Tailored Peptide Phenyl Esters Block ClpXP Proteolysis by an Unusual Breakdown into a Heptamer–Hexamer Assembly

Article abstract of DOI:10.1002/anie.201901056

The proteolytic complex ClpXP is fundamental to bacterial homeostasis and pathogenesis. Because of its conformational flexibility, the development of potent ClpXP inhibitors is challenging, and novel tools to decipher its intricate regulation are urgently needed. Herein, we present amino acid based phenyl esters as molecular probes to study the activity and oligomerization of the ClpXP complex of S. aureus. Systematic screening of (R)- and (S)-amino acids led to compounds showing potent inhibition, as well as stimulation of ClpXP-mediated proteolysis. Substoichiometric binding of probes arrested ClpXP in an unprecedented heptamer–hexamer assembly, in which the two heptameric ClpP rings are dissociated from each other. At the same time, the affinity between ClpX and ClpP increased, leading to inhibition of both enzymes. This conformational arrest is beneficial for the consolidated shutdown of ClpXP, as well as for the study of the oligomeric state during its catalytic cycle.

Full text of DOI:10.1002/anie.201901056

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Accepted Article
Title: Tailored peptide phenyl esters block ClpXP proteolysis by an
unusual breakdown into a heptamer-hexamer assembly
Authors: Markus Lakemeyer, Eva Bertosin, Friederike Möller, Dóra
Balogh, Ralf Strasser, Hendrik Dietz, and Stephan Axel
Sieber
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201901056
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10.1002/anie.201901056
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Tailored peptide phenyl esters block ClpXP proteolysis by an
unusual breakdown into a heptamer-hexamer assembly
Dr. Markus Lakemeyer, Eva Bertosin, Dr. Friederike Möller, Dóra Balogh, Dr. Ralf Strasser, Prof. Dr.
Hendrik Dietz, Prof. Dr. Stephan A. Sieber*
Abstract: The proteolytic complex ClpXP is fundamental to
bacterial homeostasis and pathogenesis. Due to its
conformational flexibility, the development of potent ClpXP
inhibitors is challenging and novel tools to decipher its intricate
regulation are urgently needed. Here, we present amino acid-
based phenyl esters as molecular probes to study the activity and
oligomerization of the ClpXP complex of S. aureus. Systematic
screening of (R)- and (S)-amino acids led to compounds showing
potent inhibition, as well as stimulation of ClpXP-mediated
proteolysis. Sub-stoichiometric binding of probes arrested ClpXP
in an unprecedented heptamer-hexamer assembly, in which the
two heptameric ClpP rings are dissociated from each other. At the
same time the affinity between ClpX and ClpP increased, leading
to inhibition of both enzymes. This conformational arrest is
beneficial for the consolidated shutdown of ClpXP, as well as for
the study of the oligomeric state during its catalytic cycle.
Phenyl esters have been identified as a new class of covalent
ClpXP inhibitors that surpass previous β-lactone inhibitors in
potency and show
a stereogenic methyl-switch for de-
oligomerization of ClpP.[7] The stereochemistry of the methyl-
group in the α-position to the ester determines, if inhibition occurs
while maintaining the ClpP14 state ((S)-ML90), or if de-
oligomerization into inactive heptameric species is induced ((R)-
ML89) (Figure 1a).[7] As the stereogenic α-methyl switch in
ML89/ML90 resembles an amino group in structurally related
aromatic amino acids, we here present customized peptide
phenyl esters as mimics of native substrates for in-depth
investigation of the catalytic activity and oligomerization of the full
ClpXP complex.
