Conserved residues in the N-domain of the AAA+
chaperone ClpA regulate substrate recognition and
unfolding
Annette H. Erbse
1,
*, Judith N. Wagner
1,
*, Kaye N. Truscott
2
, Sukhdeep K. Spall
2
, Janine Kirstein
1,3
,
Kornelius Zeth
4
,Ku
¨
rsad Turgay
1,3
, Axel Mogk
1
, Bernd Bukau
1
and David A. Dougan
1,2
1 Zentrum fu
¨
r Molekulare Biologie Heidelberg, Universita
¨
t Heidelberg, Heidelberg, Germany

Australia
Fax: +61 3 9479 2467
Tel: +61 3 9479 3276
E-mail:
B. Bukau, Zentrum fu
¨
r Molekulare Biologie
Heidelberg, Universita
¨
t Heidelberg, INF 282,
Heidelberg D-69120, Germany
Fax: +49 6221 54 5894
Tel: +49 6221 54 6795
E-mail:
*These authors contributed equally to this
work
(Received 22 November 2007, revised 10
January 2008, accepted 14 January 2008)
doi:10.1111/j.1742-4658.2008.06304.x
Protein degradation in the cytosol of Escherichia coli is carried out by a
variety of different proteolytic machines, including ClpAP. The ClpA com-
ponent is a hexameric AAA+ (ATPase associated with various cellular
activities) chaperone that utilizes the energy of ATP to control substrate
recognition and unfolding. The precise role of the N-domains of ClpA in
this process, however, remains elusive. Here, we have analysed the role of
five highly conserved basic residues in the N-domain of ClpA by monitor-
ing the binding, unfolding and degradation of several different substrates,
including short unstructured peptides, tagged and untagged proteins. Inter-
estingly, mutation of three of these basic residues within the N-domain of
ClpA (H94, R86 and R100) did not alter substrate degradation. In contrast

it has been proposed that the N-domain controls bind-
ing of ClpA to ClpP [14]. Interestingly, there is also
considerable debate regarding the role of the ClpB
N-domain (which shares a common fold with the
N-domain of ClpA) in substrate selection [15–17]. One
difficulty in understanding the role of the N-domain of
ClpA stems from the variety of activities exhibited by
various DNClpA constructs tested, each containing
different lengths of ‘linker’ residues that connect the
N-domain to the D1 domain. In order to avoid the
potential problems associated with ‘ragged’ ends of
DNClpA, we chose to create several single and double
point mutations within the N-domain to probe
N-domain function.
Here, using mutational analysis, we report the iden-
tification of a structural element composed of con-
served basic amino acids (R90 and R131), located
within the N-domain of ClpA, that dramatically alters
the ability of ClpA to degrade selected substrates. This
element, although dispensable for the recognition of
the SsrA tag per se, modulates the binding, unfolding
and subsequent degradation of SsrA-tagged protein
substrates. We propose that this element plays an
important role in the binding and subsequent release
of substrates, by triggering ‘local’ unfolding of the sub-
strate. We speculate that the ATP-dependent global
unfolding of some protein substrates is initiated
through productive binding to the substrate via two
elements in ClpA, one in the N-domain and the other
in the pore of the ClpA hexamer. In the case of short

groove (Fig. 2A). To test the role of these basic resi-
dues, we constructed a number of single (R86A, R90A
and R131A) and double (H94A ⁄ R100A and
R90A ⁄ R131A) point mutations in the N-domain of
ClpA (Fig. 2A).
First, we compared the degradation of SsrA-tagged
GFP by wild-type and mutant ClpAP complexes
(Fig. 2B). The ClpP-dependent degradation of GFP–
ssrA mediated by either the single mutant R86A
(Fig. 2B, open inverted triangles) or the double mutant
H94A ⁄ R100A (Fig. 2B, filled diamonds) was unaf-
fected. In contrast the rate of ClpP-mediated degrada-
tion by the single mutants R90A (Fig. 2B, open
diamonds) and R131A (Fig. 2B, open triangles) was
reduced approximately threefold when compared to
wild-type ClpA (Fig. 2B, open circles). Interestingly,
when we combined these two single point mutants to
create the double mutant R90A ⁄ R131A (herein referred
to as RR ⁄ AA), the degradation of GFP–ssrA was
reduced dramatically (Fig. 2B, filled circles). Although
these mutant proteins exhibited different abilities with
regard to mediation of GFP–ssrA degradation
(Fig. 2B), the basal ATPase activity was not affected
(Fig. 3E, compare lanes 1 and 4). Given that the
ATPase activity of ClpA is dependent on its oligomeri-
zation [19], as the nucleotide is bound between two
adjacent subunits, this result suggests that the overall
hexameric structure of RR ⁄ AA was maintained.
A. H. Erbse et al. Substrate recognition and unfolding by ClpA
FEBS Journal 275 (2008) 1400–1410 ª 2008 The Authors Journal compilation ª 2008 FEBS 1401

