Active site-directed proteomic probes for adenylation domains in nonribosomal peptide synthetases

Article abstract of DOI:10.1039/c4cc09412c

We describe a general strategy for selective chemical labeling of individual adenylation (A) domains in nonribosomal peptide synthetases (NRPSs) using active site-directed proteomic probes coupled to the 5′-O-N-(aminoacyl)sulfamoyladenosine (AMS) scaffold

Full text of DOI:10.1039/c4cc09412c

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Active site-directed proteomic probes for
adenylation domains in nonribosomal peptide
synthetases†
Cite this: Chem. Commun., 2015,
51, 2262
Received 25th November 2014,
Accepted 21st December 2014
Sho Konno,a Fumihiro Ishikawa,*a Takehiro Suzuki,b Naoshi Dohmae,b
Michael D. Burkartc and Hideaki Kakeya*a
DOI: 10.1039/c4cc09412c
www.rsc.org/chemcomm
We describe a general strategy for selective chemical labeling of and because of the intractability of the producer organisms to
individual adenylation (A) domains in nonribosomal peptide synthe- conventional genetic manipulation and heterologous expression.5
tases (NRPSs) using active site-directed proteomic probes coupled Profiling these enzyme family members in the proteomes of diverse
to the 50-O-N-(aminoacyl)sulfamoyladenosine (AMS) scaffold with a organisms could provide highly complementary genetic approaches
clickable benzophenone functionality. These proteomic tools can by allowing us to not only understand the activity, transcriptional
greatly facilitate the molecular identification, functional characteri- control, and post-translational events, but also facilitate the mole-
zation, and profiling of virtually any kind of A domains of NRPS cular identification and functional characterization of these enzyme
enzymes in complex biological systems.
families directly in native proteomic environments.
Several chemistry-based approaches have exploited selective
Natural products display biologically interesting activities including chemical labeling of thioesterase (TE),6 dehydratase (DH),7 and
a profound number of clinical antimicrobial, anticancer, and aryl acid adenylating enzymes8 found in any PKS and/or NRPS
immunosuppressive agents, virulence factors, and signaling mole- enzymes in bacterial proteomes. As not all domains are present in
cules.1 Many of these small molecules are biosynthesized by large, each PKS and NRPS enzymes, a more complete set of probes may
highly versatile multifunctional enzymes known as polyketide be critical to identify full modules and realize practical proteomic
synthases (PKSs), nonribosomal peptide synthetases (NRPSs), and approaches consisting of activity-based probes to natural product
PKS–NRPS hybrids.2 Although significant progress has shed light biosynthetic pathways.9,10 The adenylation (A) domain found in all
on the functions of these multifunctional enzymes at the genetic NRPS modules is an essential catalytic component and functions
level,3 their understanding at the protein level is much less. as the gatekeeper of the NRPS assembly line. The A domain
Analogous to the importance of studying the human proteome selectively incorporates a cognate amino acid into peptide-based
encoded by the genome, proteomic investigation of natural product natural products from a much larger monomer pool, including
producer microorganisms provides a level of information that all 20 proteinogenic amino acids as well as a number of non-
cannot be understood solely by genetic approaches.4 In addition, proteinogenic amino acids, aryl acids, fatty acids, and hydroxy acid
some of these multifunctional enzymes are particularly resistant to building blocks.11 Using the strict A-domain active site chemistry,
routine cloning, expression, and biochemical evaluation as intact we set out to develop chemical labeling systems for A domains in
recombinant proteins. This is partly because these enzymes are NRPS enzymes using active site-directed proteomic probes (Fig. 1).
large molecular species with a range between 200 and 1600 kDa,
We chose A domains in the gramicidin S biosynthetic enzymes,
GrsA and GrsB as the initial targets to demonstrate our strategy.13
Gramicidin S is a cyclic decapeptide antibiotic with the primary
structure cyclo (-D-Phe1-L-Pro2-L-Val3-L-Orn4-L-Leu5-)2, which is bio-
synthesized by GrsA and GrsB peptide synthetases. GrsA is a single
NRPS module containing the domain structure A1 (L-Phe)-T-E and
responsible for the incorporation of D-Phe during the gramicidin
S biosynthesis. In contrast, GrsB is a large multifunctional mega-
synthetase with a calculated molecular weight of 508 kDa, and
consists of four NRPS modules, C-A2 (L-Pro)-T-C-A3 (L-Val)-T-C-A4
(L-Orn)-T-C-A5 (L-Leu)-T-TE. Individual four A domains (A2–A5) are
housed on this single protein and selectively incorporate their
cognate amino acids, L-Pro, L-Val, L-Orn, and L-Leu, respectively,
a Department of System Chemotherapy and Molecular Sciences,
Division of Bioinformatics and Chemical Genomics, Graduate School of
Pharmaceutical Sciences, Kyoto University, Sakyo, Kyoto 606-8501, Japan.