In the phenyl ester enantiomers ML89/ML90 the prominent
3,4,5-trimethoxyphenyl moiety was shown to be critical for binding
based on extensive SAR studies.[7] We therefore selected this
motif as side chain decoration of the first amino acid phenyl
esters, yielding the unnatural amino acid 3,4,5-trimethoxy-
phenylalanine (Tmo). Both enantiomers of the respective
compounds, (R)-11 and (S)-12, were prepared following the
ClpXP is a key regulator of bacterial virulence in a variety of
pathogens including Staphylococcus aureus.[1] The proteolytic
complex consists of a peptidolytic core, formed by 14 subunits of
caseinolytic peptidase (ClpP), flanked by apical AAA+
chaperones, such as hexameric ClpX. ClpP14 exhibits only
moderate peptidolytic activity towards short peptides and requires
binding to ClpX6 to one or both apical sites of the barrel for full
proteolytic activity. Within this complex, ClpX6 recognizes and
unfolds substrate proteins for subsequent digestion by ClpP14.[2]
Previous research identified various small molecule inhibitors of
ClpP.[3-7] Among those, reversible oxazole compounds displayed
excellent potency on the ClpP peptidase but suffered from a ClpX-
mediated repulsion based on conformational selection, thus
preventing inhibition of ClpXP.[8] Irreversible b-lactone inhibitors
are limited by compound stability, but have been effective in the
reduction of virulence.[6] For a subset of these compounds,
dissociation of ClpP14 into catalytically inactive heptameric
species was observed.[9,10] It yet remains unresolved, if ClpP7 is
an artifact of inhibition with synthetic molecules, or if this state has
a biological function, e.g. during proteolysis. Thus, a detailed
understanding of the communication within the proteolytic ClpXP
complex and how it can efficiently be modulated by molecular
probes remains elusive.
Erlenmeyer-synthesis
route,
starting
from
3,4,5-
trimethoxybenzaldehyde (Scheme 1). For the synthesis of N-
terminally modified and dipeptide phenyl esters, the compounds
were subjected to standard solution-phase peptide chemistry
(Scheme 1 and supplementary information).
All phenyl esters were initially tested at three concentrations (100,
10, 1ꢀµM) using two different assay setups: 1) inhibition of ClpP’s
peptidolytic activity towards the fluorogenic peptide Ac-Ala-hArg-
2-Aoc-ACC (Figure S1),[11] and 2) inhibition of ClpXP-mediated
proteolysis of green-fluorescent protein (GFP) that was tagged
with a short SsrA degradation signal.[12]
From the first set of eight N-terminally modified Tmo phenyl
esters, the previously described stereo-preference of ClpP was
confirmed, meaning that (R)-phenyl esters were generally better
inhibitors than the respective (S)-enantiomers (Figure 1b). While
[*] Dr. M. Lakemeyer, D. Balogh, Prof. Dr. S. A. Sieber
Center for Integrated Protein Science (CIPSM) at the Department of
Chemistry, Technische Universität München, Lichtenbergstrasse 4,
85747 Garching (Germany)
Email: stephan.sieber@tum.de
E. Bertosin, Prof. Dr. H. Dietz
Physics Department and Institute for Advanced Study, Technische
Universität München, Am Coulombwall 4a, 85748 Garching (Germany)
Dr. F. Möller, Dr. R. Strasser
Dynamic Biosensors GmbH, Lochhamerstr. 15, 82152 Planegg
(Germany)
Scheme 1. Stereoselective synthesis of Tmo phenyl ester derivatives.
Reagents and conditions: i) N-acetylglycine, NaOAc, Ac2O, 125ꢀ°C, 5h; ii)
NaOMe, MeOH, rt, 1.5ꢀh; iii) H2, Pd/C, MeOH, rt, 16ꢀh; iv) Alcalase, aq. NaHCO3,
rt, 6ꢀh; v) p-TsOH, MeOH, 80ꢀ°C, 6ꢀh; vi) Boc2O, DMAP, 75ꢀ°C, 3ꢀh, then aq.
LiOH, rt, 3ꢀh; vii) methyl-4-hydroxybenzoate, EDCꢀ•ꢀHCl, HOBt, DIPEA, DCM,
0ꢀ°C à rt, 18ꢀh; viii) TFA, DCM, 0ꢀ°C, 5ꢀh.
Supporting information for this article is available on the WWW under
http://dx.doi.org/
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the biologically more relevant ClpXP protease system for
subsequent compound evaluation.
Next, we investigated, whether inhibition by (R)-amino acid
phenyl esters is a general feature or if it is an exclusive trait of the
unnatural amino acid Tmo. A collection of thirteen diverse (R)-,
i.e. D-amino acid phenyl esters was synthesized and evaluated for
ClpXP inhibition (Figure 2a). Almost all derivatives were inactive,
including phenyl esters of nonpolar alanine (20) and methionine
(22), or polar serine (27) and glutamate (29). The aromatic
phenylalanine (23) and tyrosine (24) derivatives, structurally
related to 9, also did not affect proteolysis. Solely
leucine (21) and tryptophan (25) phenyl esters showed inhibition
at high and medium concentrations, respectively.
