Allosteric activation of HtrA protease DegP by stress signals during bacterial protein quality control

Article abstract of DOI:10.1002/anie.200703273

(Figure Presented) Unleashing the proteolytic activity of DegP: A study with syntheticmimic s of cellular stress signals reveals an allosteric (chemical) activation mechanism of the bacterial HtrA protease DegP, leading to a fine-tuned amplification of protein degradation during bacterial protein quality control (see scheme: binding of a peptide sequence (circles) increases activation. The nature of the penultimate residue (red) is shown to be decisive).

Full text of DOI:10.1002/anie.200703273

Communications
DOI: 10.1002/anie.200703273
Allosteric Protease Regulation
Allosteric Activation of HtrA Protease DegP by Stress Signals during
Bacterial Protein Quality Control**
Michael Meltzer, Sonja Hasenbein, Patrick Hauske, Nicolette Kucz, Melisa Merdanovic,
Sandra Grau, Alexandra Beil, Dafydd Jones, Tobias Krojer, Tim Clausen, Michael Ehrmann,*
and Markus Kaiser*
The key cellular process of protein quality control (PQC)
ensures that all proteins are structurally and functionally
intact and properly localized.[1] Although PQC is in particular
demand under stress conditions, such as heat shock, it plays an
important role under any environmental situation as protein
misfolding occurs even under homeostatic conditions. Defi-
cient PQC often leads to the formation of protein aggregates
that are the basis for severe disorders, such as Alzheimerꢀs or
Parkinsonꢀs disease.[2] PQC is also critical for bacterial
virulence, pathogenic bacteria without these cellular factors
are unable to survive in their hosts.[3] Consequently, selective
inhibition of bacterial PQC might represent a promising
strategy for developing novel antibiotics.
In bacteria, two periplasmic HtrA (high-temperature
requirement) proteases, DegS and DegP, are key players of
PQC. The widely conserved HtrA proteases consist of a
protease domain adopting a chymotrypsin fold and one or two
PDZ domains.[4] Their proteolytic activity is controlled by
cellular stress signals that affect the reversible switch between
active and resting states. In DegS, proteolytic activity is
allosterically turned on by mislocalized outer-membrane
proteins that bind to the PDZ and protease domains.[5]
Proteolytic activation initiates a signal-transduction cascade
that ultimately leads to the expression of DegP,[6] the major
PQC factor that eliminates misfolded proteins with little
substrate specificity.
DegP consists of one protease and two PDZ domains and
assembles into complex, multimeric particles that switch
between chaperone (protein repair factor) and protease
functions in
a
temperature-dependent manner.[7] While
chaperone activity is present at low temperatures, protease
function dominates at elevated temperatures, resulting in a
distinct proteolytic activity at 378C. Although the crystal
structure of DegP in the chaperone state is available,[8] the
regulation of its catalytic activity is unclear. We hypothesized
that besides the known temperature-dependent regulation, an
additional chemical mechanism could exist. Since DegP is
already proteolytically active at 378C, this chemical activation
would result in an amplification of temperature-induced
proteolysis.
We wished to investigate the effect of chemical stress
signals on DegP activity, using a chromogenic protease assay.
However, no suitable chromogenic substrate is available and
the absence of proper peptidic substrates for any member of
the pathophysiologically important HtrA family has pre-
vented accurate enzymatic characterization of this protease
family. To overcome this limitation, we performed digests of
the known DegP substrate citrate synthase and synthesized
para-nitroaniline (pNA) derivatives of selected cleavage
products (compounds 1–3, Scheme 1). These derivatives
were obtained by a combination of 9-fluorenylmethoxycar-
bonyl (Fmoc) solid-phase peptide synthesis on 2-chlorotrityl
resin, allowing efficient synthesis of fully protected precursor
peptides, followed by solution peptide coupling with com-
mercially available H-Val-pNA. Out of these three pNA-
derivatives, 1 containing the SPMFKGV-pNA sequence was
the most suitable synthetic substrate, displaying a cooperative
binding with a K0.5 value (the half-saturation constant in the
Hill equation) of 2.7 mm, a vmax of 1.28 mmolminÀ1, a kcat of
0.56 sÀ1, and a specificity constant (kcat/K0.5) of 0.21 mmÀ1 sÀ1,
whereas 2 and 3 were not cleaved. We optimized conditions
for future protease assays by studying the effect of pH value,
buffers, and salts; 50 mm NaH2PO4 buffer at pH 8.0 was most
appropriate (for thorough biochemical characterization, see
Supporting Information).
