Disruption of oligomerization and dehydroalanine formation as mechanisms for ClpP protease inhibition

Article abstract of DOI:10.1021/ja4082793

Over 100 protease inhibitors are currently used in the clinics, and most of them use blockage of the active site for their mode of inhibition. Among the protease drug targets are several enzymes for which the correct multimeric assembly is crucial to thei

Full text of DOI:10.1021/ja4082793

Article
pubs.acs.org/JACS
Disruption of Oligomerization and Dehydroalanine Formation as
Mechanisms for ClpP Protease Inhibition
Malte Gersch,†,§ Roman Kolb,†,§ Ferdinand Alte,‡ Michael Groll,‡ and Stephan A. Sieber*,†
†Center for Integrated Protein Science at the Department of Chemistry, Institute of Advanced Studies IAS and ‡Center for Integrated
Protein Science at the Department of Chemistry, Technische Universitat Munchen, Lichtenbergstrasse 4, Garching D-85747,
̈
̈
Germany
S
* Supporting Information
ABSTRACT: Over 100 protease inhibitors are currently used
in the clinics, and most of them use blockage of the active site
for their mode of inhibition. Among the protease drug targets
are several enzymes for which the correct multimeric assembly
is crucial to their activity, such as the proteasome and the HIV
protease. Here, we present a novel mechanism of protease
inhibition that relies on active-site-directed small molecules
that disassemble the protease complex. We show the
applicability of this mechanism within the ClpP protease
family, whose members are tetradecameric serine proteases and serve as regulators of several cellular processes, including
homeostasis and virulence. Compound binding to ClpP in a substoichiometric fashion triggers the formation of completely
inactive heptamers. Moreover, we report the selective β-sultam-induced dehydroalanine formation of the active site serine. This
reaction proceeds through sulfonylation and subsequent elimination, thereby obliterating the catalytic charge relay system. The
identity of the dehydroalanine was confirmed by mass spectrometry and crystallography. Activity-based protein profiling
experiments suggest the formation of a dehydroalanine moiety in living S. aureus cells upon β-sultam treatment. Collectively,
these findings extend our view on multicomponent protease inhibition that until now has mainly relied on blockage of the active
site or occupation of a regulatory allosteric site.
binding the proteolytic subunit ClpP.16−18 In the functional
INTRODUCTION
■
ClpXP complex, hexameric ClpX stacks on top of the
tetradecameric serine protease ClpP that is built by two
heptameric rings.19−21 It has been demonstrated that the
handle domain linking the two heptameric rings of the ClpP
protease is highly dynamic22,23 and that ClpP proteins adopt
different conformations (Figure 1A).24−26 In the compressed
form, the catalytic residues are misaligned and the E helix in the
handle domain is either kinked or structurally flexible. Upon
adoption of the extended, active conformation, the catalytic
triad aligns properly.
We recently described a hydrogen-bonding network that
links the oligomeric state with the alignment of the catalytic
triad in ClpPs from Staphylococcus aureus (SaClpP)25 and
Listeria monocytogenes.27 In an active tetradecamer, Arg171 and
Asp170 engage in interactions across the heptamer−heptamer
interface that, in turn, cause a backbone motion of the catalytic
Asp172 and enable alignment of the catalytic triad (Ser98,
His123, Asp172). Similar to the case for ClpP from Escherichia
coli,28 mutation of any of these network residues as well as
mutations destabilizing the extended E helix led to catalytically
inactive, heptameric proteins.
