Engineering the substrate specificity of ADP-ribosyltransferases for identifying direct protein targets

Article abstract of DOI:10.1021/ja412897a

Adenosine diphosphate ribosyltransferases (ARTDs; ARTD1-17 in humans) are emerging as critical regulators of cell function in both normal physiology and disease. These enzymes transfer the ADP-ribose moiety from its substrate, nicotinamide adenine dinucle

Full text of DOI:10.1021/ja412897a

Communication
pubs.acs.org/JACS
Engineering the Substrate Specificity of ADP-Ribosyltransferases for
Identifying Direct Protein Targets
Ian Carter-O’Connell,‡ Haihong Jin,‡ Rory K. Morgan,‡ Larry L. David,† and Michael S. Cohen*,‡
‡Program in Chemical Biology and Department of Physiology and Pharmacology, and †Department of Biochemistry, Oregon Health
& Science University, Portland, Oregon 97210, United States
S
* Supporting Information
signaling, such as the DNA damage repair pathway. While
ABSTRACT: Adenosine diphosphate ribosyltransferases
(ARTDs; ARTD1−17 in humans) are emerging as critical
regulators of cell function in both normal physiology and
disease. These enzymes transfer the ADP-ribose moiety
from its substrate, nicotinamide adenine dinucleotide
(NAD+), to amino acids of target proteins. The functional
redundancy and overlapping target specificities among the
17 ARTDs in humans make the identification of direct
targets of individual ARTD family members in a cellular
context a formidable challenge. Here we describe the
rational design of orthogonal NAD+ analogue-engineered
ARTD pairs for the identification of direct protein targets
of individual ARTDs. Guided by initial inhibitor studies
with nicotinamide analogues containing substituents at the
C-5 position, we synthesized an orthogonal NAD+ variant
and found that it is used as a substrate for several
engineered ARTDs (ARTD1, -2, and -6) but not their
wild-type counterparts. Comparing the target profiles of
ARTD1 (PARP1) and ARTD2 (PARP2) in nuclear
extracts highlighted the semi-complementary, yet distinct,
protein targeting. Using affinity purification followed by
tandem mass spectrometry, we identified 42 direct ARTD1
targets and 301 direct ARTD2 targets. This represents a
powerful new technique for identifying direct protein
targets of individual ARTD family members, which will
facilitate studies delineating the pathway from ARTD
activation to a given cellular response.
protein targets of ADP-ribosylation in stress-signaling pathways
have been identified,5,6 in many cases it is not clear if these are
direct targets of ARTD1. This is because of the functional
redundancy and overlapping target specificities among ARTDs.
7
Non-radioactive NAD+ derivatives, such as 6-biotin-NAD+
and 6-alkyne-NAD+ (6-a-NAD+),8 for use in copper-catalyzed
conjugation to an azidoalkyl reporter (click chemistry), have
been useful for globally identifying targets of ADPr. But these
reagents are insufficient to identify the direct protein targets of
ARTDs since they are substrates for all ARTDs. Given that up
to 17 unique ARTDs are expressed within the cell at any given
time,9 identifying direct targets for each ARTD family member
remains an essential challenge toward parsing the functional
role for each of these individual enzymes.
To address this challenge, we implemented a “bump-hole”
strategy for identifying the direct protein targets of ARTDs.
This strategy has been successfully used for the identification of
the direct targets of kinases,10 acetyltransferases,11 and
methyltransferases,12 as well as for the incorporation of non-
natural amino acids via modified aminoacyl-tRNA synthe-
tases.13 We envisioned that a unique hydrophobic pocket could
be engineered in the nicotinamide-binding site of ARTDs such
that they could bind an orthogonal NAD+ variant containing a
substituent at the C-5 position on the nicotinamide moiety of
NAD+. This orthogonal NAD+ variant would also contain an
denosine diphosphate ribosylation (ADPr) is a post-
Atranslational modification that plays a major role in a wide
array of cellular processes (e.g., energy metabolism, tran-
scription, and genomic maintenance).1 In humans, ADP-ribose
transfer is catalyzed by a family of 17 diphtheria toxin-like ADP-
ribosyl-transferases (ARTDs) that share a conserved catalytic
domain.2 ARTDs catalyze the transfer of the ADP-ribose
moiety from nicotinamide adenine dinucleotide (NAD+) to
their target proteins.3 The ARTD family has been recently sub-
classified on the basis of the ability of the individual ARTD
enzymes to catalyze the transfer of a single ADP-ribose unit
(mono-ARTDs: ARTD7, 8, 10−12, 14−17) or multiple ADP-
ribose units (poly-ARTDs: ARTD1−3, 5−6) onto target
proteins.2
Figure 1. (a) Schematic for the design of modified NAD+ analogues
that are preferentially utilized by engineered ARTDs (KA-ARTD). P =
phosphate. (b) 5-Et-6-a-NAD+ (1) used in this study.
