Biochem. J. (2012) 446, 311–320 (Printed in Great Britain)
311
doi:10.1042/BJ20120649
Interaction specificity between the chaperone and proteolytic components
of the cyanobacterial Clp protease
Anders TRYGGVESSON*1 , Frida M. STÅHLBERG*1 , Axel MOGK†, Kornelius ZETH‡ and Adrian K. CLARKE*2
*Department of Biological and Environmental Sciences, Gothenburg University, Box 461, S-405 30 Göteborg, Sweden, †Zentrum fur Molekulare Biologie der Universität Heidelberg,
Im Neuenheimer Feld 282, D-69120 Heidelberg, Germany, and ‡Department of Protein Evolution, Max-Planck-Institute for Developmental Biology, 72076 Tübingen, Germany.
The Clp protease is conserved among eubacteria and most
eukaryotes, and uses ATP to drive protein substrate unfolding
and translocation into a chamber of sequestered proteolytic
active sites. In plant chloroplasts and cyanobacteria, the essential
constitutive Clp protease consists of the Hsp100/ClpC chaperone
partnering a proteolytic core of catalytic ClpP and noncatalytic
ClpR subunits. In the present study, we have examined putative
determinants conferring the highly specific association between
ClpC and the ClpP3/R core from the model cyanobacterium
Synechococcus elongatus. Two conserved sequences in the Nterminus of ClpR (tyrosine and proline motifs) and one in the
N-terminus of ClpP3 (MPIG motif) were identified as being crucial for the ClpC–ClpP3/R association. These N-terminal domains
also influence the stability of the ClpP3/R core complex itself. A
unique C-terminal sequence was also found in plant and cyanobacterial ClpC orthologues just downstream of the P-loop region
previously shown in Escherichia coli to be important for Hsp100
association to ClpP. This R motif in Synechococcus ClpC confers
specificity for the ClpP3/R core and prevents association with
E. coli ClpP; its removal from ClpC reverses this core specificity.
INTRODUCTION
in the axial loops formed by the N-terminus of EcClpP [9–13].
In the case of ClpX, the N-terminus of EcClpP binds to a region
(designated the pore-2 loop) at the bottom of the central channel
and is important in stabilizing the ClpX–ClpP interaction [11].
Clp proteins are widely distributed among various bacteria and
eukaryotes, and they exhibit considerable structural and functional
variation. This is most evident by the highly diverse Clp proteins
found in photosynthetic organisms. The model cyanobacterium
Synechococcus elongatus PCC 7942 (hereafter Synechococcus)
has three distinct ClpP paralogues (ClpP1–3) and one ClpR
protein [14]. ClpR is so far unique to photosynthetic organisms
and, despite having sequence similarity to ClpP, it lacks the
catalytic triad and is therefore proteolytically inactive [15,16].
Although its exact function remains unknown, ClpR forms part
of a heterologous Clp core complex along with the ClpP3 subunit.
The two heptameric rings each contain four ClpR and three ClpP3
subunits arranged in a defined alternating pattern [16]. The SyClp
(Synechococcus Clp) P3/R core is constitutively expressed and
is essential for cell viability [17,18], although specific protein
substrates have yet to be identified. A second heterologous core
complex also exists in Synechococcus consisting of ClpP1 and
ClpP2. The SyClpP1/2 core is non-essential for constitutive
growth but it is highly inducible under certain stress conditions,
including high light and cold [17,19]. Each of the proteolytic core
complexes has a specific Hsp100 chaperone partner, ClpC in the
case of ClpP3/R and ClpX for ClpP1/P2 [20].
The diversity of Clp proteins in photosynthetic organisms
moves to another level of complexity when considering those
in chloroplasts of vascular plants. In the model plant species
Arabidopsis thaliana, a single chloroplastic Clp proteolytic core
exists comprising five ClpP and four ClpR paralogues [21]. These
subunits are arranged to two distinct rings, the P-ring containing
ClpP3–6 and the R-ring with ClpP1 and ClpR1–4 [22]. Also
associated with the outer surface of the P-ring are two accessory
Proteolysis performs a vital homoeostatic role in all organisms,
within which energy-dependent proteases are major contributors
[1]. The best-characterized examples of such proteases include
the eukaryotic 26S proteasome and the bacterial FtsH, Lon,
HslUV and Clp proteases. All of these two-component proteases
are architecturally similar, typically with an AAA + (ATPases
associated with various cellular activities) ATPase flanking one
or both end of a barrel-shaped proteolytic core [2]. In the case
of the bacterial Clp proteases, the proteolytic core commonly
has a single ClpP subunit organized in two heptameric rings [3].
Compartmentalized within are the proteolytic active sites, with
each ClpP subunit having a catalytic triad of serine, histidine and
aspartic acid residues [4]. Access to the degradation chamber is
restricted to only short peptides by narrow axial pores. Entry of
protein substrates relies on the ATPase component, which for
the model Clp protease in Escherichia coli is either ClpA or
ClpX [2]. These members of the Hsp100 family of molecular
chaperones recognize the protein substrates and unfold them
in an ATP-dependent manner. The unfolded proteins are then
translocated into the ClpP core complex for degradation to short
peptide fragments [5].
For the Clp proteolytic machinery to operate correctly,
the ATPase chaperone component must efficiently bind to the
proteolytic core. Several interactive domains between the EcClp
(E. coli Clp) P core complex and its chaperone partners ClpA
and ClpX have been identified. One such determinant is the socalled P-loop, a short peptide sequence (IGF/L) in the C-terminal
region of ClpX and ClpA that is essential for the association to
EcClpP. Each of the P-loop domains within the ClpX hexamer
are required for strong ClpP binding, and they appear to associate
with hydrophobic clefts on the outer edge of the EcClpP ring
[6–8]. Additional motifs important for ClpA/X association exist
Key words: chloroplast, Clp protein, cyanobacterium, proteolysis.
Abbreviations used: AAA+, ATPases associated with various cellular activities; BsClpC, B. subtilis ClpC; DTT, dithiothreitol; EcClp, E. coli Clp; FtClpP,
Francisella tularensis ClpP; SyClp, Synechococcus Clp.