Over 100 different protease inhibitors are currently applied in
the clinic as therapeutic agents,1 and the most prevalent
strategy for protease inhibition is represented by blockage of
the active site with covalently or noncovalently acting
compounds.2,3 In addition, a limited number of allosteric and
exosite inhibitors has been described.4 Among those are small
molecules that inhibit the formation of the physiologically
active dimers of HIV protease5 and of the human Kaposi’s
sarcoma associated herpes virus (KSHV) protease.6 Covalent
allosteric binders have been described for caspases trapping the
enzyme in an inactive, dimeric form.7 Although they are not
proteases, HIV integrase and the C-reactive protein can be
inhibited by shifting their oligomerization equilibria.8,9
Bacterial organisms possess several multicomponent proteo-
lytic machineries such as ClpXP, FtsH, HslVU, and the Lon
protease10−13 to break down damaged and regulatory, short-
lived proteins which are of pivotal importance to cell survival
and pathogenicity.14,15 Despite considerable sequence diver-
gence, these systems all share a general topology in which
proteolytic subunits and AAA+ ATPases as chaperones act
together in barrel-shaped complexes. Chaperone and protease
may form two domains in one polypeptide chain (as in FtsH
and Lon) or may be encoded by different genes (as in ClpXP
and HslVU) to allow further flexibility: for example, with
different chaperones (ClpA, ClpC, and ClpX) being capable of
Received: August 16, 2013
© XXXX American Chemical Society
A
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Figure 1. Small-molecule-induced disruption of SaClpP oligomerization: (A) cartoon representation of SaClpP tetradecamer (extended
conformation) and SaClpP heptamer (compressed conformation), with one subunit highlighted in yellow; (B) structures of DFP, E2, and RKS09;
(C) SaClpP peptidase activity after compound treatment (1 h, room temperature; incubation carried out with 50 μM SaClpP to allow for direct SEC
analysis and then dilution of the sample to 1 μM for kinetic and MS experiments); (D) intact protein mass spectrometry after compound treatment
showing incomplete modification. with measured masses indicated above the peaks and expected masses are given in the lower left corner; (E) size-
exclusion chromatograms showing heptamer formation upon compound treatment; (F) size-exclusion chromatograms showing partial induction of
heptamer formation (1 h, room temperature); (G) chemical formulas of further compounds tested; (H) compounds with partial heptamer formation
showing remaining peptidase activity that decreased after longer incubation times and elevated temperatures.
Significant interest in ClpP inactivation started with the
discovery of its crucial role in virulence of S. aureus, a pathogen
that causes severe infections in the clinics and is difficult to treat
through the occurrence of multiresistance.29,30 For instance, a
ClpP knockout strain was severely impaired in murine infection
studies,31 indicating a major role of ClpP in virulence
regulation.32 In line with these results, covalent small-molecule
inhibitors of ClpP of the β-lactone class led to a marked
decrease of virulence factor secretion.33 While studying the
binding site of the β-lactone inhibitors, we characterized
mutants of SaClpP that were unexpectedly purely heptameric
and catalytically inactive (e.g., L150A, N151Q, T169A).34
Intrigued by the apparent sensitivity of the heptamer−
tetradecamer transition switch, we were curious whether we
could exploit this disassembly mechanism for protease
inhibition triggered by small molecules. We therefore screened
a compilation of both commercially available and custom-
synthesized, covalent serine protease inhibitors and looked for
major changes in SaClpP oligomerization upon ligand binding.
We identified several active-site-directed compounds that
induced inactive heptamers, and we thereby validated heptamer
formation as an inhibition strategy. Moreover, we characterized
the sultam-induced conversion of the active site serine into a
dehydroalanine as a complementary inhibition mechanism.
RESULTS
■
In search for compounds that disassemble tetradecameric
SaClpP into inactive heptamers, we selected molecules from
various chemical classes for testing (Supplementary Figure 1,
Supporting Information). Our focused library comprised β-
lactones, a fluorophosphonate, a fluorophosphate, a dichlor-
oisocoumarin, sulfonyl fluorides, β-sultams, and chloromethyl
ketones as reactive moieties.3,26,33,35−38 In order to obtain a
comprehensive picture of inhibitor binding to SaClpP, we
determined the size of the protein complexes using a calibrated
size exclusion chromatography column (Supplementary Figure
2A), measured peptidase activity with a fluorogenic substrate
assay, and used intact protein mass spectrometry (MS) as a
readout for covalent protein modification. Since the effect of
covalently acting compounds is time-dependent, we performed
all experiments after 1 h of incubation at room temperature.