The best-understood ARTD is ARTD1 (also known as
PARP1), which was previously thought to be the sole enzyme
responsible for poly-ADPr in cells.4 ARTD1 is involved in stress
Received: December 19, 2013
Published: March 18, 2014
© 2014 American Chemical Society
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Journal of the American Chemical Society
Communication
alkyne tag at the N-6 position on the adenosine moiety for
copper-catalyzed conjugation to a biotin−azide probe (Figure
1a). We hypothesized that a substituent (e.g., alkyl or benzyl) at
the C-5 position on the nicotinamide moiety would be
sufficient to exclude the orthogonal NAD+ variant from the
active site of wild-type ARTDs.
We first identified a position within the poly-ARTD
nicotinamide-binding site that we could mutate to a smaller
amino acid to create a hydrophobic pocket that would
accommodate orthogonal NAD+ variants containing a sub-
stituent at the C-5 position. Multiple crystal structures are
available for the poly-ARTD sub-class bound to nicotinamide
analogues, providing salient atomic details for the nicotinamide-
binding site.14−16 The crystal structure of ARTD1 bound to the
nicotinamide analogue 3-methoxybenzamide (PDB ID:
3PAX)14 reveals a lysine residue (K903) that forms van der
Waals contacts with the C-5 position of the benzamide ring
(Figure 2a). While we considered other positions within the
monitored ADP-ribose transfer to the ARTD1 target,20 Histone
H1, using the previously described clickable NAD+ derivative,
8
6-a-NAD+ (Scheme S3). 6-a-NAD+ contains an alkyne at the
N-6 position of the adenosine ring that can be conjugated to an
azide reporter via click chemistry for monitoring ADPr.8 We
sought a C-5-substituted nicotinamide analogue that was at
least 10-fold more selective for KA-ARTD1 compared to WT-
ARTD1. We found that all C-5-substituted nicotinamide
analogues were more selective for KA-ARTD1 than for WT-
ARTD1 (Figures 2c and S2). The most potent and selective
analogue of KA-ARTD1 was 5-ethyl-nicotinamide (IC50 = 165
11 μM), with 14-fold selectivity over WT-ARTD1 (IC50
=
2359 368 μM) (Figures 2c and S2). Together, these results
demonstrate that ARTD1 can be engineered to create a unique,
inhibitor-sensitizing mutation in the nicotinamide-binding site.
We next sought to prepare an orthogonal NAD+ variant that
would be a substrate for KA-ARTD1 but not WT-ARTD1.
Guided by the results of our inhibitor studies, we synthesized
the orthogonal NAD+ variant 5-Et-6-a-NAD+ (1), which
contains an ethyl substituent at the C-5 position of the
nicotinamide ring and an alkyne at the N-6 position of the
adenosine ring (Figure 1b and Scheme S4). To investigate the
substrate selectivity of 5-Et-6-a-NAD+, we monitored auto-
ADP-ribosylation of WT- and KA-ARTD1 by click conjugation
to a biotin−azide reporter. Incubation of KA-ARTD1 with
increasing concentrations of 5-Et-6-a-NAD+ resulted in auto-
ADPr of KA-ARTD1 (Figure 3a). By contrast, no modification
of WT-ARTD1 was detected with up to 250 μM 5-Et-6-a-
NAD+ (Figure 3a).
Figure 2. (a) The key residue, K903, in ARTD1 (green), near the C-5
position of 3-methoxybenzamide (yellow) (PDB ID: 3PAX).14 (b)
Sequence alignment of the nicotinamide binding site of the poly-
ARTDs. (c) Comparative inhibition of WT-ARTD1 and KA-ARTD1
by select nicotinamide analogues. IC50 values (mM) for each analogue
are plotted comparing the WT-ARTD1 (y-axis) and KA-ARTD1 (x-
axis) variants.
active site, we focused on K903 since it is conserved throughout
the poly-ARTD sub-class, thus providing a potentially general
strategy for identifying the direct targets of poly-ARTDs
(Figure 2b). Due to its established, well-characterized
enzymatic activity, we began by engineering ARTD1.17−19 To
accommodate substituents at the C-5 position of the
nicotinamide ring, we mutated K903 to an alanine (K903A)
(Figure S1). Rather than starting with a synthetically
challenging orthogonal NAD+ variant, we reasoned that we
could first test simple nicotinamide analogues as inhibitors,
since nicotinamide and the nicotinamide nucleotide of NAD+
bind in the same orientation in the active site of ARTDs. We
therefore synthesized a small panel of nicotinamide analogues
containing various substituents at the C-5 position (Figure 2c
and Schemes S1 and S2).
