a model
protein to assess the relative binding affinities. The nucleotide
binding affinity of OpuA for ATP is in the millimolar range and
cannot be probed directly. The fluorescent ATP analogue trini-
trophenyl-adenosine triphosphate (TNP-ATP) binds to OpuA
with micromolar affinity, and the relative affinity of ATP or other
analogues can be accessed via competitive nucleotide-binding
studies48 (Figure 4). Figure 4A,B,C shows the decrease in
TNP-ATP fluorescence with increasing concentration of ATP,
p-BABA-ATP, and p-BAMBA-ATP, respectively. The data were
fitted with single- and double-exponential decay functions, and
only the latter yielded excellent fits. The data are indicative of
low- and high-affinity binding of TNP-ATP, which most likely
reflects the open (low-affinity) and closed (high-affinity) con-
formation of the nucleotide-binding domains. The data and
interpretation are consistent with observations on the soluble
nucleotide-binding protein OpuAA from B. subtilis: here, the
low- and high-affinity binding correspond to the monomeric and
dimeric nucleotide-binding domains.19 In the concentration
range used, the high-affinity component of TNP-ATP binding
could not be fully chased with ATP, p-BABA-ATP, or p-BAMBA-
ATP; for economic reasons, we did not titrate at higher con-
centrations as it would have taken an impractical amount of
synthesized ATP (analogue) to chase all TNP-ATP. Despite
the complex biphasic kinetics, the data clearly show that both
p-BABA-ATP and p-BAMBA-ATP were at least 10-fold more
efficient in competing with TNP-ATP from the binding pocket of
OpuA than ATP; p-BABA-ATP displayed the highest affinity
(Figure 4D). These measurements show that the linker does
improve the binding affinity of the nucleotide analogues to the
ABC transporter OpuA.
Binding of Nucleotide-Binding Proteins. Binding of the
proteins via the ATP analogues was confirmed by SDS-PAGE
analysis using the soluble nucleotide-binding domain (GlcV) of
the glucose ABC transporter from Sulfolobus solfataricus (with a
nanomolar affinity for ATP39). GlcV bound efficiently to strep-
tactin column material in the presence of p-BABA-ATP as can be
seen in Figure 5A. Spiking of GlcV to a crude cell free extract of L.
lactis (0.2 mg total protein) at a protein concentration of 3 μM
GlcV still yielded a highly significant binding in the presence of
p-BABA-ATP (Figure 5B). Quantification of the signals on the
SDS-PAGE gel showed that about 41% of the GlcV was bound to
the column in the presence of p-BABA-ATP, which compares to
6% (unspecific binding) in the absence of the ATP analogue.
Further dilution of GlcV into the crude cell free extract showed
that specific binding could still be detected until a concentration
of 0.7 μM (140 pmol) of GlcV (Figure 5C).
Next, mixtures of three purified ABC transporters (OpuA,
GlnPQ, and Sav1866) and three non-ABC transporters (LacS,
RibU, and IIC) were compared (Figure 6A,B), where the mem-
brane proteins lacking a nucleotide-binding site (non-ABC
transporters) are used as controls for the levels of nonspecific
binding. Sav1866 and OpuA showed clear binding. The fact that
OpuAA was detected by immunoblotting indicated that the
whole complex of OpuA was bound since the His-tag is on
OpuABC subunit and p-BABA-ATP is binding to the OpuAA
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subunit. In the case of GlnPQ, the His-tag is on the ATPase
subunit (GlnQ), but no binding of GlnQ was detected. However,
GlnP (the transmembrane domain-substrate binding protein)
was detected on SDS-PAGE gels, most likely reflecting aspecific
binding. LacS and IIC did not significantly bind to the streptactin
column. Experiments using detergent-solubilized membrane
vesicles with overexpressed proteins yielded similar results (data
not shown). The results show that ABC transporters can be
enriched specifically with the ATP analogues, but the presence of
residual protein in the flowthrough and wash fractions indicates
that a further improvement in binding affinity would be desirable.
We also assessed the binding of other types of nucleotide-binding
proteins, namely, actin, myosin, and protein kinase A, for which
the probes were not designed. No specific binding was observed
for any of these proteins (Figure 6C; data for protein kinase A is
not shown), confirming the specificity of the probes for ABC-
type nucleotide-binding proteins.
Top Subsidy to BP; grant number 700-56-302 and The
Netherlands Proteomics Centre (NPC).
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’ ASSOCIATED CONTENT
S
Supporting Information. NMR spectra (Figure S1ꢀ4),
b
RP-HPLC/MS spectra (Figure S5ꢀ6), and absorbance analyses
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Corresponding Author
*Bert Poolman, Nijenborgh 4, 9747AG, Groningen, The
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’ ACKNOWLEDGMENT
We would like to thank Hjalmar Permentier for his help
with the ESI-MS/MSMS measurements, and Ruud Scheek and
Renee Otten for their help with the NMR measurements. We
would like to thank the following people for providing purified
proteins or membrane vesicles: Monika Pretz and Chris van der
Does (purified GlcV), Gea Schuurman-Wolters (GlnPQ, IIC,
Sav1866, LacY), Siva Ramadurai (LacS), Josy ter Beek (RibU),
Guus Erkens (soluble cell free extracts) and Faizah Fulyani
(soluble cell free extracts). We would like to thank Wim Huibers
and Fabrizia Fusetti for their help with the LC-MS measure-
ments. This work was supported by grants from The Netherlands
Organisation for Scientific research (NWO, Chemical Sciences
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