The analysis revealed distinct inhibition profiles so that the
compounds were classified into three categories: (a) inhibitors
that modify the active sites partially and change the
oligomerization to smaller species, (b) inhibitors that modify
B
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Figure 2. Conversion of the active site serine into dehydroalanine by β-sultams: (A) intact protein MS revealing the presence of protein species 18
Da lighter than wild-type SaClpP, with measured masses being indicated above the peaks and expected masses given in the lower left corner; (B)
efficient inhibition of SaClpP (1 μM) by RKS07 after 1 h of incubation at room temperature (IC50 = 0.98
0.01 μM); (C) size exclusion
chromatograms of β-sultam-treated SaClpP samples (data on RKS09 from Figure 1D shown for clarity); (D) time-resolved formation of SaClpP
species upon treatment with RKS07 and RKS09 via protein MS (1 μM SaClpP); (E) mechanism for the formation of Dha involving sulfonylation of
the active site serine and subsequent elimination; (F) generation of the SaClpP-RKS07 adduct (1 min, 10 μM SaClpP), dilution into buffers with
different pHs, and quantification of Dha-SaClpP formation after 2 h by MS (1 μM SaClpP); (G) CID fragmentation spectrum of the peptide
A76IYDTIQHIKPDVQTICIGMAADhaMGSFLL104, showing the presence of a dehydroalanine moiety at position 98 (y ions, blue; b ions, red). For
a complete list of identified fragments, please refer to Supplementary Table 6.
all active sites and do not change the oligomerization, and (c)
nonbinders.
heptamer (Figure 1E). We further confirmed the size of DFP-
treated SaClpP by static light scattering that yielded masses of
304 kDa for unmodified SaClpP and 150 kDa for DFP-treated
SaClpP, showing clearly the presence of a heptameric species.
Although up to 65% of the active sites were unmodified in these
samples, enzyme activity was completely blocked (Figure 1C).
In the presence of the tetradecamer-stabilizing agent glycerin,
higher occupancies by E2 were obtained, while glycerin alone
did not change SaClpP peptidase activity (Supplementary
Figure 3). These results strongly support the view of
tetradecamers as active and heptamers as inactive species.25
Furthermore, the data demonstrate a mechanism in which
inhibitor binding at some but not all active sites destabilizes the
Inhibition of SaClpP by DFP, RKS09, and E2 through
Heptamer Formation despite the Presence of Unmodi-
fied Active Sites (Category a). To our surprise, we identified
a total of seven compounds that led to only partial modification
of the active sites but efficiently altered the oligomerization to
smaller species (category a). In an MS experiment, diisopropyl
fluorophosphate (DFP), β-sultam RKS09, and β-lactone E2
showed modification of 57%, 63%, and 35% of the active sites,
respectively, but caused complete inhibition of SaClpP (Figure
1B−D). Interestingly, size exclusion chromatography revealed
an almost quantitative shift from the tetradecamer to the
C
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tetradecamer and is sufficient for the dissociation into
heptamers, thereby causing complete inhibition.
lighter than the unmodified protein (Figure 2A). To resolve the
identity of the 18 Da lighter SaClpP species formed upon
treatment with RKS07, we studied the reaction of SaClpP with
β-sultams in more detail. Time-dependent measurements
showed that both RKS07 and RKS09 induced the formation
of the 18 Da lighter SaClpP after 20 h of incubation as the sole
species (Figure 2A,D). Formation occurred at different
velocities and seemed to proceed through the sulfonylated
enzyme, which decreased to the same extent as the 18 Da
lighter species increased. Notably, partially modified SaClpP by
RKS09 was heptameric, while incubation for 20 h led to the 18
Da lighter, inactive species and a change of the oligomerization
back to the tetradecamer (Figure 2C). This is in accordance
with the tetradecameric size of the RKS07-treated SaClpP
(Figure 2C).
