A mechanism-based aryl carrier protein/thiolation domain affinity probe

Article abstract of DOI:10.1021/ja069201e

The design, synthesis, and biochemical characterization of a mechanism-based aryl carrier protein (ArCP) affinity probe that selectively modifies the terminal thiol of the aryl carrier protein phosphopantetheine (Ppant) prosthetic group is described. Labe

Full text of DOI:10.1021/ja069201e

Published on Web 05/01/2007
A Mechanism-Based Aryl Carrier Protein/Thiolation Domain Affinity Probe
Chunhua Qiao, Daniel J. Wilson, Eric M. Bennett, and Courtney C. Aldrich*
Center for Drug Design, Academic Health Center, UniVersity of Minnesota, Minneapolis, Minnesota 55455
Received December 21, 2006; E-mail: aldri015@umn.edu
Carrier proteins (CPs) play a central role in the biosynthesis of
polyketide synthase (PKS), nonribosomal peptide synthetase (NRPS),
and fatty acid synthase (FAS)-derived natural products. CPs, also
referred to as thiolation domains, are responsible for transporting
the substrate and chain intermediates to the catalytic centers of the
PKS, NRPS, and FAS assembly lines. The biosynthetic chain
intermediates are tethered as thioesters on the terminal thiol of a
phosphopantetheine (Ppant) prosthetic group that is covalently
attached to an invariant serine residue of the CP.2 CPs are either
freestanding or embedded in these multifunctional proteins and exist
as three variants: an acyl carrier protein (ACP) found in PKSs
and FASs, a peptidyl carrier protein (PCP) found in NRPS systems,
and an aryl carrier protein (ArCP) commonly found in siderophore
NRPS synthetases.2
An understanding of how CPs recognize their upstream and
downstream partner proteins is essential to “deciphering the logic”
for assembly of these natural products.1 CPs also constitute a
potential target for the development of a new class of antibiotics
since they are involved in the synthesis of several bacterial virulence
factors and essential primary metabolites. For example, the natural
antibiotic platensimycin acts by disrupting interactions between a
CP and a ketosynthase (KS) domain in the bacterial type II FAS.2
Figure 1. Carrier protein (CP) modifications. (A) The Ppant prosthetic
group on an invariant serine residue is installed post-translationally by a
The groups of Burkart, Walsh, and Johnsson demonstrated the utility
of CPs as low molecular weight protein fusion tags that can be
easily modified by exploiting the promiscuity of phosphopanteth-
einyl transferases (PPTase) to incorporate fluorescent and affinity
tags onto the conserved serine residue of the carrier domain.3
Mechanism-based affinity probes that specifically modify the
terminal thiol of the Ppant prosthetic group of CPs could serve as
new reagents for the site-specific labeling of proteins, lead to the
development of novel antibacterial agents, and provide powerful
chemical probes to study the interactions of CPs with their partner
enzyme domains. In this latter regard, Burkart and co-workers
recently detailed a pantetheine analogue, which cross-links KS and
ACP domains.4 We report herein our complementary efforts at the
design and biochemical characterization of a mechanism-based CP
affinity probe that selectively modifies ArCPs.
The ArCP affinity probe design is based on the reaction
mechanism for ArCP loading, catalyzed by an adenylation enzyme
illustrated in Figure 1B. In the first half-reaction, the substrate aryl
acid and ATP are condensed to form a tightly bound acyladenylate
with the release of pyrophosphate (see Figure 1B). In the second
half-reaction, the adenylation enzyme binds ArCP and transfers the
acyl group onto this protein. Consequently, analogues that mimic
the essential features of the acyladenylate intermediate to confer
sufficient binding to the adenylating enzyme, but incorporate a
reactive functional group that can either reversibly or irreversibly
bind the terminal thiol of the Ppant prosthetic group of the CP, are
expected to provide selective mechanism-based inactivators. We
conceived of a vinylsulfonamide as an acyladenylate surrogate that
contains a Michael acceptor at the precise position of the incoming
nucleophile (Figure 1C). Roush and co-workers recently ranked
PPTase. (B) Loading of CPs mediated by adenylation domains. (C) A
mechanism-based CP affinity probe for CP domains incorporates a Michael
acceptor. (D) Reversible bisubstrate inhibitors of MbtA.8
the relative activities of Michael acceptors: enone > vinylsulfone
> vinylsulfonate > enoate > vinylsulfonamide.5 The vinylsulfona-
mide was chosen initially since this is the least reactive member in
the series to minimize nonspecific thiol addition. Additionally,
molecular modeling showed that the vinylsulfonamide adopted the
required folded conformation of the bound acyladenylate (Figure
S11, Supporting Information).