Previous studies revealed that ClpP contains a large
hydrophobic S1 pocket tailored for the accommodation of long
alkyl chains.[13] Correspondingly, β-lactones with alkyl chains of
various lengths,[6,13] as well as fluorogenic peptide substrates,
containing the unnatural amino acid (S)-2-aminooctanoic acid (2-
Aoc) in P1 position,[11] are good inhibitors and substrates,
respectively. This preference was not seen for phenyl esters, as
the Boc-(R)-2-Aoc analog 31 did not inhibit ClpXP. Likewise, the
amide analog of phenyl ester 9, compound 17, as well as an
activated tetrafluoroanilide (18), did not affect ClpXP-mediated
proteolysis (Figure S3a). Overall, these results emphasize the
importance of (R)-Tmo phenyl esters at the C-terminal position.
To further converge to natural peptide substrates of ClpXP,
dipeptide Tmo-phenyl esters were synthesized. First, the stereo-
preference for the P2 position was evaluated by testing all four
diastereomers of Boc-Met-Tmo-OAr and Boc-Leu-Tmo-OAr. The
(S,S)-dipeptides (32, 36) as well as the (S,R)- and (R,S)-
diastereomers (33, 34, 37, 38) failed to efficiently inhibit
proteolysis (Figure S3b). Conversely, the (R,R)-phenyl esters 35
and 39 were potent inhibitors, with 35 showing full inhibition at
10 μM (Figure S3b). Having identified the (R)-stereo-preference
of ClpXP for the P2-position, a representative set of Boc-(R,R)-
dipeptide Tmo-phenyl esters was synthesized and tested in the
GFP degradation assay (Figure 2b). In contrast to the P1 position,
ClpXP tolerated various amino acid side chains in P2, ranging
from hydrophobic groups, such as alanine (41) or methionine (35),
to polar side chains including serine (44), glutamate (45) or
arginine (48). This finding is in accordance with the lack of a well-
defined S2 pocket, which allows ClpP to digest peptides with
diverse amino acids in the P2 position.[11] However, as Boc-Gly-
Tmo-OAr (40) did not inhibit ClpXP, a certain degree of sidechain
interaction in the correct conformation is required for efficient
binding to the protein.
We also identified structurally diverse compounds that did
not inhibit, but rather stimulated the GFP-degradation by ClpXP.
While the dipeptide Boc-(S)-Met-(R)-Tmo-OAr (34) led to a
moderate increase of proteolysis up to a maximum of 150% at
10 μM, the acetylated (R)-Tmo phenyl ester 49 and Boc-(R)-
Glu(OBn)-(R)-Tmo-OAr 50 activated the ClpXP complex to about
250% at 100 μM and 10 μM, respectively (Figure 2c).
Interestingly, compound 51, the S-enantiomer of 49, hardly
affected ClpXP (Figure S3c), and a benzyl-deprotected derivative
of 50 (compound 45) was a potent inhibitor of ClpXP (Figure 2b).
In stark contrast to the activation of proteolysis, the three
compounds did not stimulate the peptidase activity of ClpP alone,
but rather showed moderate-to-good inhibition (depending on the
buffer system used, compare Figure S3d-f), thus differentiating
them from non-covalent peptide agonists of Mycobacterium
Figure 1. Biochemical evaluation of N-terminally capped Tmo phenyl esters.
a) Reaction of phenyl esters with the active site Ser98 traps ClpP in the acyl-
enzyme state. b) Inhibition of S. aureus ClpP peptidase and ClpXP protease
activity by the respective enantiomers. c) Apparent IC50 of both enantiomers of
Boc-Tmp-OAr in the protease assay. Data are normalized to the DMSO
control (100% activity). Data depict mean and standard deviation (SD) (n≥6).
