Communications
the absence of free 2 or 1. The protein was extracted from the
gel, trypsin digested, and subjected to mass spectrometry.
Proteomic analysis identified it as ClpC1 (Rv3596c), an
ATPase belonging to the Clp/Hsp100 AAA + superfamily
(ATPases associated with various cellular activities). The
ClpC proteins function as regulatory subunits of the casein-
olytic Clp protease. It is a chaperone that unfolds protein
substrates in an ATP-dependent manner before channeling
them to the ClpP protease subunit of the complex.[5] The
Clp complex typically consists of a sandwich of two hepta-
meric rings of ClpP protease units flanked by two hexameric
rings of Clp ATPases, thus structurally and functionally
resembling the eukaryotic proteasome. In bacteria, AAA +
proteins play a crucial role in protein quality control by
removing unfolded proteins from the cell. In addition,
AAA + proteins have been described to specifically control
the turnover of regulatory proteins and thus play a key role in
developmental processes and virulence in pathogenic bacteria
such as S. aureus.[6] In Mtb the Clp complex has not been
studied extensively.[7] Activators and inhibitors of the pro-
teolytic subunit ClpP have been described in S. aureus and
other Gram-positive bacteria.[8] To the best of our knowledge
cyclomarin A is the first small molecule described to date to
modulate the activity of the regulatory subunit ClpC of the
Clp complex.
The specificity of the protein–compound interaction was
analyzed by affinity chromatography combined with compe-
tition experiments using free compounds preincubated with
cell lysate. Both free 1 and 2 reduced the amount of captured
protein by 50% at a concentration of 10 nm, thus suggesting a
specific and high affinity binding of both compounds (Fig-
ure 1a). Unrelated cyclic peptides like thiopeptide GE2270
and cyclosporin had no influence even at concentrations of
10 mm. Neither ATP nor the nonhydrolyzable g-S-ATP
competed for binding at a concentration of 10 mm. To the
contrary, g-S-ATP increased the amount of captured
ClpC1 protein, probably owing to stabilization of the hex-
americ complex in the protein lysate.[7] From these experi-
ments it can be concluded that 1 binds specifically and with
high affinity to ClpC1 and does not interfere with ATP bind-
ing by the two ATPase domains of ClpC1.
Figure 1. Cyclomarin A (1) binds specifically and with high affinity to
ClpC1. a) SDS-PAGE gels of mycobacterial protein lysate stained with
SYPRO Ruby dye. Lysate (1 mg), prepared from M. bovis BCG culture
with OD600 value of 1.5, was incubated for 2 h at 48C with different
concentrations of free 1, 2, thiopeptide GE2270E (GE), cyclosporin A
(CsA), ATP, and g-S-ATP prior to incubation with immobilized 2
(1.4 mmolmLÀ1 NHS-sepharose beads, GE Healthcare). b) Isothermal
titration calorimetry experiments were performed using an AutoITC
(MicroCal) at 308C, with 10 mm ClpC1 (in 20 mm Tris-HCl pH 7.8,
100 mm NaCl, 0.5 mm TCEP) in the sample cell. 28 aliquots of 10 mL
of 100 mm compound solutions (90% DMSO) were injected into the
sample chamber at 6 min intervals. The data were fitted to a single-
site binding equation. DMSO=dimethyl sulfoxide, SDS-PAGE=so-
dium dodecylsulfate–polyacrylamide gel electrophoresis.
The compound–protein interaction was confirmed using
purified, recombinant ClpC1 in isothermal titration calorim-
etry experiments (Figure 1b). The data fitted to an equilib-
rium dissociation constant of 16 nm, thus indicating that the
aminoalcohol 2 binds to ClpC1 with high affinity. The
stoichiometry of the interaction was 1:1 (Table 2 in the
Supporting Information). Importantly, the antimycobacterial
activity of cyclomarin-derivatives correlated well with binding
to ClpC1. Binding to ClpC1 was not detected for the inactive
methyl-phenylamine derivative 3.
To further study the effect of 1 on the Clp complex we
employed a protein degradation assay in Mycobacterium
smegmatis. This fast growing mycobacterium is sensitive to 1
(MIC50 = 0.6 mm) and possesses Clp proteins exhibiting
> 90% identity to the Mtb Clp proteins. The Clp ATPases
recognize their substrates by binding to an 11 amino acid
peptide sequence, which was used to tag green fluorescent
protein (GFP-LDD).[9] A reduction of fluorescence by 40%
was observed in the bacteria expressing GFP-LDD in the
presence of cyclomarin, whereas bacteria expressing untag-
ged GFP showed a decrease of only 10% (Figure 2). These
results suggest that cyclomarin specifically increases proteol-
ysis mediated by the caseinolytic protease inside the cell.
Previously, clpC1 has been indicated as an essential gene
in Mtb using a genome-wide transposon mutagenesis.[10] This
supports our finding that a clpC1 knock-out in Mtb was
successful only when a plasmid-born copy of the clpC1 gene
was provided in trans (Figure 2 in the Supporting Informa-
tion). Taken together this observation suggests that ClpC1 is
indeed essential for bacterial growth.
In conclusion, ClpC1 is the protein target of the natural
product antibiotic cyclomarin in Mtb. By using chemical
proteomics it could be demonstrated that interference with
the function of the ClpC1 ATPase with a noncompetitive
small molecule is bactericidal in actively growing and in
hypoxic nongrowing mycobacteria. The frequency of sponta-
ꢀ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2011, 50, 5889 –5891
Schmitt, Esther K.