[*] Dipl.-Biol. M. Meltzer, Dr. S. Hasenbein, Dipl.-Biol. N. Kucz,
Dr. M. Merdanovic, Prof. Dr. M. Ehrmann
Zentrum für Medizinische Biotechnologie
FB Biologie und Geographie
Universität Duisburg-Essen, 45117 Essen (Germany)
Fax: (+49)201–183–3315
E-mail: michael.ehrmann@uni-due.de
Dipl.-Chem. P. Hauske, Dr. M. Kaiser
Chemical Genomics Centre der Max-Planck-Gesellschaft
Otto-Hahn-Strasse 15, 44227 Dortmund (Germany)
Fax: (+49)231–9742–6479
E-mail: markus.kaiser@cgc.mpg.de
Dr. S. Grau, Dr. A. Beil, Dr. D. Jones, Prof. Dr. M. Ehrmann
Cardiff School of Biosciences
Cardiff University
Museum Avenue, Cardiff CF10 3US (UK)
Dr. T. Krojer, Dr. T. Clausen
Research Institute for Molecular Pathology (IMP)
Dr. Bohrgasse 7, 1030 Vienna (Austria)
After having established a suitable assay, we investigated
our hypothesis. Initially, we added casein as a model
compound for misfolded proteins and monitored (by photo-
metry) degradation of substrate 1 by DegP as we reasoned
that some short cleavage products derived from casein might
amplify DegP proteolysis. Indeed, 2.5-fold activation was
[**] This work was supported by Deutsche Forschungsgemeinschaft, the
Fonds der Chemischen Industrie and BBSRC (M.E.), Austrian
Science Fund FWF P17881-B10 (T.K.) and by BBSRC (D.J., M.E.)
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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Angewandte
Chemie
Figure 1. Sequences of activating peptides 4–16 and results derived
Scheme 1. Synthesis of chromogenic substrates by solid-phase syn-
thesis on 2-chlorotrityl resin. a) Acetic acid/trifluoroethanol/CH2Cl2
(2:2:6); b) H-Val-pNA·HCl (2 equiv), EDC·HCl (1.5 equiv), HOBt
(2 equiv), DIEA (3.5 equiv); c) TFA/H2O/ethandithiol/triisopropylsilane
(94:2.5:2.5:1). Boc=tert-butoxycarbonyl, EDC=N’-(3-dimethylamino-
propyl)-N-ethylcarbodiimide, HOBt=1-hydroxy-1H-benzotriazole,
DIEA=N,N-diisopropylethylamine, TFA=trifluoroacetic acid.
from proteolysis experiments with 1. The varied penultimate residue is
highlighted in bold. Assay conditions: 0.5 mm 1, 50 mm activating
peptides 4–16, 0.13 mm DegP, 50 mm NaH2PO4 pH 8.0 normalized to
activity without addition of activating peptide. Each assay was repeated
five times with duplicate samples, and the resulting standard devia-
tions are shown as error bars.
observed, supporting our assumption of an additional allos-
teric activation. We then used a native substrate, periplasmic
a-amylase (MalS) and found that full-length denatured MalS,
as well as MalS fragments and the C-terminus of MalS,
amplified DegP proteolysis (Supporting Information).
activator, leading to 3-fold activation. Importantly, these
experiments supported our hypothesis of an additional
allosteric activation of DegP activity. Moreover, a good
correlation between sequences activating DegS and DegP was
observed with peptides 4–16, indicating a physiologically
relevant mode of regulation.