the α-methyl compounds (R)-ML89/(S)-ML90 and the unmodified
Tmo phenyl esters (R)-11/(S)-12 only showed moderate inhibition
in both assays, capping the amine with a pivaloyl- (13 and 14) or
Boc- (9 and 10) group led to a significant improvement in
inhibition. (R)-Boc-Tmo-OAr (9) had an apparent IC50 of 0.37 μM
in the protease assay, which distinguishes it as the most potent
ClpXP inhibitor reported to date. Contrarily, its (S)-enantiomer 10
is a 25-times weaker binder (Figure 1c) that arrests the protein in
the acyl-enzyme state (Figure S2a-d). Importantly, the average
grade of active site modification for 9 at the IC50 concentration was
only ≈10% (Figure S2e). Hence, sub-stoichiometric binding of 9
facilitates an overall conformational rearrangement that leads to
the inactivation of all remaining active sites. The sterically more
demanding Cbz-group was only well-tolerated for the (R)-
enantiomer 15 in the peptidase assay, but failed to inhibit the full
ClpXP complex (Figure 1b). In general, effective inhibition of
ClpP's peptidolytic activity did not necessarily correlate with
efficient inhibition of the proteolytic ClpXP complex. This finding
is in accordance with previous studies, confirming that ClpX
exerts conformational control over ClpP and is able to boost
peptidolysis and revoke inhibitor binding.[8,10] We thus focused on
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Figure 2. Inhibition of ClpXP activity by a set of
Boc-protected (R)-amino acid- (a), and (R,R)-
dipeptide- (b) phenyl esters. c) Stimulation of
ClpXP-mediated proteolysis by selected phenyl
ester compounds (mean and SD, n≥6).
formation of the ClpX6P7 complex
(Figure S4b,d). This indicates that
breakdown of the ClpXP complex is
indispensable for full proteolytic arrest,
providing important principles for
inhibitor design.
To gain further insights into the
origin of ClpX6P7 complex formation,
we investigated the affinity between
ClpP and ClpX. So far, apparent
dissociation constants determined via
activity-based assays have been the
standard
complexes.[15,17] All our efforts to
directly quantify the interaction
procedure
for
ClpP
between ClpX and ClpP by isothermal
titration calorimetry (ITC) and surface
plasmon resonance (SPR) failed. We
thus applied the switchSENSE®
technology.[18] For the study of the
ClpP-ClpX interaction, ClpP14 was
conjugated to a short DNA-strand and
immobilized on a biochip. ClpX was
tuberculosis ClpP1P2.[14] This again highlights the conformational
control of ClpX on the catalytic activity of ClpP. Of note, a similar
small molecule-induced enhancement solely of proteolysis was
previously described for the ClpXP2 complex from Listeria
monocytogenes.[15]
titrated to the complex and the switching dynamics upon
application of alternating current potentials were measured
(Figure 3d). Applying this technology allowed, for the first time,
the direct determination of the Kd-value of the ClpX and ClpP
interaction (19.5ꢀ±ꢀ3.2 µM, Figure 3e). This rather low affinity
becomes reasonable when considering that in the bacterial cell
ClpP is required to dynamically exchange between various
cognate chaperones (in S. aureus ClpC and ClpX). Accordingly,
the related HslUV protease complex, which does not have
additional chaperone partners, has a lower Kd-value of 1ꢀµM.[19] In
line with compound-triggered formation of a stable ClpX6P7
complex, its corresponding dissociation constant was five-times
lower compared to the native complex (Kdꢀ=ꢀ3.9ꢀ±ꢀ0.4ꢀµM) (Figure
3e). We next studied if the increased affinity between ClpP and
ClpX influences the ATPase activity of the chaperone. Previous
comprehensive studies showed that the ATPase activity of E. coli
ClpX (EcClpX) is markedly reduced upon binding of ClpP.[17,20]
We, however, found that S.ꢀaureus ClpX displays an intrinsically
low activity, which is drastically enhanced in the presence of
increasing equivalents of ClpP (Figure 3f). Hence, the catalytic
activities of EcClpX and SaClpX are inversely regulated.