A mass decrease of 18 Da is consistent with the loss of a
water molecule in the course of an elimination reaction of the
sulfonylated active site serine to furnish a dehydroalanine
moiety (Figure 2E). Remarkably, the addition reaction of
RKS07 and SaClpP reached completion (>95%) within 1 min
of incubation at room temperature (Figure 2A). We diluted this
adduct into different buffers, showing a clear pH dependence of
the second reaction step that proceeded to completion only at
neutral or basic pH (Figure 2F and Supplementary Figure 7A).
We next digested the modified enzyme with trypsin and
chymotrypsin and analyzed the resulting peptides by nano-LC-
MS/MS (sequence coverage of SaClpP 100%; see Supple-
mentary Tables 4−6). We identified peptides that upon
collision-induced dissociation (CID) fragmentation showed
the mass corresponding to dehydroalanine (Dha) instead of the
catalytic serine 98 (Figure 2G). A dehydroalanine on a protein
surface was recently used to install posttranslational mod-
ifications on purified proteins by addition of thiols.40 Despite
extensive attempts, we were unable to observe complete
covalent addition of several sulfur nucleophiles to Dha-SaClpP.
Heat denaturation in the presence of thiols yielded only 30%
modified protein (Supplementary Figure 8). This indicates that
the Dha in the ClpP active site is either sterically or
electronically distinct from Dha moieties in loops on protein
surfaces. To confirm the existence of the Dha residue and to
investigate putative cross links between the electrophilic
Michael system and a nearby nucleophile, we solved the crystal
Next, we characterized the fluorosphosphonate probe FP, the
dichloroisocoumarin DCI, the β-sultam RKS02, and the
chloromethyl ketone Z-LY-CMK (Figure 1G) and recorded
partial active site modification by MS (Supplementary Figure
2C). In these cases, we detected two peaks in the size exclusion
chromatogram corresponding to a tetradecameric and a
heptameric species, with the latter being more prominent
(Figure 1F). We then analyzed the degree of active site
modification in the peaks of Z-LY-CMK-treated SaClpP by MS
and found that the degree of modification was larger in the
heptameric peak (33%) than in the tetradecameric fraction
(17%), fitting well to an overall modification of 29%
(Supplementary Figure 2C). As expected from the presence
of unmodified active sites in the tetradecameric species, minor
enzymatic activity could be detected by a fluorogenic substrate
assay (Figure 1H). Longer incubation times (2 h) and higher
temperatures (37 °C) led to full inhibition (Figure 1H) but not
to complete modification by Z-LY-CMK (Supplementary
Figure 2C). These results suggest a dynamic equilibrium
between tetradecamers and heptamers. In this equilibrium,
reassociation of heptamers into tetradecamers would be
accompanied by a conformational change by which inhibitor
binding at the remaining unmodified active sites can occur. The
resultant higher occupancy would shift the equilibrium toward
the heptameric population. This mechanism explains the
increased modification in the presence of the stabilizing agent
glycerin, the delayed complete modification by some
compounds, and the increased degree of modification in the
heptameric fraction.
Occupation of Each Active Site by D3, RM448, and
RKS07 without a Change in the Oligomeric State
(Category b). As described previously,34 β-lactones D3 and
RM448 result in an almost instant covalent modification of all
14 active sites and complete inhibition of peptidase activity.
These small molecules retain the tetradecameric state
(Supplementary Figure 2B), thus belonging to category b.
Another compound of this category, the β-sultam RKS07, also
abolished enzyme activity completely (Figure 2B) without
changing the oligomerization (Figure 2C). Since we found the
IC50 value of RKS07 to depend on the enzyme concentration
(Supplementary Figure 4), we concluded an irreversible
mechanism of inhibition.39 We thus measured kobs/[I] values
as quantitative measures of potency suitable for the character-
ization of covalent inhibitors. While RKS07 reacted with
structure of the modified enzyme to 2.3 Å resolution (Rfree
=
0.221). Consistent with MS data, a Dha could be refined at the
position of all 14 active site serine residues (Figure 3,
velocity comparable to that of β-lactones D3 and E2 (140
9
M−1 s−1 (RKS07); 78 6 M−1 s−1 (D3); 64 3 M−1 s−1 (E2)),
RKS09 reacted much faster with a kobs/[I] value of 1320 82
M−1 s−1 (Supplementary Figure 6), thus representing the fastest
ClpP inhibitor known to date.