E-mail: fishika@pharm.kyoto-u.ac.jp, scseigyo-hisyo@pharm.kyoto-u.ac.jp
b RIKEN Global Research Cluster, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198,
Japan
c Department of Chemistry and Biochemistry, University of California, San Diego,
9500 Gilman Drive, La Jolla, California 92093-0358, USA
† Electronic supplementary information (ESI) available: Supporting figures;
procedures for the syntheses of L-Phe-AMS-BPyne 1, L-Pro-AMS-BPyne 2, L-Orn-
AMS-BPyne 3, L-Pro-AMS 5, L-Orn-AMS 7, and L-Leu-AMS 8; complete gel images;
full experimental details; and copies of 1H and 13C-NMR spectra. See DOI:
10.1039/c4cc09412c
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Fig. 1 Methods for proteomic analysis of A domains in NRPS biosynthetic
enzymes using active site-directed proteomic probes for A domains.
Modules are comprised of thiolation (T), adenylation (A) [A1: L-Phe; A2: L-Pro;
A3: L-Val; A4: L-Orn; A5: L-Leu specific A domains], epimerization (E), condensa-
tion (C), and thioesterase (TE) domains. GrsA and GrsB are depicted as
examples. The rectangles represent nonspecific proteins. In a gel-based
analysis, recombinant NRPS enzymes or proteomes treated with individual
probes are incubated with a rhodamine (Rh)-azide reporter tag under click
chemistry (CC) conditions and separated by SDS-PAGE, and labeled A
domains in NRPS enzymes are visualized by in-gel fluorescence imaging.12
in the NRPS assembly line. Briefly, A domains selectively recognize
the cognate amino acids and convert them to aminoacyl adenylate
intermediates. The adenylated substrates in turn undergo a
nucleophilic attack by the terminus thiol group of the
40-phosphopantetheine arm of a downstream T domain, forming
a thioester bound aminoacyl-S-T (Fig. 2a). Ligand design exploiting
tight binding characteristics for the active sites of A domains was
Fig. 2 (a) Adenylation reaction in NRPS. (b) Structures of active site-directed
proteomic probes and inhibitors for A domains described in this study.
based on the reliable bisubstrate inhibitor scaffold by conjugating Kai pp values of 1.20 ꢀ 0.14 nM for GrsA16 and 431 ꢀ 42.0 nM for
amino acids with 50-O-[N-(aminoacyl)sulfamoyl]adenosine (AMS) TycB1, respectively (Fig. S2, ESI†). These experiments demonstrated
wherein the highly reactive acylphosphate linkage has been sub- that the attachment of a bulky photoreactive benzophenone and a
stituted by a bioisosteric and chemically stable non-hydrolysable clickable alkyne at the 20-OH group of the adenosine skeleton have
acylsulfamate group.14 A complex structure of the GrsA A domain no effect on the binding affinity of the A domains present within the
with L-Phe and AMP revealed that chemical modification at the NRPS enzymes tested.
20-OH group of the adenosine skeleton would be tolerated.15
Our labeling studies began by examining the ability of probes 1
Indeed, attachment of a long pegylated biotin to L-Phe-AMS 4 at and 2 to specifically label recombinant proteins, GrsA and TycB1,
this position showed no effect of binding for the GrsA A respectively. Probes 1 (1 mM) and 2 (1 mM) were incubated with GrsA
domain.14a,16 We therefore decided to incorporate a clickable (1 mM) and TycB1 (1 mM) in either the absence or presence of excess
benzophenone functionality at this position. We selected L-Phe, 4 and 5, respectively, for 10 min at room temperature and pH 8.0.
L-Pro, and L-Orn as ligands to demonstrate the versatility of this These samples were then photoactivated with ultraviolet light
scaffold as labeling reagents for A domains. To this end, active (365 nm) for 30 min at 0 1C, treated with Rh-azide under standard
site-directed proteomic probes, L-Phe-AMS-BPyne 1, L-Pro-AMS- click chemistry (CC) conditions,12 and monitored by SDS-PAGE
BPyne 2, and L-Orn-AMS-BPyne 3 were synthesized by the coupled with in-gel fluorescence scanning. SDS-PAGE analysis
assembly of the AMS scaffold, a photoreactive benzophenone and demonstrated the clear labeling of GrsA and TycB1. The labeling
a clickable alkyne functionality (Fig. 2b, and Schemes S1 and S2, processes of GrsA with 1 and TycB1 with 2 were completely inhibited
ESI†). In addition, L-Phe-AMS 4,16 L-Pro-AMS 5, L-Val-AMS 6,17 L-Orn- by pre-treatment of GrsA with 4 and TycB1 with 5 (Fig. 3a). We next
AMS 7, and L-Leu-AMS 8 were prepared to provide analogs of 1–3 for performed time course experiments by evaluating the labeling
comparative activity studies (Fig. 2b and Scheme S3, ESI†).