1
These authors contributed equally to this work.
2
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2012 Biochemical Society
312
A. Tryggvesson and others
proteins, ClpT1 and ClpT2, that appear to regulate the assembly
of the core complex [23]. ClpC is the principle chaperone partner,
two near-identical paralogues of which exist in A. thaliana
(ClpC1 and ClpC2). Like the homologous ClpCP3/R protease
in cyanobacteria, the chloroplast counterpart is an essential
constitutively expressed protease in plants, with up to 25 putative
substrates identified to date [22,24].
Although much progress has been made in identifying
determinants in the interaction between ClpP and ClpA/X in E.
coli, little is yet known about such defining factors between a
ClpR-containing proteolytic core and its ClpC chaperone partner.
We have recently shown that certain specificity does exist in
the interaction between ClpC and ClpP3/R in cyanobacteria,
in that SyClpC does not associate with EcClpP and EcClpA
does not bind SyClpP3/R [16]. The fact that several of the
interactive determinants identified for the E. coli orthologues
are also conserved in the cyanobacterial Clp proteins, such as
the P-loop in ClpC, suggests that other as yet unknown factors
must be involved. In the present study, we have used specific
chimaeric recombinant versions of the Synechococcus ClpP3,
ClpR and ClpC proteins to identify domains important in their
association. We show that motifs in both the N-terminus of ClpP3
and ClpR are essential for the interaction with ClpC. Moreover,
a motif unique to ClpC orthologues located just downstream of
the C-terminal P-loop is shown to define the specificity between
ClpC and the ClpP3/R core complex.
EXPERIMENTAL
Purification of Synechococcus ClpP3/R complexes
SyClpP3 and SyClpR proteins were co-expressed in E. coli as
described previously [16]. The modified versions of SyClpP3 and
SyClpR were made by PCR and confirmed by DNA sequencing.
For each construct, the sequence corresponding to a His6 tag
was added to the 3 -end of the clpP3 gene to aid purification.
Co-expression of SyClpP3 and SyClpR was performed in E.
coli BL21-STAR cells (Invitrogen) grown at 37 ◦ C to a D600
of approximately 0.5. Protein overexpression was induced by
addition of IPTG (isopropyl β-D-thiogalactopyranoside) to a
final concentration of 0.4 mM. After 2 h, cells were pelleted and
washed once in buffer A [20 mM Tris/HCl (pH 7.5), 300 mM
NaCl, 40 mM imidazole and 1 mM DTT (dithiothreitol)]. Cells
were then ruptured using a French Press [1000 psi (1 psi = 6.9
kPa)] followed by centrifugation to remove cell debris (14 000 g
for 30 min). The soluble protein fraction was loaded on to a 5 ml
Ni2 + -affinity column (HisTrap HP, GE Healthcare) and washed
with buffer A. Bound proteins were then eluted with buffer B
[20 mM Tris/HCl (pH 7.5), 300 mM NaCl, 300 mM imidazole
and 1 mM DTT]. The recombinant proteins were further purified
by gel filtration (HighLoad 16/60 Superdex, GE Healthcare) in
buffer C [20 mM Tris/HCl (pH 7.5), 75 mM NaCl and 1 mM
DTT]. Collected fractions were concentrated using VivaSpin 500
columns (GE Healthcare), and stored in buffer C with 20 %
(w/v) glycerol. Protein concentrations were determined using the
Bradford assay (Thermo Scientific). Protein concentrations used
in the experiments were based on the oligomeric size of ClpC and
the ClpP3/R core.
Purification of SyClpCA
The modified version of SyClpC (called SyClpCA) was
constructed by synthesizing the full-length clpC gene (Invitrogen)
but with the P-loop region changed to replace that coding for
c The Authors Journal compilation c 2012 Biochemical Society
EFSGVDEAENQYNRIRSLVN in ClpC to the corresponding
part in ClpA which is IHQDNSTDAM. The sequence
corresponding to a His6 tag was also added to the 3 -end of the
gene to aid purification. The SyClpCA construct was ligated into
the pMAL-C2E vector (New England Biolabs) and transformed
into E. coli MC4100 cells lacking EcClpP [25]. Overexpression
of SyClpCA in MC4100 was performed as described for
SyClpP3/R. The purified SyClpCA protein was stored in buffer
C with 30 % (w/v) glycerol, 2 mM ATP and 4 mM MgCl2 .
Degradation of α-casein
All of the proteolytic assays were performed with the various Clp
proteins diluted in buffer E [20 mM Tris/HCl (pH 7.5), 25 mM
NaCl and 5 mM MgCl2 ] together with an ATP-regeneration
system [26]. When analysing the various SyClpP3/R chimaerics,
SyClpC was added in excess (0.6 μM) relative to the core
complexes (0.2 μM). When comparing the different Hsp100
proteins (SyClpC, SyClpCA and EcClpA), an excess of the core
complexes (0.5 μM) was used in comparison with 0.2 μM of
the chaperones. All of the assays were performed at 33 ◦ C and
samples were taken every 3 min over the 12 min time course.
Reactions were stopped by addition of 4× NuPAGE sample
buffer [1 M Tris/HCl (pH 8.5), 8 % lithium dodecyl sulfate,
40 % (w/v) glycerol and 2 mM EDTA] and incubated at 75 ◦ C
for 5 min. Samples were analysed by PAGE on 12 % BisTris
gels using the NuPAGE gel system (Invitrogen). Proteins were
stained using the colloidal Coomassie Blue method.
ATPase activity
The ATPase activity of the different Hsp100 proteins was
measured by the release of inorganic phosphate using a Malachite
Green assay [27]. Assays were done in buffer F [40 mM Tris/HCl
(pH 7.5), 50 mM NaCl, 10 mM MgCl2 and 1 mM DTT] with
2 mM of ATP at 37 ◦ C. If not otherwise stated, 0.3 μM of
chaperone and 0.6 μM of core were used in the assays. Standards
were made by dissolving K2 HPO4 in buffer E and diluting to a
range of 5–50 nmol of inorganic phosphate.