Both 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF)
and phenylmethylsulfonyl fluoride (PMSF) as well as Z-L-
CMK and N-p-tosylphenylalanyl chloromethyl ketone (TPCK)
did not inhibit SaClpP (category c), as judged from peptidase
activity measurements (Supplementary Figure 5).
Dehydroalanine Formation at the Active Site Serine
Caused by β-Sultams. Unexpectedly, we found by MS two
peaks in the RKS07-treated sample (Figure 2A): the larger peak
corresponded to the joint masses of protein and inhibitor that
can be rationalized by a simple addition reaction upon attack at
the sulfur by the active site serine, fission of the S−N bond, and
ring opening. The second, smaller species, however, was 18 Da
Figure 3. Crystal structure of Dha-SaClpP: stereoview on the active
site of Dha-containing SaClpP in the crystal structure with 2.3 Å
resolution. The electron density (blue mesh) is consistent with a
dehydroalanine moiety (for comparison, the gray mesh denotes Ser98
in the wild type structure). The 2Fo − Fc electron density maps are
contoured at 1.0 σ; amino acids have been excluded prior to phase
calculations.
D
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Supplementary Table 3). The electron density clearly depicted
the lack of the hydroxyl group and provided no evidence for an
intramolecular Michael-type attack. No other structural changes
were observed by comparison of Dha-SaClpP with the wild
type protein. Collectively, these data further confirm the
formation of a dehydroalanine residue through elimination of
the sulfonylated active site serine upon treatment with β-
sultams.
inactive heptamers in which also the acyl ester hydrolysis is
impaired. ClpP incubated with RKS07 and Z-LY-CMK
displayed no enzyme activity even after 1 week (see
Supplementary Figure 7C,D for data at 25 and 4 °C).
Accordingly, purified Dha-SaClpP was inactive at all time
points.
The generality of the approach was investigated by screening
ClpP proteins from Listeria monocytogenes (LmClpP2) and
Escherichia coli (EcClpP) for compound-induced changes in
their oligomerization state. E2 and RKS09 were able to induce
heptamer formation in LmClpP2, as observed with SaClpP
(Supplementary Figure 9A). In contrast, EcClpP only showed a
slight shift after E2 and Z-LY-CMK treatment, while D3
induced partial formation of heptamers (Supplementary Figure
9B). Moreover, we observed the respective dehydroalanine
proteins upon RKS07 treatment by mass spectrometry
(Supplementary Figure 10).
Figure 4. (A) Reversal of inhibition of SaClpP by β-lactones (D3, E2)
by hydroxylamine treatment. (B) Allowance of prolonged inhibition by
both dehydroalanine formation and disruption of oligomerization
(incubation at 37 °C; see Supplementary Figure 7B for DMSO
controls). In both experiments, incubation was carried out with 10 μM
SaClpP and 10× excess of inhibitor and aliquots were diluted to 1 μM
SaClpP for activity measurements.
Figure 5. (A) Structures of the synthesized alkyne-free β-sultams. (B)
Competitive labeling experiment in which living S. aureus cells were
pretreated with DMSO or the compounds shown in (A) (1000 μM)
and then labeled with ClpP-specific probe D3 (100 μM). (C) ABPP-
labeling experiment in which living S. aureus cells were incubated with
the probes indicated at 100 μM and, following cell lysis and click
chemistry mediated rhodamine attachment to the probe, the proteome
was separated by SDS-PAGE and imaged for fluorescence. See
Supplementary Figure 11 for coomassie loading controls and images of
the entire gels. (D) Western blot against SaClpPs after treatment of S.
aureus cells with the indicated compounds for 2 h (see Supporting
Figure 13 for an uncropped image).