the ClpP-mediated degradation of FR-linker–GFP
by wild-type ClpA required the addition of ClpS
(Fig. 2D). Importantly, like wild-type ClpA, RR ⁄ AA
was also able to support the ClpS-dependent degrad-
ation of FR-linker–GFP (Fig. 2D), demonstrating a
functional interaction between ClpS and the N-domain
of RR ⁄ AA, and this result suggests that neither the
local nor the overall structure of the RR ⁄ AA mutant
was compromised.
Mutation of the conserved arginine residues has
only a moderate effect on degradation of short
unstructured peptides and the model unfolded
protein, casein
To determine whether RR ⁄ AA also demonstrated an
inability to degrade other known ClpAP substrates, we
examined the ClpP-dependent degradation of several
model ClpA substrates, including the N-terminal
domain of the k repressor fused to the SsrA tag (kR–
ssrA) [21], two short peptides, and the model unfolded
protein a-casein [22]. As for GFP–ssrA, the rate of
fluorescein-labelled kR–ssrA degradation mediated by
RR ⁄ AA was dramatically reduced when compared to
wild-type ClpA (Fig. 3A, filled circles and open
circles). Interestingly, the rate of RR ⁄ AA-mediated
degradation was not significantly altered for an SsrA-
tagged peptide (Fig. 3B), indicating that recognition of
the SsrA tag is not affected by RR ⁄ AA. Moreover,
two other unfolded substrates, a-casein (Fig. 3C) and
a 21-amino-acid polypeptide derived from r
32

wished to compare the ability of the RR ⁄ AA mutant
to bind to the various substrates tested. To do this, we
constructed a ClpA variant in which the glutamic acid
residue within the Walker B motif of each AAA
domain (E286, E565) was changed to alanine. This
double Walker B mutant (herein referred to as dWB)
Fig. 2. Two conserved arginine residues flanking a hydrophobic groove are essential for N-domain function. (A) Structure of the ClpA N-
domain. ClpA is shown as a ribbon diagram (dark grey), and the side chains of R86, R90, H94, R100 and R131 are represented as a ball and
stick (blue) flanking the putative peptide-binding groove (orange). The surface of the N-domain is shaded light grey, and R86, R90, H94,
R100 and R131 are highlighted in blue. (B) The ClpP-mediated degradation of GFP–ssrA was monitored by fluorescence in the presence of
wild-type ClpA (open circles), R86A (inverted open triangles), H94A ⁄ R100A (filled diamonds), R90A (open diamonds), R131A (open triangles)
and RR ⁄ AA (filled circles). (C) The interaction between wild-type ClpA (lane 3) or RR ⁄ AA (lane 4) with ClpP, assessed by co-immunoprecipita-
tion using a-ClpP antiserum, was visualized by staining of the protein bands using Coomassie brilliant blue following separation by SDS–
PAGE. In the absence of added ATPcS (lane 1) or ClpP (lane 2), ClpA was not co-precipitated. The relative amount of ClpA binding to ClpP
was determined from quantification of three independent experiments. Error bars represent the standard error of the mean. A non-specific
protein band is indicated by an asterisk. (D) The functional interaction between ClpS and ClpA (wild-type and RR ⁄ AA) was observed by moni-
toring the ClpS-dependent degradation of FR-linker–GFP (in the presence of ClpP).
A. H. Erbse et al. Substrate recognition and unfolding by ClpA
FEBS Journal 275 (2008) 1400–1410 ª 2008 The Authors Journal compilation ª 2008 FEBS 1403
and the corresponding mutant in RR⁄ AA (referred to
as RR ⁄ dWB) were used to monitor substrate binding
as determined by co-elution of substrate–ClpA
complexes during gel filtration. Initially, we tested the
ability of dWB and RR ⁄ dWB to interact with FITC-
casein. As a control, in the absence of ClpA,
FITC-casein eluted in a single peak at 21.5 mL
(Fig. 4A, open circles). However, upon addition of
ATP and dWB (Fig. 4A, open triangles) or RR ⁄ dWB
(Fig. 4A, filled diamonds), the FITC-casein peak
shifted and formed two new peaks, the largest of