These data indicated that DegP is activated by oligopep-
tides. We imagined that the underlying mechanism might be
similar to DegS activation. The crystal structure of the DegS-
activator complex revealed that activating peptides bind to
the PDZ domain and that residue X of the YXF C-terminal
motif of activating peptides additionally interacts with a loop
of the protease domain, thereby triggering proteolytic activ-
ity. The activation of DegS critically depends on the chemical
nature of residue X. We therefore prepared decapeptide
derivatives of the known stress signal consensus sequence
DNRDGNVYXF by solid-phase peptide synthesis, where X
was substituted by various residues. We synthesized ten
peptides and tested their effect on proteolytic activation of
DegP and DegS. In addition, we analyzed decapeptides
derived fromnative C-termini of cell envelope proteins, that
is, outer membrane porin C (OmpC), periplasmic alkaline
phosphatase A (PhoA), and MalS. Of these, the C-terminus
of OmpC is a known DegS activator while PhoA served as a
negative control. The proteolytic activity of DegP was
measured with substrate 1 (Figure 1) and denatured MalS to
ensure that the observed effects were not due to the use of a
synthetic substrate (Supporting Information). DegS activity
was determined using its native substrate, the anti-sigma
factor RseA (Supporting Information). Peptides 4 and 5,
carrying YFF or YWF C-termini, activated DegP by about
2.5- and 2-fold, respectively. Peptide 7 with a C-terminal YQF
sequence, corresponding to the C-terminus of the natural
stress signal OmpC, induced a 1.5-fold activation. Interest-
ingly, a decamer of the C-terminus of OmpC (15) was the best
Thermodynamic binding affinities of peptides 4 and 6,
carrying an activating YFF or a weakly activating YLF C-
terminus, were determined by isothermal titration calorim-
etry. The data could be fitted with a three sequential binding
sites model and revealed cooperative binding. For all three
binding sites, only slight differences in the dissociation
constant Kd between 4 and 6 could be observed (Supporting
Information), suggesting an important role of the penultimate
residue of the activating peptides.
To elucidate if DegP activation is based on an interaction
of the activating peptide with PDZ domains, we constructed
two point mutations in PDZ domain 1. Arg262, which is
involved in binding of the C-terminus carboxy group of the
activating peptide ligands, was replaced by alanine. In the
second mutant, Val328 was substituted by serine, thereby
changing the spatial arrangement of the ligand binding
pocket.[8] These mutations completely abolished proteolytic
activation of even the strongest activating peptide 15,
suggesting that the allosteric binding site is indeed located
on PDZ domain 1 (Figure 2, and Supporting Information
Figure S5). Furthermore, isothermal titration calorimetry
revealed that peptide 4 did not bind to either of these PDZ
mutants (data not shown). Therefore, PDZ 1 of DegP is able
to transmit information to the protease domain. Given the
structural differences between DegP and DegS (different
oligomeric states, activation domains, and sensor loops)
future studies are required to understand the precise activa-
tion mechanism. In addition, peptides 7 and 15, both carrying
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Communications
Figure 2. Schematic model of the allosteric activation mode of DegP.
Activating peptides (blue circles) bind to PDZ domain 1 and the
chemical nature of the penultimate residue (red circle) determines the
extent of activation.
the OmpC C-terminus YQF, differed in their activation
potential (Figure 1). This indicates that while the last three
residues of the ligand are essential, upstreamsequences are
also contributing to activation; however, the underlying
structural basis is not yet understood.
Finally, we asked if activating peptides and upregulated
protease activity result in a loss of chaperone function at low
temperatures. To address this question, we performed refold-
ing assays with MalS and activating peptide 4 (Supporting
Information Figure S6). Interestingly, we detected an
increased degradation of MalS and almost no refolding
when 4 was added, showing that activating peptides can
switch DegP fromchaperone to protease at low temperatures.
Therefore, chemical activation dominates over regulation by
temperature.