of GrsA and TycB1. Maximum fluorescence band intensity was
Our biochemical studies began with an examination of the observed at 30 min for both reactions (Fig. 3b). Subsequent charac-
specific inhibitory activities of L-Phe-AMS-BPyne 1, L-Phe-AMS 4, terization allowed us to identify the lower detection limit for each
L-Pro-AMS-BPyne 2, and L-Pro-AMS 5 against recombinant protein labeling. As shown in Fig. 3c and d, we found that probes 1
holo-GrsA for 1 and 4 and holo-TycB1 for 2 and 5. TycB1 is and 2 attained highly sensitive detection as low as 10 fmol of GrsA
derived from the tyrocidine biosynthetic enzymes, incorporates and 5 fmol of TycB1. We finally asked whether probes 1 and 2 could
L-Pro, and contains the domain structure C-A (L-Pro)-T.18 Apparent Ki discriminate the cognate A domain activity by ligand-directed
values were determined by a coupled hydroxamate-MesG continuous protein labeling. AusA1 is the excised didomain (A1-T1) of the
spectrophotometric assay (Fig. S1, ESI†).19 Probes 1 and 2 exhibited aureusimine synthase AusA (A1-T1-C-A2-T2-R) from Staphylococcus
tight-binding properties against the A domains of GrsA and TycB1, aureus.17 The A1 domain of AusA incorporates L-Val during the
respectively, with calculated Kaipp values of 1.43 ꢀ 0.19 nM for GrsA biosynthesis of aureusimines. Labeling experiments of GrsA, TycB1,
and 327 ꢀ 17.0 nM for TycB1 (Fig. S2, ESI†). Inhibitors 4 and 5 gave AusA1, and BSA with 1 mM 1 resulted in selective labeling of the
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Fig. 3 Labeling of recombinant holo-GrsA and holo-TycB1 with probes 1
and 2. (a) Labeling of GrsA and TycB1 and competitive inhibition studies with
excess inhibitors 4 and 5. GrsA (1 mM) and TycB1 (1 mM) were individually
preincubated in either the absence or presence of 100 mM of inhibitors 4 and
5 and treated with 1 mM of the individual probes 1 and 2. (b) Ultraviolet
photolysis time course studies of the labeling of GrsA with probe 1 (left) and
TycB1 with probe 2 (right). SDS-PAGE analysis denoting the labeling of 1 mM
of GrsA and TycB1 with 1 mM probes 1 and 2, respectively. (c and d) Limit
of detection of GrsA and TycB1 labeling. GrsA (1–500 fmol) and TycB1
(1–500 fmol) were individually incubated with probes 1 and 2. (e and f)
Labeling specificity of probes 1 and 2. GrsA (1 mM), TycB1 (1 mM), AusA1 (1 mM),
and BSA (1 mM) were individually treated with 1 mM of probes 1 and 2 in either
the absence or presence of 100 mM of the inhibitors 4, 5, and 6. For each
Fig. 4 Proteomic applications of active site-directed proteomic probes
1–3 for A domains. (a) Labeling of endogenous GrsA in an A. migulanus
ATCC 9999 cellular lysate by using probe 1. The A. migulanus ATCC 9999
lysate (1.4 mg mLꢁ1) was treated with 1 mM 1 in either the absence or
presence of 100 mM 4. (b and c) Labeling of endogenous GrsB in the
A. migulanus DSM 5759 cellular lysate by probes 2 and 3. The A. migulanus
DSM 5759 lysate (1.5 mg mLꢁ1) was individually treated with 100 mM 5 and 7
before the addition of individual members of 1 mM probes 2 and 3. (d) Individual
labeling of A domains and profiling of A domain functions using a combination
of probes 1–3 and inhibitors 4–8. In order to investigate GrsA labeling, the
A. migulanus ATCC 9999 lysate (1.5 mg mLꢁ1) was preincubated with individual
members of inhibitors 4–8 (100 mM) before the addition of 1 mM of probe 1. To
evaluate the labeling of GrsB, the A. migulanus DSM 5759 lysate (1.5 mg mLꢁ1
)
panel, F depicts the fluorescence observed with lex = 532 nm and lem
=
580 nm, and S displays the total protein content by staining with Coomassie
Blue (a, b, e, and f) or a silver staining method (c and d).