respectively) or in the presence of GFP–ssrA (grey bars; lanes 2, 5 and 8, respectively) or a-casein (black bars; lanes 3, 6 and 9, respec-
tively). The ATPase activity (relative to ClpA in the absence of substrate) was determined from three independent experiments. Error bars
represent the standard error of the mean.
Substrate recognition and unfolding by ClpA A. H. Erbse et al.
1404 FEBS Journal 275 (2008) 1400–1410 ª 2008 The Authors Journal compilation ª 2008 FEBS
SsrA-tagged substrate co-eluted with RR ⁄ dWB when
compared to dWB ClpA (Fig. 4B,C). These data are
consistent with the notion that RR ⁄ AA is able to bind
each substrate but exhibits a change in the release of
some substrates (e.g. kR–ssrA). This lack of release is
expected to hinder unfolding and ultimately reduce
degradation of the substrate.
To further test the possibility that RR ⁄ AA has a
compromised unfolding activity, we compared the abil-
ity of wild-type ClpA and RR ⁄ AA to unfold SsrA-
tagged GFP in the presence of the GroEL trap [24].
As expected wild-type ClpA, in the absence of ClpP,
was able to unfold GFP–ssrA (Fig. 5A, open circles)
but the unfolding ability of RR ⁄ AA (Fig. 5A, filled
circles) was strongly compromised. Surprisingly, the
kinetics of unfolding by RR ⁄ AA measured using the
GroEL trap were slower than expected. As this
method does not directly measure the change in sub-
strate conformation and may be affected by rapid
refolding of the substrate, we chose to validate this
finding using a more sensitive and direct approach.
Thus, hydrogen–deuterium exchange was used to mea-
sure the unfolding of GFP–ssrA in the presence and
absence of either wild-type ClpA or RR⁄ AA. Follow-
ing incubation of GFP–ssrA (28 954 Da) in deuterated

without (lane 4) or with addition of 2 mM ATP (lane 5) as described in (A). (C) GFP–ssrA (990 pmol) was separated by gel filtration
without the addition of ATP (lane 1), in the presence of 160 pmol dWB ClpA
6
without (lane 2) or with addition of 2 mM ATP (lane 3), or in
the presence of 160 pmol RR ⁄ dWB ClpA
6
without (lane 4) or with addition of 2 mM ATP (lane 5) as described in (A).
A. H. Erbse et al. Substrate recognition and unfolding by ClpA
FEBS Journal 275 (2008) 1400–1410 ª 2008 The Authors Journal compilation ª 2008 FEBS 1405
presence of ClpA and ATP, we observed a further
peak (Fig. 5B, asterisk), which arises from incorpora-
tion of deuterium into the core region of GFP–ssrA as
a result of its unfolding. With time, the relative
amount of this heavier species (29 130 Da) increased,
reflecting complete unfolding of all GFP–ssrA by ClpA
(Fig. 5D, open circles). In contrast, the rate of
RR ⁄ AA-mediated unfolding (Fig. 5D, filled circles)
was significantly slower than that of wild-type ClpA,
with more than half of the GFP–ssrA still folded after
30 min (Fig. 5C, asterisk). Taken together, these data
suggest that the change in degradation of GFP–ssrA
mediated by RR⁄ AA stems from a delayed release of
substrate, which results in reduced unfolding of the
substrate.
Discussion
As for most AAA+ proteases, ClpA utilizes the
hydrolysis of ATP to drive substrate unfolding and
translocation into the associated peptidase (ClpP). To
date, however, the role of the N-domains in this pro-
cess has not been well defined as several conflicting

substrates (including an SsrA-tagged peptide) as deter-
mined by rapid degradation of these peptides
(Fig. 3B,D). RR ⁄ AA also exhibited a reduced rate of
GFP–ssrA unfolding as measured by hydrogen–deute-
rium exchange or in the presence of the GroEL trap
(Fig. 5). Collectively, these data confirm that the SsrA
tag does not bind to the N-domain of ClpA, and sug-
gest that these basic residues influence substrate release
from the N-domain, which in turn allows substrate
unfolding to proceed.
Importantly, in contrast to previous studies on the
N-domain of ClpA (which examined the effect of
removing the entire domain and resulted in dramatic
affects on ATPase activity or ClpP binding [14,26]),
our site-directed mutagenesis approach has allowed us
Fig. 5. Mutations in the N-domain reduce
substrate unfolding. Unfolding of GFP–ssrA
was monitored (A) in the presence of the
GroEL trap D87K upon the addition of ClpA
(open circles) or RR ⁄ AA (closed circles), (B)
by hydrogen–deuterium exchange in D
2
O
buffer in the presence of ClpA and ATP, or
(C) by hydrogen–deuterium exchange in
D
2
O buffer in the presence of RR ⁄ AA and
ATP. (D) The relative amount of ‘unfolded’
GFP–ssrA (29 130 Da) was determined in