PAGE
Separation of the recombinant SyClpP3/R proteins under
denaturing conditions was performed on 12 % BisTris gels as
described above. Quantification of the Coomassie-Blue-stained
SyClpR and SyClpP3 bands was done according to Peltier et al.
[21]. Separation of purified SyClpP3/R under non-denaturing
conditions was done using a Tris Borate gel system [20]. Purified
proteins (10 μg) were resolved on 4–13 % polyacrylamide gels
electrophoresed for 18 h at 4 ◦ C at a constant current (14 mA).
Gels were stained using the colloidal Coomassie Blue method.
Modelling of SyClpP3/R and SyClpC
We have used the HHpred server to identify ClpP models of
the current PDB as a basis for structure modelling [27a]. The
monomeric SyClpR and SyClpP models were constructed using
the program Modeller [27b]. The PDB entry 3P2L was used
to assemble the 14 subunits by simple superimposition of the
SyClpR and SyClpP monomers on to the symmetric complex in
an RPRPRPR arrangement. Further energy minimization of the
model using the CNS program package [27c] was performed
to remove bumps between inter- and intra-molecular atoms.
Assembly of the Clp protease in cyanobacteria
Figure 1
313
Importance of conserved N-terminal motifs in SyClpR for SyClpC association
(A) An amino acid alignment of the N-terminal domain from several cyanobacterial ClpR orthologues against that from SyClpP3 and EcClpP. Functionally conserved amino acids are shaded in black
or grey depending on the degree of conservation in the different sequences. The conserved tyrosine (Tyr) and proline (Pro) motifs in ClpR proteins are indicated, with arrows showing which parts
of the N-terminus have been replaced in the chimaerics R-N1, R-N2 and R-N3. (B) Degradation of α-casein (2 μM) by SyClpC with wild-type SyClpP3/R, R-N1, R-N2 or R-N3 as visualized by
SDS/PAGE and colloidal Coomassie Blue staining. Quantification of the degraded α-casein was performed relative to the time 0 control, which was set to 100 %. Shown are mean values +
− S.E.M.
(n = 6). (C) Stimulation of SyClpC ATPase activity by the addition of wild-type SyClpP3/R or one of the three chimaerics R-N1, R-N2 or R-N3. SyClpC ATPase activity was measured by the linear
release of inorganic phosphate and plotted as means +
− S.E.M. (n = 3).
Modelling of SyClpC was achieved using the closest structural
homologue from Bacillus subtilis currently available in the PDB
database [BsClpC (B. subtilis ClpC), PDB code 3PXI]. The
monomeric SyClpC model was constructed using the program
Modeller [27b]. The trimeric template model of BsClpC was
further extended to generate the hexameric structure on the basis
of crystallographic symmetry operations. Monomeric models of
SyClpC were superimposed on monomers of BsClpC derived
from the complex structure to finally construct a hexameric
SyClpC model. Residues 303–310 and 604–612 were removed
from all of the model chains due to strong loop clashes inside
the SyClpC cavity. Inter- and intra-molecular main- and sidechain clashes were removed through energy minimization using
the CNS program package [27c].
RESULTS
Conserved motifs in the N-terminus of SyClpR
To investigate the observed specificity between SyClpC and
SyClpP3/R, we first focused on the main difference between
this type of Clp proteolytic core and those in non-photosynthetic
organisms, i.e. the inclusion of the non-catalytic subunit ClpR.
Our search for conserved motifs concentrated on the N-terminal
region of ClpR since it is this region in EcClpP that is important
for the association with ClpA and ClpX [9–10,12,13]. Moreover,
earlier modelling of SyClpR also pointed to the N-terminus
protruding further from the main core structure than the Nterminus of EcClpP [16], again suggesting that this region in
ClpR might be involved in ClpC association. By performing
extensive multiple sequence alignments of cyanobacterial ClpR
against SyClpP3 and EcClpP, a minimized example of which is
shown in Figure 1(A), two conserved motifs specific for ClpR
were identified in the N-terminus. The first of these (YYGD)
was situated 12–15 amino acids from the N-terminus of SyClpR,
whereas the second (RTPPP) was 19–23 amino acids; these
two domains were designated the tyrosine and proline motifs
respectively.
To test if these conserved motifs were important for the
interaction with SyClpC, a series of chimaeric SyClpR variants
were prepared. In these chimaerics, different lengths of the
ClpR N-terminus were substituted with the corresponding amino
acid sequence in SyClpP3. In the chimaeric R-N1, the first
23 amino acids of SyClpR were replaced with the corresponding
21 amino acids in SyClpP3, thereby eliminating both the tyrosine
and proline motifs. In the chimaeric R-N2, only 15 amino acids
from SyClpR were replaced with the matching region from
SyClpP3, thereby removing the tyrosine motif, but leaving the
proline motif intact. In the last chimaeric R-N3, only the first
nine amino acids in SyClpR were changed to those in SyClpP3,
leaving both tyrosine and proline motifs unchanged. All of these
SyClpR variants were then co-expressed with wild-type SyClpP3
(including a C-terminal His tag) in E. coli cells and later purified
by sequential affinity and size-exclusion chromatography.
Once the various Clp core complexes containing the different
SyClpR variants were purified, their proteolytic activities
with SyClpC were determined using the unstructured model
substrate α-casein. In our degradation assay, wild-type SyClpP3/R
c The Authors Journal compilation c 2012 Biochemical Society
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Figure 2
A. Tryggvesson and others
Importance of the N-terminus of cyanobacterial ClpP3 for ClpC association
(A) An amino acid alignment of the N-terminal domain from several cyanobacterial ClpP3 orthologues against that from SyClpR and EcClpP. Functionally conserved amino acids are shaded in black
or grey depending on the degree of conservation in the different sequences. The conserved motif MPIG in ClpP3 orthologues is highlighted, with arrows showing which parts of the N-terminus
have been substituted in the chimaerics P3-N1 and P3-N3. (B) Degradation of α-casein (2 μM) by SyClpC with wild-type SyClpP3/R, P3-N1 or P3-N3 as visualized by SDS/PAGE and colloidal
Coomassie Blue staining. Quantification of the degraded α-casein was performed relative to the time 0 control, which was set to 100 %. Shown are mean values +
− S.E.M. (n = 6). (C) Stimulation of
SyClpC ATPase activity by the addition of wild-type SyClpP3/R or one of the two chimaerics P3-N1 or P3-N3. SyClpC ATPase activity was measured by the linear release of inorganic phosphate and
plotted as means +
− S.E.M. (n = 3).
degraded almost all of the α-casein by 12 min (Figure 1B).