was individually treated with 100 mM of inhibitors 4–8 before the addition of
individual members of 1 mM probes 2 and 3. For each panel, F depicts the
fluorescence observed with lex = 532 nm and lem = 580 nm, and S displays the
total protein content by staining with Coomassie Blue.
cognate A domain of GrsA with virtually no labeling of the non-
cognate A domains of TycB1 and AusA1 and BSA (Fig. 3e). Probe 2 sample, further demonstrating the specificity of this probe. These
also showed similar selective labeling of the cognate A domain of results validate that probe 1 is a highly specific labeling agent for
TycB1 with no detectable labeling of GrsA, AusA1, and BSA (Fig. 3f). L-Phe activating A domains that shows no cross-reactivity with
These results indicate that the strict substrate recognition systems other proteins and markedly low levels of background signals
of A domains allow us to selectively label the A domains with sub- within the proteomic environment.
strate specificity identical to an attached amino acid moiety.
As an ultimate application of proteomic activity, we evaluated
After demonstrating the compatibility of probes 1 and 2 for the availability of probes 2 and 3 by applying them to A domains
selective labeling of A domains in recombinant enzyme systems, housed on a multifunctional megasynthetase, GrsB in a proteomic
we sought to demonstrate the applicability of probes 1–3 by context. The DSM 5759 proteome was individually incubated with
applying them to the analysis of natural product producers’ NRPS 1 mM of probes 2 and 3 for 10 min at room temperature, irradiated
systems. Two strains of Aneurinibacillus migulanus known as for 5 min at 0 1C and subsequently treated with Rh-azide under the
Bacillus brevis, ATCC 9999 and DSM 5759 have been demonstrated CC conditions. In-gel fluorescence analysis showed only labeled
as gramicidin S producing strains.20 We first evaluated the avail- fluorescence bands at B500 kDa in the individual probes applied
ability of probe 1 to detect endogenous GrsA. The cellular lysate of (Fig. 4b and c). Significantly, this labeling completely disappeared
producing strain ATCC 9999 was adjusted to pH 8.0, incubated by pre-treatment with the corresponding inhibitors 5 and 7 (Fig. 4b
with 1 mM of 1 for 10 min at room temperature, irradiated for and c). In particular, the high-molecular-weight (HMW) band was
30 min at 0 1C and subjected to the click reaction with Rh-azide. the only species labeled by 2 and 3 in this proteome. Collectively,
In-gel fluorescence analysis showed a distinguishable fluorescence these labeling experiments led us to identify the labeled protein as
signal at B120 kDa corresponding to the size of labeled standard GrsB with the knowledge of the molecular weight and the presence
recombinant GrsA (Fig. 4a).16 In addition, the observed single of two A domains with substrate specificity for L-Pro and L-Orn on
band was abrogated by pre-treatment with 4 (Fig. 4a). Notably, the this single protein. A labeled HMW band was visualized with silver
GrsA was the only specific labeled protein in this proteomic staining, excised, analyzed by LC-MS/MS, and found to contain
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the endogenous GrsB protein with 40% peptide coverage low-abundance enzymes in proteomic samples, and we are cur-
(Fig. S5 and S6, ESI†). rently applying cocktails of these probes for studying biosynthetic
With proteomic tools in hand, we finally attempted to demon- pathways in a variety of natural product producer organisms.
strate individual labeling and profiling of substrate preference of This work was partly supported by the Sasakawa Scientific
A domains in GrsA and GrsB by a combination of probes 1–3 with Research Grant from The Japan Science Society (S.K.), a Grant-in
inhibitors 4–8. In order to investigate GrsA labeling, ATCC 9999 Aid for Young Scientists (B) 26750370 (F.I.), and the research
lysates were preincubated with inhibitors 4–8 (100 mM) before the grant from the Japan Society of the Promotion of Science and the
addition of 1 mM of probe 1, and the samples were exposed to Ministry of Education, Culture, Sports, Science and Technology
ultraviolet light for 30 min and treated with an Rh-azide. To in Japan (MEXT) (H.K.). We thank Prof. Mohamed Marahiel
¨
evaluate the labeling of GrsB, DSM 5759 lysates were individually (Philipps-Universitat Marburg, Germany) for providing the GrsA
treated with 100 mM of inhibitors 4–8 before the addition of 1 mM and TycB1 expression constructs.
probes 2 and 3. As shown in Fig. 4d, the labeling of GrsB by
probes 2 and 3 disappeared only by the addition of 5 and 7,
respectively. These results validated that probes 2 and 3 selectively
Notes and references
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because the A domain of GrsA is known to recognize L-Leu as a
miscognate substrate with lower catalytic efficiency than that of
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cant differences with the substrate specificity of individual A
domains by comparing the competitive activity-based protein
profiling (ABPP) of the A domains.
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