Figure 3. Orthogonal auto-ADPr of sensitized ARTD(s) using
modified NAD+ variants. The concentration of the NAD+ analogue
is indicated. Samples were subjected to immunoblot detection with
streptavidin (biotin) to detect modified protein. His-tag (His)
detection served as a loading control. Poly-ADPr and mono-ADPr-
modified fractions are indiciated: (a) ARTD1, (b) ARTD2, and (c)
ARTD6cat.
Whereas extensive poly-ADPr of WT-ARTD1 was observed
using 6-a-NAD+, as evidenced by the biotinylated smear above
the molecular weight of WT-ARTD1, only mono-ADPr of KA-
ARTD1 was observed using both 6-a-NAD+ and 5-Et-6-a-
NAD+, as evidenced by the single biotinylated band at the
molecular weight of KA-ARTD1 (Figure 3a). Using a poly-
ADP-ribose specific antibody (10H) and the native NAD+
To determine if nicotinamide analogues with substituents at
the C-5 position are more selective for K903A ARTD1 (KA-
ARTD1) than for wild-type ARTD1 (WT-ARTD1), we
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Communication
substrate, we confirmed that KA-ARTD1 functions as a mono-
ARTD (Figure S3). We cannot rule out the possibility that the
altered activity of KA-ARTD1 may complicate efforts to
identify all of the ARTD1 substrates. However, the apparent
activity of the mono-ARTD is sufficient to transfer modified
ADP-ribose onto target substrates for initial identification. In
fact, the exclusive mono-ADPr activity of KA-ARTD1 provides
an unexpected benefit: lack of poly-ADPr actually decreases the
complexity of samples submitted for liquid chromatography
tandem mass spectrometry (LC-MS/MS) analysis, decreasing
noise in the fragmentation pattern. Indeed, a previous study
demonstrated that converting ARTD1 to a mono-ARTD by
mutating the catalytic glutamate to a glutamine greatly
facilitated the identification of ADP-ribosylation sites by LC-
MS/MS.21
To further establish the generalizability of our approach, we
next sought to determine if our engineered ARTD enzyme−5-
Et-6-a-NAD+ pair could work for other poly-ARTD family
members. We focused on ARTD2 and ARTD6 (also known as
Tankyrase 2) (Figure S1) because of their well-characterized
enzymatic activity. Similar to WT-ARTD1, no modification of
WT-ARTD2 and WT-ARTD6 was detected by up to 250 μM
5-Et-6-a-NAD+ (Figure 3b,c). Mutation of the conserved lysine
in the nicotinamide binding pocket of ARTD2 and ARTD6 to
alanine (KA-ARTD2 and KA-ARTD6, respectively) conferred
sensitivity to 5-Et-6-a-NAD+, as evidenced by the single
biotinylated band at the molecular weight of the KA-ARTDs
(Figure 3b,c). Taken together, these experiments demonstrate
that we have successfully created a poly-ARTD wide-
engineered enzyme−modified substrate pair.
Having demonstrated that we can engineer poly-ARTDs to
accept an orthogonal NAD+ variant that is not used by their
WT counterpart, we next determined if we could label direct
targets of ARTD1 in a cellular context. We treated nuclear
extracts from human embryonic kidney (HEK) 293T cells with
5-Et-6-a-NAD+ (250 μM) alone or in the presence of KA-
ARTD1. As a positive control for ADP-ribose detection, we
treated nuclear extracts with 6-a-NAD+ alone. Protein targets of
ADP-ribosylation were detected by click chemistry with
biotin−azide. In contrast to 6-a-NAD+ (100 μM) treatment,
which resulted in labeling of several proteins due to the
endogenous activity of various ARTDs (predominately
ARTD1),8 no labeling was detected with 5-Et-6-a-NAD+
treatment (Figure 4a). Treatment of nuclear extracts with
both 5-Et-6-a-NAD+ (250 μM) and KA-ARTD1 resulted in
extensive labeling (Figure 4a). The band pattern produced is
similar to that seen with endogenous enzymes and 6-a-NAD+,
though there are notable differences between total ADP-
ribosylation and direct ADPr attributable to KA-ARTD1.
As previous studies suggest that ARTD2 is capable of
compensating for the loss of ARTD1,22 we applied our method
to compare the direct protein targets of ARTD1 and ARTD2.
Nuclear extracts treated with 5-Et-6-a-NAD+ (250 μM) and
KA-ARTD2 resulted in a band pattern that was similarwith
notable differences in band presence and intensityto the
pattern produced from KA-ARTD1 (Figure 4a). These results
suggests that, while ARTD1 and ARTD2 share redundant
functions in the cell,22 they do have distinct protein targets.