In comparison, the core complex containing the chimaeric RN1 lacking both the tyrosine and proline motifs exhibited no
proteolytic activity throughout the time course. In contrast, the
core with the R-N2 chimaeric displayed wild-type levels of αcasein degradation, whereas the core with the least modified
chimaeric R-N3 had a degradation rate twice that of the wildtype (Figure 1B).
We next tested if the above changes in proteolytic activity were
affected by changes in the association between the chimaeric
core complexes and the chaperone partner SyClpC. Because the
formation of the intact Clp protease is difficult to observe by
native PAGE or gel filtration [14,20], we instead determined the
relative binding affinity between the proteolytic and chaperone
components by the level of enhanced ATPase activity of the
Hsp100 chaperone by the addition of the ClpP core [28]. As shown
in Figure 1(C), the addition of wild-type SyClpP3/R stimulated
SyClpC ATPase activity approximately 3-fold, consistent with
previous observations [16]. In comparison, addition of the R-N1
core showed no significant increase in SyClpC ATPase activity,
suggesting that the removal of the tyrosine and proline motifs in
SyClpR impairs association of the core complex with SyClpC.
The loss of association between these two components would
also explain the lack of proteolytic activity seen in Figure 1(B).
In contrast, the R-N3 chimaeric core stimulated the ATPase
activity of SyClpC almost twice as much as the wild-type core,
suggesting that the short N-terminal SyClpP3 sequence in each
c The Authors Journal compilation c 2012 Biochemical Society
chimaeric SyClpR subunit enhanced the association between the
core complex and SyClpC. A near doubling of SyClpC ATPase
activity by R-N3 association is also consistent with the 2-fold
increase in proteolytic activity observed in Figure 1(B). As for the
R-N2 chimaeric core, it also stimulated SyClpC ATPase activity,
but to a lesser extent than the wild-type core complex, suggesting
an impairment in the SyClpC association. Given that the R-N2
chimaeric core degraded α-casein at the same rate as the wild-type
core, it is likely that this activity was the net effect of enhanced
proteolysis as observed for the R-N3 chimaeric core, but with
lower affinity to SyClpC.
The SyClpP3 N-terminus is crucial for SyClpC association
Given the dramatic effects on core activity by the various changes
to the N-terminus of SyClpR, particularly between the R-N1
and R-N3 chimaerics, we next investigated similar changes to
the N-terminus of SyClpP3. We also performed an extensive
multiple sequence alignment of available cyanobacterial ClpP3
orthologues against SyClpR and EcClpP to search for distinct
motifs in this region (Figure 2A). Overall, the N-terminal region
of ClpP3 is more highly conserved than that in ClpR, in particular
the first six amino acids (MPIGVP, designated MPIG motif). As
such, two chimeras were constructed with different lengths of
the SyClpP3 N-terminus replaced with the corresponding regions
from SyClpR. In the P3-N3 chimaeric, the first eight amino acids
of SyClpP3 were changed to the first 10 amino acids in SyClpR,
Assembly of the Clp protease in cyanobacteria
Figure 3
Stability of the chimaeric SyClpP3/R core complexes
Wild-type (Wt) SyClpP3/R and the various chimaeric core complexes were separated on
denaturing 12 % BisTris (A) or non-denaturing 4–13 % Tris-borate (B) polyacrylamide gels.
Shown are the two batches of each recombinant protein purified. (A) For those chimaeric core
complexes in which the ClpR/ClpP3 ratio differed from that of the wild-type (4:3), the altered
ratio is shown underneath. (B) The sizes of the native molecular mass markers are shown on
the left-hand side.
thereby eliminating the MPIG motif. In the chimaeric P3-N1, the
first 21 amino acids of SyClpP3 were replaced with the matching
23 amino acids in SyClpR, thereby not only removing the MPIG
motif, but also adding both the tyrosine and proline motifs. All
of these SyClpP3 variants (including a C-terminal His tag) were
co-expressed with wild-type SyClpR in E. coli cells and then
purified as described above. When assayed for proteolytic activity,
both the P3-N1 and P3-N3 constructs were unable to degrade αcasein (Figure 2B). Both of the constructs also exhibited reduced
stimulation of SyClpC ATPase activity (Figure 2C), suggesting
that impaired association with SyClpC was involved in the loss of
degradation activity. This implies that the MPIGV motif in ClpP3
is important for ClpC association.
Core subunit composition, stability and peptidase activity
Because of the disparate functional effects of the various
chimaeric constructs, we next investigated if the changes to the
N-terminus of either SyClpP3 or SyClpR affected the structural
stability of the core complex. We first examined if the wild-type
ratio of ClpR/ClpP3 (4:3) had changed in any of the chimaeric core
complexes. The relative amounts of SyClpP3 and SyClpR were
quantified from the Coomassie-Blue-stained proteins separated
by denaturing PAGE according to Peltier et al. [21]. Separation
of both batches of each chimaeric construct showed that two
had indeed changed subunit ratios, with R-N1 and P3-N1 having
subunit ratios of 7:3 and 3:4 respectively (Figure 3A).