As a model system, we selected the ArCP domain in MbtB and
its cognate adenylating enzyme MbtA that are responsible for
incorporating salicylic acid into mycobactin, a siderophore produced
by Mycobacterium tuberculosis.6 MbtA and MbtB are independent
proteins enabling us to dissect the specificity and mechanism of
the affinity probe. In order to facilitate subsequent MS analysis,
the N-terminal ArCP domain (∼10 kDa) of mbtB was subcloned
into pET28b and coexpressed in E. coli BL21 (DE3) with sfp7 to
afford holo-MbtB-ArCP as a C-terminal His6-tagged fusion protein.
Syntheses of affinity probes 1 and 2 (Figure 1C) are described in
the Supporting Information. Since the mechanism-based inactivator
1 requires binding to MbtA, we first demonstrated that 1 is able to
interact with this protein using an ATP-[32P]PPi exchange assay.8
Affinity probe 1 displayed competitive inhibition with respect to
both ATP (appKi(ATP) ) 288 ( 56 µM) and salicylic acid (appKi(Sal)
) 143 ( 9 µM) (Figures S1 and S2, Supporting Information).
Previously, we designed and synthesized analogues 3-6 as potent
reversible inhibitors of MbtA, which provided important compara-
tive SAR data (Figure 1D).9 The approximately 5 orders of
magnitude difference in activity between 1 and 5 can be attributed
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C O M M U N I C A T I O N S
(EntE: appKi(2,3-DHBA) ) 639 ( 114 µM), but is a noncompetitive
inhibitor with respect to ATP of both YbtE (appKi(ATP) ) 91 ( 43
µM, R ) 7.9) and EntE (appKi(ATP) ) 535 ( 236 µM, R ) 1.7)
(Figures S3-S6, Supporting Information). The attenuated activity
of 1 toward EntE is consistent with the preference of EntE for a
3-hydroxy group on the aryl ring. Thus, the lack of in trans
modification of MbtB-ArCP by either YbtE or EntE is likely a result
of improper protein-protein interactions between these heterologous
protein pairs. This establishes another level of selectivity of affinity
probe 1, which requires not only binding to the adenylating enzyme
but also proper interdomain recognition between the ArCP and
cognate adenylation enzyme.12
Finally, we investigated the ability of affinity probe 1 to promote
an interaction between the adenylating enzyme MbtA and MbtB-
ArCP using an electrophoretic mobility shift assay with a nonde-
naturing polyacrylamide gel. Western blot analysis of MbtB
revealed that 1 indeed did stabilize the protein-protein interaction
between MbtA and MbtB-ArCP as the His6-tagged MbtB-ArCP
coalesced to a sharp band only in the presence of MbtA and affinity
probe 1 (Figure S8, Supporting Information).
Figure 2. MALDI-TOF of the ArCP domain of MbtB modified with
affinity probe 1. MS: calcd for [C7S holo-ArCP] 10376; calcd for [C7S
holo-ArCP + 1] 10843.
to the removal of the carbonyl group which interacts with Lys519
of MbtA (∼100-fold loss, compare 3 vs 4 in Figure 1D) and the
central nitrogen atom (∼1000-fold loss, compare 5 vs 6 in Figure
1D).9a However, the modest micromolar activity of 1 toward MbtA
was deemed adequate to ensure binding to MbtA before channeling
onto MbtB-ArCP.