We conclude that the bacterial PQC factor DegP is
allosterically regulated by model peptides mimicking cellular
stress signals. This result implies that besides the known
transcriptional activation, an additional allosteric activation
of DegP occurs. The simultaneous allosteric regulation of
DegP and DegS by peptides significantly contributes to a
rapid and robust stress response and a fine-tuned amplifica-
tion of the stress signal (Figure 3). Consequently, synthesis of
non-activating peptidomimetics binding to the allosteric
regulation pocket should induce a distinct disturbance of
bacterial PQC and could therefore represent a novel strategy
for the development of antimicrobials. Moreover, we propose
that allosteric activation is a common feature of all members
of the HtrA family. This conclusion is in line with other
studies reporting binding of short peptides to human HtrA
proteases HtrA1 or HtrA2.[9] Although peptidic stress signals
have not yet been identified for human HtrAs, the conserved
activation mode might be relevant for a better understanding
of protein-folding diseases as HtrA1 has been described as a
risk factor for amyloid-diseases including age-related macular
degeneration.[10]
Figure 3. Amplification of bacterial stress response by allosteric activa-
tion. DegS senses unfolded proteins and initiates a proteolytic cascade
that leads to elevated expression of DegP. In addition, these activating
sequences also lead to an allosteric activation of DegP, causing an
amplification of the original stress signal by transcriptional and
allosteric activation.
Keywords: allosterism · proteases · protein quality control ·
.
proteins · stress signal
[1] B. Bukau, J. Weissman, A. Horwich, Cell 2006, 125, 443 – 451.
[2] D. J. Selkoe, Nat. Cell Biol. 2004, 6, 1054 – 1061.
[3] T. L. Raivio, Mol. Microbiol. 2005, 56, 1119 – 1128.
[4] T. Clausen, C. Southan, M. Ehrmann, Mol. Cell 2002, 10, 443 –
455.
[5] a) H. Hasselblatt, R. Kurzbauer, C. Wilken, T. Krojer, J. Sawa, J.
Kurt, R. Kirk, S. Hasenbein, M. Ehrmann, T. Clausen, Genes
Dev. 2007, 21, 2659 – 2670; b) C. Wilken, K. Kitzing, R.
Kurzbauer, M. Ehrmann, T. Clausen, Cell 2004, 117, 483 – 494;
c) N. P. Walsh, B. M. Alba, B. Bose, C. A. Gross, R. T. Sauer, Cell
2003, 113, 61 – 71.
[6] S. Hasenbein, M. Merdanovic, M. Ehrmann, Genes Dev. 2007,
21, 6 – 10.
[7] C. Spiess, A. Beil, M. Ehrmann, Cell 1999, 97, 339 – 347.
[8] T. Krojer, M. Garrido-Franco, R. Huber, M. Ehrmann, T.
Clausen, Nature 2002, 416, 455 – 459.
[9] a) Murwantoko, M. Yano, Y. Ueta, A. Murasaki, H. Kanda, C.
Oka, M. Kawaichi, Biochem. J. 2004, 381, 895 – 904; b) S. Gupta,
R. Singh, P. Datta, Z. Zhang, C. Orr, Z. Lu, G. DuBois, A. S.
Zervos, M. H. Meisler, S. M. Srinivasula, T. Fernandes-Alnemri,
E. S. Alnemri, J. Biol. Chem. 2004, 279, 45844 – 45854; c) L. M.
Martins, B. E. Turk, V. Cowling, A. Borg, E. T. Jarrell, L. C.
Cantley, J. Downward, J. Biol. Chem. 2003, 278, 49417 – 49427.
[10] a) Z. Yang, N. J. Camp, H. Sun, Z. Tong, D. Gibbs, D. J.
Cameron, H. Chen, Y. Zhao, E. Pearson, X. Li, J. Chien, A.
Dewan, J. Harmon, P. S. Bernstein, V. Shridhar, N. A. Zabriskie,
J. Hoh, K. Howes, K. Zhang, Science 2006, 314, 992 – 993; b) A.
Dewan, M. Liu, S. Hartman, S. S. Zhang, D. T. Liu, C. Zhao, P. O.
Tam, W. M. Chan, D. S. Lam, M. Snyder, C. Barnstable, C. P.
Pang, J. Hoh, Science 2006, 314, 989 – 992.
Received: July 21, 2007
Revised: September 19, 2007
Published online: January 3, 2008
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Angew. Chem. Int. Ed. 2008, 47, 1332 –1334