To determine their oligomeric size, all of the chimaeric cores
along with the wild-type complex were then separated by nondenaturing PAGE. As shown in Figure 3(B), the wild-type core
separated to a size of 270 kDa, consistent with previous size
determinations [16]. The R-N2, R-N3 and P3-N3 chimaerics
all formed stable cores of approximately the same size as the
315
wild-type SyClpP3/R. In contrast, the P3-N1 core showed a
significant degree of instability, with at least half of the stainable
protein resolving as a smaller-than-expected smear on the gel
(Figure 3B). This suggests that the altered ClpR/ClpP3 ratio
in this chimaeric construct (i.e. 3:4) results in core instability,
which would also explain, in part, the lack of proteolytic
activity exhibited by the P3-N1 chimaeric. Interestingly, the R-N1
chimaeric formed in approximately equal amounts two different
cores, one which matched the size of the wild-type core and
another approximately 90 kDa larger, which would be consistent
with a double nonomeric complex. Taking into account the overall
ClpR/ClpP3 ratio of this chimaeric core complex (7:3; Figure 3A),
and assuming the one matching the wild-type size has the wildtype ratio (4:3), then the larger core complex would have a
ClpR/ClpP3 ratio of 10:3. On the basis of a double nonomeric
configuration, this would suggest a 7:2 ratio of ClpR/ClpP3.
Overall, the N-terminal changes in the R-N1 chimaeric affects
the assembly of the ring structure of the core, which results in
complete loss of ClpC association as described above (Figure 1C).
One of the unusual features of the wild-type SyClpP3/R
complex is the lack of degradation of synthetic peptides
commonly used to measure such peptidase activity of ClpP
complexes from other organisms [16]. One possible explanation
for this came from modelling of the SyClpP3/R core structure,
which revealed that the axial entrance pores appear to be unusually
narrow and are essentially closed [16]. Given the proximity of the
N-termini of both SyClpP3 and SyClpR to the pore entrance,
we next tested if the changes in the various chimaeric cores
affected their ability to degrade synthetic peptides. However,
testing of several synthetic peptides, including the standard
N-succinyl-Leu-Tyr-7-amido-4-methylcourmarin revealed that
none of the chimaeric cores exhibited peptidase activity (results
not shown).
ClpC has a unique core specificity region
All of the Hsp100 chaperone partners to ClpP have a conserved
motif (IGL/L[I]GF), known as the P-loop, in the C-terminal region
that is essential for binding of ClpP [6]. Although SyClpC also
possesses the P-loop, it also has specificity for the interaction with
the SyClpP3/R core complex, as demonstrated by the inability of
SyClpC to bind EcClpP and EcClpA to associate with SyClpP3/R
[16]. To search for the underlying cause for this specificity,
we aligned the C-terminal regions of ClpC orthologues from
several cyanobacteria and plants with the corresponding region
from various bacterial ClpA proteins. As shown in Figure 4(A),
using Synechococcus and Arabidopsis ClpC along with EcClpA
for demonstration purposes, a short region just downstream of
the P-loop was identified in ClpC, but absent in ClpA. This
unique extension contained an eight amino acid sequence we
now term the R motif that is rich in basic residues and is highly
conserved in all of the ClpC orthologues from photosynthetic
organisms. To investigate the potential importance of this R motif,
we prepared a modified form of SyClpC (SyClpCA) in which
the extension in SyClpC (amino acids 695–714) was replaced
with the much shorter sequence from EcClpA. The SyClpCA
protein was overexpressed in an E. coli strain lacking EcClpP
and purified as described above. The activity of SyClpCA was
then tested using the α-casein degradation assay and compared
with that of the control reactions using the unmodified proteins. As
previously shown [16], SyClpC promoted SyClpP3/R degradation
activity, but not that of EcClpP, whereas EcClpA functioned with
EcClpP, but not SyClpP3/R (Figure 4B). In comparison, SyClpCA
was unable to function with SyClpP3/R, with no observable
proteolytic activity. Instead, SyClpCA enabled significant
c The Authors Journal compilation c 2012 Biochemical Society
316
Figure 4
A. Tryggvesson and others
Identification of a SyClpP3/R core specificity factor in the C-terminal domain of SyClpC
(A) An amino acid alignment of the C-terminal domain surrounding the P-loop motif in EcClpA, SyClpC and the two ClpC paralogues in A. thaliana . Functionally conserved amino acids are
shaded in black or grey depending on the degree of conservation in the different sequences. The conserved P-loop and R motif are indicated above the sequences. (B) Degradation of α-casein
(2 μM) by SyClpC + wild-type SyClpP3/R (䊉), SyClpC + EcClpP (), SyClpCA + EcClpP (䉲), EcClpA + EcClpP (䊐), EcClpA + SyClpP3/R (䊏) and SyClpCA + SyClpP3/R (䊊) as visualized
by SDS/PAGE and colloidal Coomassie Blue staining. Quantification of the degraded α-casein was done relative to the time 0 control, which was set to 100 %. Shown are mean values +
− S.E.M.
(n = 6). (C) Stimulation of the ATPase activity of SyClpC, SyClpCA or EcClpA by the addition of either SyClpP3/R or EcClpP. ATPase activities are measured by the linear release of inorganic
phosphate and plotted as means +
− S.E.M. (n = 3).
α-casein degradation by EcClpP with rates faster than that of
SyClpC with SyClpP3/R, but somewhat slower than EcClpA
with EcClpP. This change in core specificity by SyClpCA was
also reflected in ATPase activity measurements (Figure 4C).
Addition of SyClpP3/R stimulated the ATPase activity of wildtype SyClpC as expected, but not that of SyClpCA, Moreover,
addition of EcClpP failed to stimulate the ATPase activity of
SyClpC as expected, but did stimulate that of SyClpCA. Overall,
this confirms that the R motif in SyClpC is crucial for the specific
interaction with SyClpP3/R and prevents association with EcClpP.
It should be noted that the SyClpCA exhibited significantly higher
basal ATPase activity than SyClpC, suggesting that the R motif
also restricts somehow this activity in wild-type SyClpC.