Improved Inhibition by both Induction of Heptamers
and Dehydroalanine Formation. The high propensity to
hydrolysis of the acyl ester intermediate is the major drawback
of β-lactone-based inhibitors. This feature is commonly
observed with hydrolases inhibited by β-lactones, since the
hydrolysis of an ester is part of their catalytic cycle and
reactivation times of as low as 2 min are possible.41,42
Hydroxylamine cleaves thioesters and, to a lesser extent, esters
in aqueous solutions. As expected, β-lactone-treated SaClpP
was found susceptible to hydroxylamine treatment, restoring
the catalytic activity, while RKS07 and Z-LY-CMK treated
samples were unaffected, proving the different inhibition
mechanisms (Figure 4A). To test for sustained inhibition, we
incubated SaClpP with several inhibitors (D3, E2, RKS07, Z-
LY-CMK) at different temperatures (4, 25, 37 °C) and
measured peptidase activity at several time points (Figure 4B,
Supplementary Figure 7B−D). While the D3-treated enzyme
regained its activity almost completely within 20 h at 37 °C, the
E2-treated sample still showed 90% inhibition after 1 week at
37 °C normalized to the untreated control (Supplementary
Figure 7B). This result was found to be in accordance with
mass spectrometry results. It supports the view of E2-induced
Dehydroalanine Formation in Living Bacteria. To test
if this reaction occurs in vivo, we synthesized alkyne-free
analogues of the previous β-sultams termed RKS13, RKS14,
and RKS15 (Figure 5A). We employed a competitive activity-
based protein profiling (ABPP)43−46 experiment in which we
preincubated living S. aureus cells with alkyne-free β-sultams
followed by addition of alkyne-functionalized D3 as an SaClpP-
selective probe.33 After cell lysis, a rhodamine fluorophore was
attached to the probe via click chemistry,47−49 the proteome
was separated by SDS-PAGE, and the gel was imaged for
fluorescence (Figure 5B, Supplementary Figure 11). In
agreement with our in vitro data, no band in the case of
E
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RKS13 and only weak bands in the case of RKS14 and RKS15
were observed in the pretreated samples, indicating either the
complete formation of dehydroalanine or the complete
conversion of SaClpP into inactive heptamers owing to partial
modification with inhibitor. To rule out the latter possibility, we
treated living S. aureus cells with alkyne-functionalized probes,
where we could not detect a fluorescent band in the molecular
weight range of ClpP (Figure 5C; see Supplementary Figure 12
for the result of a time-dependent labeling experiment).
Moreover, we showed by Western blot that the compounds
do not alter the cellular concentration of ClpP (Figure 5D).
These data suggest the formation of dehydroalanine-modified
SaClpP in living cells.
reported for β-lactamases.55 This reaction proceeds rapidly
through the sulfonylated serine.35 Subsequent spontaneous
elimination of this metastable intermediate results in an
increase of the dehydroalanine species. The RKS09 compound
displayed a unique behavior, since binding to ClpP caused
disassembly of the holo-enzyme into heptamers that upon
dehydration reassociate back into tetradecamers. The identity
of the dehydroalanine was unequivocally confirmed by mass
spectrometry and protein crystallography. Furthermore,
activity-based protein profiling experiments suggest the
formation of a dehydroalanine moiety in living S. aureus cells
upon β-sultam treatment. These results are a significant
advance over the previously reported dehydroalanine formation
in isolated enzymes.55,56
DISCUSSION
Incubation of ClpP with β-sultams and subsequent
dehydroalanine formation represents an elegant strategy to
introduce a site-directed mutation into a protease active site
without touching any other serine residue of the protein.
Alternative methods to install a dehydroalanine have either
been restricted to cysteine residues57 or have been impaired by
tedious optimization of reaction conditions37 and, possibly,
incomplete turnover.58 With SaClpP having failed to react with
PMSF, this reactivity is no longer a prerequisite for
dehydroalanine formation in serine proteases. Moreover,
inhibition by dehydroalanine formation is irreversible and
thus long lasting in comparison to acyl ester intermediates
formed by β-lactones and β-lactams that can be hydrolyzed.