Incubation of ClpXP with the inhibitor 35 reduced the ATPase
activity of ClpX in a concentration-dependent manner, converging
to the rate of ClpX alone (Figure 3g). Vice versa, compound 50, a
stimulator of ClpXP proteolysis, activated the ATPase activity of
ClpX within the ClpXP complex in a similar way (Figure 3g). Both
compound-induced alterations in ATP-turnover were solely
mediated by binding to ClpP, as the compounds did not have an
effect on ClpX alone (Figure S6). Accordingly, the ClpXP complex
inhibited by 35 did not have any residual GFP-unfolding activity
beyond the background level, as compared to the proteolytically
inactive complex of the active site mutant ClpP(S98A) and ClpX
As binding of compounds to ClpP can either retain the
tetradecameric state or to induce disassembly into heptameric
species, we determined the oligomeric state of the full ClpXP
complex upon incubation with phenyl esters. Analytical size-
exclusion chromatography (SEC) with ClpXP composed of an
ATP-hydrolysis deficient ClpX(E183Q) mutant (to facilitate a
stable complex assembly)[16] revealed that the strongly activating
compounds 49 and 50 retain the native complex stoichiometry,
namely ClpX12P14 (Figure S4a,c). In contrast, an unprecedented
peak corresponding to a molecular weight of about 400 kDa was
detected after incubation with saturating concentrations of
inhibitor 9 (Figure 3a). Intact protein mass spectrometry (MS) of
the peak fraction revealed the presence of ClpP modified by
compound 9 and ClpX (Figure S5). Incubation of ClpP alone with
compound 9 led to the formation of heptameric ClpP species
(Figure S4d). Hypothesizing that the new complex might
correspond to an unusual ClpX6P7 stoichiometry with a molecular
weight of 435 kDa, we visualized the protein species of the peak
fraction by negative-stain electron microscopy (EM). Indeed, 2D
classification
and
3D-reconstruction
of
the
particles
unambiguously revealed the presence of an unprecedented
complex that consisted of one ClpX hexamer stacked on top of a
catalytically inactive ClpP heptamer (Figure 3b,c). The disruption
of both the peptidolytic and proteolytic system was observed for
all potent amino acid phenyl esters tested, including the (S)-Boc-
Tmo-OAr (10), which - although 25 times weaker than its (R)-
enantiomer 9 - also induced heptamerization of ClpP and partial
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Figure 3. Investigation of an
unprecedented
ClpX6P7
complex. a) SEC revealed
a
novel complex upon incubation
of ClpX(E183Q)12P14 with 9.
Negative-stain EM images of
selected 2D class averages (b)
and 3D reconstruction (c)
verified the ClpX6P7 complex. d)
Schematic representation of the
switchSENSE®
determine the
setup
dissociation
to
constants of ClpX and ClpP. e)
Dissociation constants of ClpX
and unmodified ClpP in its 14-
mer, as well as in its 35-treated
heptameric state (mean and
SD, n=5). f) Stimulation of the
ATPase activity of S. aureus
ClpX6 (0.33 μM) upon titration of
ClpP. g) Influence of inhibitor 35
as well as activator 50 on ATP
turnover by ClpXP. Data for f-g:
mean and SD, n=6. h) Residual
GFP-unfolding activity of 35-
treated ClpXP as compared to
unfolding by the proteolytically
inactive ClpXP-S98A mutant
(100% GFP unfolding).
#) Increased value due to GFP-
degradation by proteolysis, not
solely unfolding.
(Figure 3h). As a result, covalent binding of phenyl esters corrupts
the interaction of the heptameric ClpP rings, inhibits proteolysis
and at the same time strengthens the ClpP-ClpX interaction, while
impairing chaperone activity.
Intrigued by the conformational arrest of ClpXP by
compounds that are related to its natural substrates, we
hypothesized that the trapped ClpX6P7 complex could resemble a
proteolysis of SsrA-tagged GFP. Untagged ClpP did not co-elute
with Strep-ClpP, neither under the peptidase (Figure 4b) nor the
protease assay (Figure 4c) conditions, suggesting that a temporal
collapse of Clp(X)P in the context of its catalytic activity can most
likely be excluded.