Modelling of SyClpC and SyClpP3/R
Using the SyClpP sequence, three PDB entries 3QWD (ClpP
from Staphylococcus aureus), 1YG6 (EcClpP) and 3P2L [FtClpP
(Francisella tularensis ClpP)] comprising 60–64 % sequence
identity for 189–194 aligned residues were identified and
considered for model building. However, although the models
1YG6 and 3P2L show essentially the same topology with the
intercalating β9 and helix E motifs (assignment according to
the EcClpP [4]), the 3QWD structure was different, as the
two heptameric ClpP rings were only weakly connected and
the β9/E motifs fold back on to the individual subunits. This
back-folded conformation also denoted as possible breathing
c The Authors Journal compilation c 2012 Biochemical Society
mechanism of ClpP causes a significant deviation for the oligomer
superimposition and therefore this structure was rejected from the
modelling. For the SyClpR sequence the same template structures
showed significant, but weaker, identity of 40–44 %. The 14meric SyClpP3/R models of the present study were developed
in two different modes: (i) with all N-termini elongated and
sticking outwards of the core complex (Figure 5B) and (ii) with
the ClpP termini located inside the cavity (Figure 5A). These two
models are supported by the secondary structure assignment of
SyClpR predicting the initial 24 residues including the tyrosine
and proline motifs to be largely disordered (see Supplementary
Figure S1 at http://www.BiochemJ.org/bj/446/bj4460311add.htm
for the alignment). The proline motif is located nearby the cavity
entrance pore of the complex, while the tyrosine motif is located
further apart and the connecting random coil structure may adopt
a variety of different conformations (see Figure 5C). The distance
of this tyrosine motif from the cavity entrance is about 2.5 nm
which results in a maximum distance between two tyrosine motifs
of neighbouring subunits of ∼ 5 nm if two termini would assume
strongly bent conformations (see the model in Figure 5A). In
contrast with SyClpR, the structure organization of the SyClpP3
N-terminus may vary with the N-termini either exposed outside or
buried inside the protease core (see Figures 5A or 5B). Secondary
structure prediction, as well as structure analysis, suggests the
presence of the short N-terminal β-sheet which is also present
in EcClpP and FtClpP (Supplementary Figure S1). In the latter
model the SyClpP3/R pore is partially covered by the N-terminal
Assembly of the Clp protease in cyanobacteria
Figure 5
317
Modelling of SyClpC and the SyClpP3/R complex
(A) Model of the SyClpP3/R complex in cartoon representation with the SyClpP3 N-termini located inside the cavity. One SyClpR subunit is marked in red, whereas one SyClpP3 molecule is
colour-coded in blue. Two tyrosine motifs (Tyr-motif) of SyClpR are marked with circles and the potential conformational flexibility of these termini is indicated by two bent conformations with an
approximate distance of 5 nm. (B) Model representation of SyClpP3/R with all N-termini exposed on the protease surface. Colour coding of the individual subunits is identical with (A). (C) Enlarged
view of two subunits in the same orientation as (B). Individual interacting motifs observed in SyClpR and SyClpP3 are marked accordingly, including sequence numbers. (D) The hexameric complex
of SyClpC was modelled using the PDB entry of the structure of the MecA108–ClpC complex from B. subtilis (PDB code 3PXI). The model is shown as a side view in cartoon representation with
monomers colour-coded in grey, except one monomer which is colour-coded in rainbow colours from the N- to the C-terminus [blue, N-terminus (NT) and red, C-terminus (CT)]. The C-terminal
domain which comprises the motif mutated in the present study is marked by a box and the residues deleted are marked in ball-and-stick representation. (E) Close-up of the SyClpC C-terminal
domain in the same orientation as shown in (D). The conserved residues YNRIRSLV mutated are located at the C-terminal end of an extended loop structure (residues 678–706). (F) Superimposition
of the C-terminal domains of EcClpA (PDB code 1R6B, marked in yellow) and SyClpC (marked in orange) [32]. The P-loop in EcClpA indicated by a broken line is not resolved, presumably due to
flexibility and disorder in the crystal structure. The loop in SyClpC is significantly extended in sequence than the P-loop observed in EcClpA (see also Figure 4A).
extensions of SyClpP3 and the N-terminal MPIG motif would not
be accessible for the interactions with the SyClpC chaperone.
In order to visualize the biochemical results on a threedimensional basis, we constructed a model of SyClpC based
on the recently published structure of the MecA–ClpC complex
from B. subtilis (BsClpC; Figure 5D). This BsClpC structure,
although solved at lower resolution, is currently the only complete
hexameric structure of a ClpC-like chaperone. The two sequences
SyClpC and BsClpC are 62 % identical with only a small number
of sequence gaps in the alignment (insertions in SyClpC) and the
SyClpC model was generated showing the additional extended
loop structures such as the P-loop and R motif modelled in
random conformations (Figure 5E). In comparison with the ClpA
structure homologue, SyClpC shows an elongated loop based
on the insertion of the R motif into the P-loop structure (see
Figures 4A and 5F). This P-loop is located within the C-terminal
of SyClpC and forms molecular contacts with SyClpP3/R leading
to an enhanced overall stability and activation of the entire
c The Authors Journal compilation c 2012 Biochemical Society
318
A. Tryggvesson and others
complex. The P-loop fingerprint of SyClpC is very similar to
EcClpA and located at the outermost extended tip of this loop
structure (see Figures 4A, 5E and 5F). By contrast, the R motif
is close to the C-terminal domain structure and therefore less
mobile due to the rather tight physical connection with the domain
backbone.
DISCUSSION
In the present study, we have revealed that N-terminal motifs
in both the ClpP3 and ClpR subunits of the cyanobacterial
ClpP3/R proteolytic core play an important interactive role
with the ClpC chaperone partner. Previous studies have shown that
the N-terminus of EcClpP is essential for the association to the
chaperone partners EcClpA and EcClpX [9,12], in that removal of
the first 22 amino acids from the N-terminus of EcClpP prevents
binding to EcClpA or EcClpX. Early crystal structures of EcClpP
failed to resolve the N-terminus [4,25] due to the flexible nature
of this region. More recent structures, however, with improved
resolution have revealed that the N-terminus has two distinct
configurations, the so-called ‘up’ and ‘down’ formations [10]. In
the up conformation, the N-terminus of six of the seven subunits in
one heptameric ring protrudes out from the entrance pore, leading
to the closing of the access channel. In the down configuration,
the N-terminus of all seven subunits face towards the interior
of the complex producing an open-access pore [10]. It remains
unclear how these two N-terminal configurations affect chaperone
association, although it might influence the local symmetry match
between the ClpA and ClpP, if only six of the N-terminals at the
same time have the same structure and associate with ClpA [10].