In summary, we have designed, synthesized, and biochemically
characterized an exquisitely selective aryl carrier protein affinity
probe. The ability to cross-link CPs with adenylation domains
located in cis may provide a means to crystallize such didomain
pairs and provide insight into the elusive second half-reaction
catalyzed by adenylation domains. Finally, this affinity probe of
MbtB-ArCP could serve as a prototype for a new class of
antitubercular agents since a mbtB disruption mutant of M.
tuberculosis exhibited attenuated virulence in macrophages, the
primary site of infection in pulmonary tuberculosis.13
Incubation of 1 with MbtB-ArCP (1.0 mM 1, 10 µM MbtB-
ArCP, 50 mM Tris, pH 8.5, 1.0 mM TCEP, 10 mM MgCl2, 37 °C,
24 h) did not lead to any covalent modification, demonstrating that
the affinity probe does not react nonspecifically. However, addition
of catalytic MbtA (2 µM MbtA) afforded a molecular ion peak
corresponding to covalent modification at m/z 10838 [MbtB-ArCP
+ 1]+ as determined by MALDI-TOF (Figure 2). Labeling of
MbtB-ArCP by 1 could be completely suppressed by addition of
3, which is a reversible nanomolar inhibitor of MbtA. By contrast,
unsubstituted vinylsulfonamide 2 reacted nonspecifically with
MbtB-ArCP, consistent with the greater reactivity of this uncon-
jugated Michael acceptor. These results validate the design strategy
and serve to highlight the requirement for the adenylating enzyme
that must first bind 1 then channel this inactivator onto the ArCP.
The pseudo first-order rate constant for reaction of 1 with
N-acetylcysteamine was found to be (1.85 ( 0.8) × 10-3 min-1 at
25 °C and pH 8.0, illustrating the low intrinsic reactivity of 1 toward
thiols.
Acknowledgment. This research was supported by a grant from
the NIH (R01AI070219) and funding from the Center for Drug
Design, University of Minnesota to C.C.A. We thank the Minnesota
Supercomputing Institute VWL lab for computer time.
Supporting Information Available: Experimental procedures,
supplementary data, and the complete ref 2. This material is available
free of charge via the Internet at http://pubs.acs.org.
In order to confirm that modification of MbtB-ArCP occurred
on the Ppant prosthetic group of S39, the protein was subjected to
trypsin digestion. After proteolysis, the modified peptide fragment
ADALHPGANLVGQGLDS*IR (A23-R41) with m/z 2692.0 [A23-
R41 + Ppant + 1]+ was observed (Figure S9, Supporting
Information). Tandem mass sequencing (ESI+) of the 4+ charge
state of this modified peptide yielded two major ions due to Ppant
elimination at m/z 992.4788 [A23-R41 + PO3]2+ and 355.1137
[Ppant + 1 - PO3]2+, which unequivocally proved the affinity probe
1 was linked to Ppant (Figure S10 and Schemes S1 and S2,
Supporting Information).10
The ability of other noncognate adenylation domains to transfer
affinity probe 1 to MbtB-ArCP was assessed to determine the
specificity of this process. The adenylating enzymes YbtE and EntE
were evaluated since these are responsible for transferring salicylic
acid and 2,3-dihydroxybenzoic acid (2,3-DHBA) to their cognate
aryl carrier domains YbtB and EntB involved in biosynthesis of
the siderophores yersinabactin and enterobactin, respectively.11
Incubation of either YbtE or EntE with MbtB-ArCP and 1 did not
result in any detectable modification of MbtB-ArCP. We confirmed
that vinylsulfonamide 1 is a competitive inhibitor with respect to
salicylic acid (YbtE: appKi(Sal) ) 133 ( 18 µM) and 2,3-DHBA
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