Leu109 and Val134) located in close proximity to R90
and R131 and a substrate (data not shown). Impor-
tantly, these variants were able to crosslink to ClpS
and mediated the degradation of GFP–ssrA and FR-
linker–GFP by ClpP (data not shown). Thus it remains
unclear whether these arginine residues are directly
involved in substrate binding. Of note, although the
N-domains of ClpA and p97 are not structurally
related, mutations in several basic residues (R95G,
R155C, R155H) within the N-domain of p97 have
been implicated in the inclusion body myopathy asso-
ciated with Paget’s disease of bone and fronto-tempo-
ral dementia [30]. Interestingly, in the crystal structure
of p97, these basic residues are not surface-exposed
but face the AAA domain, in close proximity to the
Walker A motif. Hence, it is appealing to speculate
that the arginine residues in ClpA do not regulate sub-
strate unfolding directly through interaction with the
substrate, but instead coordinate substrate bind-
ing ⁄ release via an interaction elsewhere in ClpA. Inter-
estingly, although RR ⁄ AA has a dramatic inhibitory
effect on the degradation of SsrA-tagged GFP, it does
not affect binding or delivery of the model N-end rule
substrate (FR-linker–GFP) by ClpS (Fig. 2), which
suggests one of two possibilities. Firstly, that the defect
in RR ⁄ AA-mediated unfolding is not dependent on
the global thermodynamic stability of the substrate,
but rather correlates with local unfolding of the sub-
strate (i.e. unfolding of the N- or C-terminus). This
can be understood by examining the N- and C-termi-

fied from the clarified lysates as described previously [8].
All ClpA mutant proteins were purified as for wild-type
ClpA. kR–ssrA was labelled with fluorescein as described
previously [21]. Purification of FR-linker–GFP was per-
formed as previously described [20]. FITC-casein was
obtained from Sigma (St Louis, MO, USA). All proteins
were > 95% pure as determined by Coomassie-stained
SDS–PAGE. Protein concentrations were determined using
a Bradford assay system (Bio-Rad, Munich, Germany)
using BSA purchased from Pierce (Rockford, IL, USA) as
a standard, and refer to the protomer.
Unfolding and protein degradation assays
GFP–ssrA degradation was monitored by changes in
fluorescence (excitation at 400 nm and emission at
510 nm). Degradation of fluorescein-labelled kR–ssrA and
A. H. Erbse et al. Substrate recognition and unfolding by ClpA
FEBS Journal 275 (2008) 1400–1410 ª 2008 The Authors Journal compilation ª 2008 FEBS 1407
FITC-labelled a-casein was monitored by changes in fluo-
rescence (excitation at 490 nm and emission at 520 nm)
using a Perkin-Elmer fluorescence spectrometer LS50B
(Waltham, MA, USA). The reactions were carried out as
described previously [24]. Unfolding assays for GFP–ssrA
were performed in the presence of GroEL trap D87K as
previously described [24]. Non-fluorescent degradation
assays (GFP–ssrA, a-casein, FR-linker–GFP and peptides)
were preformed as previously described [8,20]. Unless other-
wise stated, 1 lm ClpA (wild-type and all mutant ClpA
proteins) and 1 lm ClpP were used. Samples were removed
from the reactions at the indicated time points and degra-
dation was stopped by the addition of sample buffer. Pro-

5% glycerol with or without 2 mm ATP. Fractions of
250 lL were collected in a 96-well plate and samples analy-
sed by fluorescence and 15% SDS–PAGE.
ATPase assay
The ATPase activity of wild-type and mutant ClpA
(0.5 lm) was measured at 660 nm, in degradation buffer
(25 mm Tris ⁄ HCl pH 7.5, 100 mm NaCl, 100 mm KCl,
20 mm MgCl
2
, 0.05% Triton X-100, 10% glycerol) in the
absence or presence of unlabelled substrate (5 lm). The
reaction was started by the addition of 2 mm ATP and
stopped by the addition of 800 lL malachite green solution
(0.034% malachite green, 0.1% Triton X-100 and
10.5 gÆ L
)1
ammonium molybdate in 1 N HCl) and 100 lL
of 34% citrate.
Hydrogen–deuterium exchange and mass
spectrometry
GFP–ssrA (2 lm) was diluted 75· into deuterated buffer
(50 mm Tris ⁄ HCl pH 7.5, 300 mm NaCl, 10% glycerol,
0.5 mm dithiothreitol, 10 mm ATP). Where appropriate,
ClpA or RR ⁄ AA (2 lm) was added at the start of the
exchange reaction (t = 0 min); for the control experiment,
equal volumes of non-deuterated buffer were added. At
indicated time points, samples were removed from the reac-
tion. The hydrogen–deuterium exchange was stopped by
rapidly lowering the pH to 2.4 at 4 °C. All subsequent steps
were carried out on ice to minimize back exchange. The pro-

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