Because of the lack of crystal structures for a cyanobacterial
ClpP3/R core, it is unclear if the N-termini of the two different
subunits exist in the two different configurations. Although the
first 20 amino acids of ClpP3 shares moderate sequence similarity
to that of EcClpP, that of ClpR has none whatsoever. Despite this,
the modelling of the SyClpP3/R core shown in the present study
based on the currently available ClpP structures suggests that all
of the N-termini protrude further out from the access pore, in
particular that of the ClpR subunit. As a consequence, it is not
surprising that this extended N-terminus plays an important role
in the association with the chaperone partner ClpC.
The N-terminal domain of SyClpR has two conserved
motifs that are important for the association with SyClpC; the
tyrosine and proline motifs. Changing these motifs, such as to
the corresponding region in SyClpP3 in the R-N1 chimaeric,
eliminated SyClpC association and thereby proteolytic activity.
The structural significance of the proline motif is particularly
intriguing given that it is essential for SyClpC association.
Modelling places the proline motif outside of the main core
structure along the extended N-terminus and thereby likely
accessible for direct binding to SyClpC. The proline motif also
appears to influence the ring structure of the core complex.
Removal of the proline motif causes two distinct cores to form,
one with the expected size of the wild-type complex and another
considerably larger corresponding to a double nonomeric ring
configuration. Despite the lack of crystal structure, it is known that
each heptameric ring of the wild-type SyClpP3/R core consists
of alternating R/P3/R/P3/R/P3/R subunits [16]. This suggests
that each SyClpP3 subunit interacts with the adjacent SyClpR
subunit within the annular structure. In the EcClpP configuration,
a hydrogen bond between Arg12 of one subunit and Ser21 of
another is important for ring formation [10]. Interestingly, all
of the cyanobacterial ClpP3 orthologues have a matching arginine
residue (position 11 in SyClpP3) to that in EcClpP, but the ClpR
c The Authors Journal compilation c 2012 Biochemical Society
orthologues do not, whereas the ClpRs have a corresponding
serine residue (position 27 in SyClpR) to that in EcClpP, but the
ClpP3 orthologues do not. This suggests that hydrogen bonding
between Arg11 in ClpP3 and Ser27 in ClpR might play a role
in heptameric ring assembly. If so, it is then plausible that the
proline motif in ClpR could affect this subunit interaction; an
effect that was perturbed by the removal of the proline motif in
the R-N1 chimaeric. Such an interaction between the ClpP3 and
ClpR subunits would also explain the instability of the P3-N1
chimaeric core complex since all the Arg11 residues in SyClpP3
were lost by the substitution with the corresponding SyClpR Nterminal sequence, which lacks this residue.
Like SyClpR, the SyClpP3 subunit also contains an N-terminal
motif important for binding of SyClpC. This MPIG motif at the
very start of the sequence is highly conserved in all cyanobacterial
ClpP3 orthologues. In addition to ClpC association, the MPIG
motif also has a positive effect on the proteolytic activity of the
core complex. When all of the subunits have the MPIG sequence
as in the case of the R-N3 chimaeric, both the ATPase activity
of SyClpC and the overall degradation activity of the protease
are significantly higher than that of the wild-type proteins. This
stimulation of both activities suggests that the MPIG motif
promotes translocation of unfolded substrates from SyClpC into
the SyClpP3/R core. From structures of the ClpP core from E.
coli, Streptococcus pneumonia and humans, the first seven amino
acids of the N-terminus line the axial pore [9,10,29]. These seven
amino acids in the N-terminus appear to perform a gating function,
controlling substrate access into the degradation chamber [12].
They also appear important for the actual proteolytic activity,
helping to stabilize the acyl-enzyme intermediate [12]. A more
recent study proposes that charged amino acids within the Nterminus of ClpP which line the channel determine the maximal
rate of degradation [13]. Modelling of SyClpP3/R revealed that
the axial pore is narrower than that of EcClpP and is essentially
closed [16]. It is possible that the first few amino acids of SyClpR
contribute mostly to this closed structure and thereby restrict
substrate access into the degradation chamber. Such a role would
be consistent with the enhanced proteolytic activity (due to more
efficient substrate access) of the R-N3 chimaeric in which all of the
subunits possess the first seven amino acids of SyClpP3. However,
such a position of the SyClpR N-terminus would be inconsistent
with modelling prediction. Moreover, given the highly conserved
nature of the MPIG motif in ClpP3 orthologues and the lack
of such conservation for the first seven amino acids of ClpR
orthologues, it is likely that the enhanced activity of the R-N3
chimaeric is also due to a direct role of the ClpP3 N-terminal
sequences. Like the N-terminus of EcClpP, the MPIG motif
in SyClpP3 might contribute to the catalytic efficiency of the
proteolytic active sites, especially given that SyClpP3 is the only
active subunit within the SyClpP3/R core complex.
Although the N-terminal sequences of both SyClpP3 and
SyClpR are clearly important for SyClpC binding, the question
arises as to with which sequences in SyClpC do they interact? For
the ClpXP protease in E. coli, a short domain just downstream of
the single Walker B site has been shown to influence ClpP association [11]. Since mutations of this so-called pore-2 loop produced
far less deleterious effects on ClpX–ClpP binding than N-terminal
mutations of ClpP, it is likely that other regions in ClpX also
interact with the ClpP N-terminus. Modelling of the pore-2 loop
places it close to the N-terminus of ClpP that forms the axial pore.
Interestingly, analysis of EcClpA and various ClpC sequences
reveals that the region downstream of both Walker B domains
shares no obvious similarity to that of the ClpX pore-2 loop.
The sequence downstream of the second Walker B site is almost
identical in ClpA and ClpC, and if involved in ClpP binding would
Assembly of the Clp protease in cyanobacteria
not explain the specificity between SyClpC and SyClpP3/R. The
sequence downstream of the first Walker B motif is much less
conserved between ClpA and ClpC and is adjacent to the beginning of the linker region that separates the two AAA domains. The
length of the linker in ClpC is characteristically longer than that
in ClpA. It should also be noted that ClpP binding inhibits ClpX
ATPase activity [6], whereas it stimulates that of ClpA and ClpC,
also suggesting a difference in the ClpP-binding characteristics
between ClpX and the larger Hsp100 chaperone partners.