In conclusion, this work identified tailored chemical tools
that efficiently probe the activity and oligomerization of the
S. aureus ClpXP protease and unraveled a novel mechanism of
inhibition. Screening of a comprehensive amino acid- and
dipeptide phenyl ester library revealed potent inhibitors which
possess a mirror-inverted stereochemistry as compared to
ClpXP's natural protein substrates. In addition, we identified
compounds that activate proteolytic turnover by binding to the
state of biological relevance. While the pathway of substrate
delivery is well understood, the mechanism by which cleaved
peptide fragments are liberated from ClpXP is still unclear. Two
main hypotheses for egress have been proposed based on the
temporal kinking of the central E-helices of ClpP: 1) formation of
equatorial pores, or 2) opening of the sequestered ClpP barrel by
the collapse of the ClpXP complex.[21-24] The detection of the novel
ClpX6P7 state demonstrates that disassembly of the protease into
ClpX6P7-species, and not full disruption, would be sufficient for
release. ClpX could thus remain bound to each heptamer, which
would facilitate re-assembly of the catalytically active ClpX12P14
complex. With tools in hand that facilitate a controlled ClpXP
disassembly (and slow re-assembly via hydrolysis of the acyl-
enzyme intermediates), we established a pulldown-assay to
determine, if the catalytic activity of ClpP or ClpXP was associated
with a temporal collapse of the ClpP7-ClpP7 interaction. In this
assay, we used two different ClpP14 complexes either exclusively
consisting of Strep-tagged ClpP (Strep-ClpP) or untagged ClpP.
A possible exchange of both ClpP forms was analyzed by Strep-
Tactin-based affinity purification and subsequent intact protein
MS (Figure 4a). After demonstrating that mixed ClpP complexes
can be formed by compound 9 and detected (Figure S7),
experiments were conducted on the potential temporal de-
oligomerization of 1) ClpP upon incubation with the peptide
substrate Ac-Ala- hArg-2-Aoc-ACC and 2) ClpXP during
catalytic
site,
which
differentiates
them
from
acyldepsipeptide(ADEP) activators that bind into apical
hydrophobic pockets.[25] In-depth biochemical investigations
revealed that inhibitor-binding induces the formation of an
unprecedented ClpX6P7 complex. Within this complex, active site
modification of ClpP is linked to an abrogation of the ClpP7-ClpP7
interaction and, more importantly, to an increased affinity between
ClpP and ClpX. Significantly, binding of phenyl esters to ClpP
subunits within ClpX6P7 not only stalls protein degradation, but
also suppresses ClpX-mediated turnover of ATP (Figure 4d). This
simultaneous inhibition of both enzyme activities leads to a
sustainable blockage of the whole complex, surpassing previous
strategies and guiding future design. While a biological function of
the ClpX6P7 complex, including its hypothesized existence as an
intermediate during proteolysis, could not be verified within the
scope of this work, it is intriguing to speculate that bacterial
metabolites regulate the activity of ClpXP in a similar manner as
phenyl esters.
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Keywords: activation • caseinolytic protease • conformational
selection • inhibitors • structure-activity relationships
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Figure 4. Putative temporal collapse of ClpP14 and ClpX12P14 during peptidase
and protease activity, respectively. a) Schematic representation of the
experimental setup. b-c) Relative MS-intensities of untagged and tagged ClpP
forms for respective fractions after affinity purification. For each experiment,
one representative dataset from at least two replicates is shown. FT: Flow-
through, Elu: Elution. d) Summary of the phenyl ester-induced structural and
catalytic alterations of ClpXP.
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The work was funded by the Deutsche Forschungsgemeinschaft
(SFB1035) and the Center for Integrated Protein Science (CIPSM).
M.L. was supported by the German National Academic
Foundation. We thank Katja Bäuml, Katja Gliesche and Mona
Wolff for technical assistance. We are grateful to Dr. Stephan
Hacker for critical revision of the manuscript.
This article is protected by copyright. All rights reserved.

10.1002/anie.201901056
Angewandte Chemie International Edition
COMMUNICATION
Entry for the Table of Contents
COMMUNICATION
Tight and loose: Covalent (R)-amino
acid modifiers are novel tools for
probing the activity and
oligomerization of the bacterial ClpXP
protease. Sub-stochiometric binding
strengthens the ClpX-ClpP
Dr. Markus Lakemeyer, Eva Bertosin,
Dr. Friederike Möller, Dóra Balogh, Dr.
Ralf Strasser, Prof. Dr. Hendrik Dietz,
Prof. Dr. Stephan A. Sieber
Page No. – Page No.
interaction. Depending on the
compound’s substitution, proteolysis
is either stimulated, or efficiently
inhibited by formation of an
Tailored peptide phenyl esters block
ClpXP proteolysis by an unusual
breakdown into a heptamer-hexamer
assembly
unprecedented complex assembly.
This article is protected by copyright. All rights reserved.