We next sought to use our strategy to identify the direct
protein targets of ARTD1 and ARTD2 in nuclear lysates using
LC-MS/MS. HEK 293T nuclear extracts were treated with
either 6-a-NAD+ alone (total ADP-ribosylated proteins), 5-Et-
6-a-NAD+ alone (background control), or 5-Et-6-a-NAD+ in
Figure 4. (a) Lysate labeling by ARTDs and modified NAD+
analogues. The faint bands observed in the 5-Et-NAD+-only lane
correspond to endogenous biotinylated proteins. The membrane
stained with Ponceau S serves as a loading control. The asterisks mark
the KA-ARTD1 (upper) and KA-ARTD2 (lower) bands. (b)
Immunoblot detection of the LC-MS/MS-identified targets
(XRCC5, Catenin-δ-1, hnRNP Q/R) following NeutrAvidin enrich-
ment.
the presence of either KA-ARTD1 (ARTD1-specific protein
targets) or KA-ARTD2 (ARTD2-specific protein targets).
Following conjugation with biotin−azide, biotinylated proteins
were enriched using NeutrAvidin agarose and proteolyzed, and
eluted peptides were subjected to LC-MS/MS (Figure S4). We
identified 339 proteins across the multiple conditions (Tables
S1−S5). Among the 339 proteins, 42 proteins were uniquely
identified as direct ARTD1 targets, and 301 were direct ARTD2
targets (thresholds discussed in Supporting Information).
There is a 52% and 7% overlap between direct ARTD1 or
ARTD2 targets, respectively, and total ADP-ribosylated targets
(Figure S5), with an additional 25 and 17 targets identified as
ADP-ribosylated but not directly by ARTD1 or ARTD2.
Twenty-two of the ARTD1 targets and 133 of the ARTD2
targets were previously identified as targets of ADP-ribosylation
under cell stress paradigms,5,6,8,23−25 suggesting that we have
identified bona fide ARTD1 and ARTD2 targets.
To confirm our LC-MS/MS results, we selected the ARTD1
and ARTD2 direct target, XRCC5 (also known as Ku80),23 and
the ARTD2-specific targets, Catenin-δ-1 and hnRNP Q/R, for
identification using NeutrAvidin enrichment (Figure S6)
followed by immunoblot detection with target specific
antibodies. Nuclear lysate treated with 5-Et-6-a-NAD+ in the
absence of KA-ARTD1 or KA-ARTD2 resulted in a complete
lack of enrichment for the selected targets (Figure 4b).
Enrichment of XRCC5 was observed with both KA-ARTD1
and KA-ARTD2 (Figure 4b). By contrast, enrichment of
Catenin-δ-1 and hnRNP Q/R was observed only with KA-
ARTD2 (Figure 4b). Taken together, these results highlight our
ability to identify unique, direct targets of a given ARTD family
member in a complex mixture.
In this study we developed new orthogonal NAD+ analogue-
engineered ARTD pairs to efficiently label and identify family-
member-specific ARTD protein targets. By initially focusing on
simple C-5-substituted nicotinamide analogues as selective
inhibitors of the engineered ARTD1 mutant, KA-ARTD1, we
were able to quickly identify a complementary interaction,
which guided the design of the orthogonal NAD+ variant, 5-Et-
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6-a-NAD+. The results from the nicotinamide inhibitor assay
mirrored the results achieved when the modified 6-a-NAD+
analogue was tested for specificity in auto-ADP-ribosylation
reactions of ARTD1. By enriching for tagged proteins in
experiments with either KA-ARTD1 or KA-ARTD2 and the
modified NAD+ analogue, we identified sets of direct, bona fide
ARTD1 and ARTD2 targets via LC-MS/MS.
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Our strategy proved applicable to multiple members of the
poly-ARTD sub-class and revealed how two closely related
poly-ARTD family members, namely ARTD1 and ARTD2,
exhibited distinct targeting patterns in nuclear lysate. Although
we focused primarily on the well-characterized poly-ARTDs, we
envision that the strategy described here could be applicable for
the identification of the direct targets of mono-ARTDs, as the
nicotinamide binding site is conserved throughout the family.
Given the difficulties in delineating the targeting specificities for
this highly homologous enzyme class, our method provides a
powerful new tool toward advancing our understanding of
ARTD function in the cell.
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ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental procedures, synthesis of compounds, and addi-
tional data. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
cohenmic@ohsu.edu
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Jeffrey Huang for assistance in cloning the ARTD2
gene and John Kilmek for the MS/MS analysis. We thank
Samie Jaffrey, Tom Scanlan, and members of the Cohen
laboratory for many helpful discussions and for advice on the
manuscript.
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