DOI: 10.1002/anie.201104391
Heterooligomeric Complexes
Vibralactone as a Tool to Study the Activity and Structure of the
ClpP1P2 Complex from Listeria monocytogenes**
Evelyn Zeiler, Nathalie Braun, Thomas Bçttcher, Andreas Kastenmꢀller, Sevil Weinkauf, and
Stephan A. Sieber*
Nature provides a rich source of bioactive compounds
comprising a diverse set of electrophilic core structures that
are poised to react with corresponding nucleophilic residues
such as serine and cysteine in enzyme active sites.[1–3] These
residues are usually relevant for catalysis and therefore
display fine-tuned reactivity towards their dedicated sub-
strates.[4] We and others previously investigated the dedicated
targets of monocyclic b-lactones which turned out to be
potent and selective inhibitors of diverse disease-associated
enzyme classes.[2,3,5–8] Covalent inhibition of the caseinolytic
peptidase ClpP, for instance, resulted in a dramatic attenu-
ation of bacterial virulence.[3] ClpP is an important, highly
conserved heat shock protein with additional regulatory
functions in many pathogens.[9–11] Some organisms such as
Listeria monocytogenes genetically encode for two function-
ally and structurally uncharacterized ClpP isoforms (ClpP1
and ClpP2). So far, all b-lactones were reported to target
solely ClpP2 and not ClpP1,[2] raising the question whether
monocyclic lactones lack suitable reactivity to interact with
the ClpP1 active-site nucleophile.
We herein expand the scope of natural-product-derived b-
lactones to strained bicyclic ring systems which may exhibit
enhanced reactivity profiles. The natural products omuralide,
salinosporamide, and vibralactone (VL) represent such
desired scaffolds and have been reported to be potent
proteasome or lipase inhibitors.[12–14] We utilized a chemical
proteomic strategy termed “activity-based protein profiling
(ABPP)”[15–17] to demonstrate that vibralactone (VL), con-
trary to monocyclic b-lactones, binds to both ClpP1 and ClpP2
in L. monocytogenes. Moreover, by combining transmission
electron microscopy (TEM) and homology modeling/struc-
ture predictions, we were able to determine the quaternary
structure of the hetero-oligomeric complex (Figure 1).
VL was synthesized as described by Zhou and Snider[18]
(Scheme 1 in the Supporting Information) and modified with
an alkyne handle in the final step for target discovery by
ABPP (Figure 1).[19] Target analysis started by the incubation
of this vibralactone probe (VLP) with intact living cells of
L. welshimeri and its pathogenic counterpart L. monocyto-
genes. Upon cell lysis, the proteome was treated under click
chemistry (CC)[20–22] conditions with rhodamine azide and the
targets were visualized by fluorescent SDS-PAGE analysis
(Figure 1 in the Supporting Information). Two strong fluo-
rescent bands for approximately 20 kDa proteins were
present at comparable intensities in L. welshimeri as well as
in L. monocytogenes (Figure 2A) down to a VLP concen-
tration of 3.4 mm (Figure 1C in the Supporting Information).
Pre-incubation with various concentrations of unmodified VL
gradually abolished the labeling of these bands, demonstrat-
ing that the natural product exhibits comparable target
selectivity (Figure 2B). Mass spectrometric (MS) analysis
revealed that the lower band corresponds to ClpP2 which has
been labeled by monocyclic b-lactones before.[2] Interestingly,
the upper band corresponds to ClpP1 which could not be
addressed by any other b-lactone probe (Figure 2A, Table 1
in the Supporting Information). While ClpP2 orthologues
from various organisms exhibit a high sequence homology
(77% identity between L. monocytogenes and S. aureus) with
a tetradecameric barrel-shaped assembly in crystal struc-
tures,[23–26] ClpP1 shares only 41% identity with ClpP2
(Figure 2 in the Supporting Information). This raises the
question whether ClpP1 exhibits a different fold and function
and assembles with ClpP2 in mixed complexes, as previously
suggested for hetero-oligomeric complexes of different ClpP
isoforms.[27]
[*] E. Zeiler, Prof. Dr. S. A. Sieber
Department Chemie, Center for Integrated Protein Science CIPSM,
Institute of Advanced Studies IAS, Technische Universitꢀt Mꢁnchen
Lichtenbergstrasse 4, 85747 Garching (Germany)
E-mail: stephan.sieber@tum.de
Dr. N. Braun, A. Kastenmꢁller, Prof. Dr. S. Weinkauf
Center for Integrated Protein Science Munich CIPSM
Department of Chemistry, Technische Universitꢀt Mꢁnchen
Lichtenbergstrasse 4, 85747 Garching (Germany)
Dr. T. Bçttcher[+]
AVIRU, EXIST Transfer of Research, OC II
Lichtenbergstrasse 4, 85747 Garching (Germany)
[+] Current address:
Department of Biological Chemistry and Molecular Pharmacology,
Harvard Medical School, Boston, MA (USA)
[**] We thank Mona Wolff for excellent scientific support and Wolfgang
Steglich for helpful discussions. E.Z. was supported by the SFB749
and by the TUM-GS. E.Z. thanks Quan Zhou for helpful discussions.
T.B. was supported by the German National Academic Foundation
and by an EXIST technology transfer grant of the Federal Ministry of
Economics and Technology (BMWi); S.A.S. was supported by the
Deutsche Forschungsgemeinschaft (Emmy Noether), SFB749,
FOR1406, an ERC starting grant, and the Center for Integrated
Protein Science Munich CIPSM. S.W. and N.B. were funded by the
DFG (SFB594) and CIPSM.
Supporting information for this article (including details on the
synthesis and characterization of compounds, bioassays, electron
microscopy, image processing/3D reconstruction, structure pre-
diction, homology modeling as well as proteome preparation and
To address these questions, we recombinantly overex-
pressed ClpP1 and ClpP2 independently as well as by means
of a co-expression vector system in Escherichia coli. Co-
expressed ClpP1P2 was isolated through a C-terminal strep
Angew. Chem. Int. Ed. 2011, 50, 11001 –11004
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
11001