Collectively, we report two mechanisms for improved
inhibition of multicomponent proteases. These findings extend
our view on protease inhibition that until now has mainly relied
on blockage of the active site or occupation of a regulatory
allosteric site.
■
ClpP is a crucial pathogenesis-associated enzyme in several
bacterial pathogens such as Staphylococcus aureus, Mycobacte-
rium tuberculosis, and Listeria monocytogenes,33,50,51 which
demands novel ways for sustained inhibition. Here, we report
the discovery of several covalently acting, active-site-directed
compounds that lead to partial or complete dissociation of the
proteolytic complex into catalytically inactive heptamers. Three
compounds (DFP, RKS09, and E2) caused complete
heptamerization, while other compounds shifted the oligome-
rization equilibrium toward the heptameric population. We
have shown that this mechanism is not limited to SaClpP but
could also be observed with other ClpP proteins. These results
likely explain observations in the literature that DFP induces
structural heterogeneity,52 that DFP shows incomplete
inhibition,53 and that DFP displays nonstandard inhibition
kinetics.54 This is, to our knowledge, the first report in which a
multimeric protease is inhibited by a ligand-induced dis-
assembly triggered by an active-site-directed molecule.
It was previously speculated that transient fluctuations in the
ClpP handle region might result in the formation of equatorial
pores contributing to product release. This view is based on
NMR data as well as on the observation of a functional ClpP
enzyme where both axial pores are blocked by two ClpX
hexamers.17,22 These fluctuations would most likely be caused
by a transition from the extended state into the compressed
state, though the mechanism triggering this conformational
switch remains to be elucidated. However, our results reveal
that certain steric bulk at the active site, e.g. introduced by the
peptidic inhibitor Z-LY-CMK, destabilizes the extended
conformation and causes the formation of heptamers. In
analogy, native peptide fragments with spacious side chains
might serve as triggers for the conformational switch generating
equatorial pores for product release next to the active site.
Examination of the crystal structures of E. coli ClpP with bound
Z-LY-CMK36 as well as Bacillus subtilis ClpP with DFP26 has
not provided a clear explanation as to why ClpP proteins
dissociate in solution. Our results suggest that steric bulk at the
active site might shift the catalytic His123, thereby moving the
catalytic Asp172 that would disrupt the Arg171−Asp170
interaction across the ring interface. This view is supported
by the observation that small changes in this region (e.g.,
T169A) also led to heptamerization and by results that show a
coupling of the catalytic triad with oligomerization.25,27 Further
experiments will have to clarify the molecular basis for this
mechanism of ligand-induced oligomerization defects.
ASSOCIATED CONTENT
* Supporting Information
■
S
Text, figures, and tables giving experimental procedures,
additional biochemical and kinetic data, proteomic data,
compound characterization data, and 1H and 13C NMR spectra.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
E-mail: stephan.sieber@tum.de.
■
Author Contributions
§These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful for funding from the Deutsche Forschungsge-
meinschaft, SFB 749, SFB 1035, CIPSM, and the European
Research Council (ERC starting grant). We thank Mona Wolff
and Burghard Cordes for technical assistance, Lisa Meixner and
Jakob Staab for help with protein assays, Christoph Kaiser for
assistance with native protein purification, Arie Geerlof for help
with SLS measurements, and Maria Dahmen for comments on
the manuscript. We thank the staff of the beamline X06SA at
the Paul Scherrer Institute, Swiss Light Source, Villigen,
Switzerland, for their help with data collection, and we thank
the Waldman lab (MPI Dortmund) for compound RM448.
M.G. acknowledges Ph.D. fellowships from the Fonds der
A mechanistically different inhibition principle for ClpP is
represented by the selective conversion of the active site serine
into a dehydroalanine moiety by β-sultams, as previously
F
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(31) Frees, D.; Qazi, S. N.; Hill, P. J.; Ingmer, H. Mol. Microbiol.
2003, 48, 1565.
(32) Frees, D.; Sorensen, K.; Ingmer, H. Infect. Immun. 2005, 73,
chemischen Industrie (FCI) and the German National
Academic Foundation.
8100.
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