It is now clear that the R motif in SyClpC specifies
the chaperone interaction with the SyClpP3/R core complex.
Removal of this domain not only loses the association with
SyClpP3/R, but enables binding of EcClpP and subsequent
proteolytic activity, which normally does not occur with wild-type
SyClpC. The position of the R motif in SyClpC just downstream
of the P-loop suggests it is this interaction site with the proteolytic
core that is modified. The P-loop in EcClpX supposedly docks
with high affinity to hydrophobic depressions on the periphery of
the ClpP ring forming a stable ClpXP complex [10]. Each
of the six P-loops within the ClpX hexamer is important for
both tight ClpX binding and efficient ClpXP proteolytic activity
[11]. The hydrophobic depression within each ClpP subunit is
formed by six distinct residues (Tyr60 , Tyr62 , Phe82 , Ile90 , Phe112
and Leu189 for EcClpP). All but one (Phe112 ) is conserved in
the cyanobacterial ClpP3 and ClpR orthologues, in ClpR the
phenylalanine residue is changed to an alanine, and in ClpP3
to leucine or valine. Interestingly, the Phe112 residue in EcClpP
is crucial for EcClpA association, and changing it to an alanine
residue strongly destabilizes the ClpAP complex [10]. Again,
this emphasizes the difference in the specific interaction between
SyClpC and SyClpP3/R relative to their E. coli counterparts.
Another interesting observation from the present study was the
increased basal rate of ATP hydrolysis exhibited by the SyClpCA
protein. Similar increases were shown for EcClpX when the pore2 loop was mutated [11]. This was explained by the proximity of
the pore-2 loop to the Walker B portion of the AAA domain and
possible conformation changes resulting from the loop mutations.
Although situated further away from the Walker AB domains
in SyClpC, the R motif might well cause similar conformation
changes that stimulate basal ATPase activity.
One of the fascinating architectural principles of ATPdependent proteolysis in both the proteasomal and Clp
machineries is the symmetry mismatch between the unfoldase
and the protease. This symmetry mismatch requires significant
flexibility of the individual interacting modules and their
connecting elements (P-loop/R motif of SyClpC, N-termini of
SyClpR and SyClpP3) to adapt to binding sites of the associated
complex. This conformational freedom is provided either by
extended and flexible loop structures such as the P-loop of
EcClpA, EcClpX and the P-loop/R motif of SyClpC or by
flexible and extended terminal elements such as the N-terminus
of SyClpR and SyClpP3. This principle system is also maintained
by the ATP-dependent PAN/proteasome complex system which is
connected through flexible C-termini of the ATPases. In SyClpC
two such elements were identified by sequence analysis, both
of which are located in an extended loop structure which is
longer than the P-loops of the ClpA and ClpX orthologues due
to the insertion of additional residues. For SyClpR the tyrosine
motif is located far apart from the protease core and presumably
flexible for docking into binding sites on SyClpC as random coil
extension. Both SyClpP3 and SyClpR have extended terminal
sequences when compared with ClpP species from the PDB.
The identification of specificity domains for the cyanobacterial ClpCP3/R association also has direct relevance for the
plastid Clp protease in plants. According to the endosymbiotic
319
theory, cyanobacteria are the progenitors of plant chloroplasts, and
the ClpCP3/R protease is clearly the ancestor of the chloroplast
Clp protease [30]. Both are constitutively expressed and are essential for cell viability. The ClpC chaperone partners are highly
conserved in cyanobacteria and chloroplasts and all contain
the R motif identified in the present study. With regard to
the proteolytic core, however, the chloroplast Clp protease
has diversified significantly from the cyanobacterial orthologue.
The model chloroplast Clp protease in Arabidopsis has a core
consisting of nine different subunits, five ClpP (ClpP1 and
ClpP3–6) and four ClpR (ClpR1–4). These subunits are arranged
in two distinct heptameric rings; the P-ring containing the
ClpP3–6 subunits and the R-ring with ClpP1 and ClpR1–4.
Also bound to the periphery of the P-ring are two accessory
proteins (ClpT1 and ClpT2) unique to plants that appear to
regulate the assembly of the core complex [23]. It is clear
from sequence comparisons that the chloroplast ClpP1 subunit
is the orthologue to the cyanobacterial ClpP3, and includes the
MPIG motif in the N-terminus. Similarly, chloroplastic ClpR1
and ClpR3/4 match the cyanobacterial ClpR and contain both
the N-terminal tyrosine and proline motifs. ClpR2 is unusual
in that it lacks the signature extension region typical of all
of the other ClpR proteins, as well as lacks the tyrosine and
proline motifs identified in the present study. Indeed, ClpR2
has more sequence similarity to ClpP1, particularly with the
presence of the MPIG motif in the N-terminus. It also has the conserved Arg11 characteristic of ClpP3-like subunits and not the Ser27
commonly found in ClpR orthologues. On the basis of a similar
subunit configuration to that in cyanobacterial ClpP3/R, therefore,
the R-ring is likely to have three ClpP3-like subunits (ClpP1 and
ClpR2) and four ClpR (ClpR1 and 3/4) possibly arranged in the
same alternating pattern. A recent study, however, has proposed
that the R-ring contains a subunit conformation directly matching
that of the cyanobacterial ClpP3/R, with three ClpP1 subunits and
one of each ClpR protein [31].
Whatever its actual subunit configuration, it is the R-ring
that is most likely to associate with ClpC. The P-ring contains
four nuclear-encoded ClpP paralogues (ClpP3–6) and no ClpR
subunits. None of these ClpP subunits have any of the three
N-terminal motifs identified in the present study necessary for
ClpC binding. Indeed, the N-terminal sequences of ClpP3–
6 proteins are highly diverse with little obvious conservation.
As a consequence, it is unlikely that ClpC binds to the
P-ring side of the core complex. Although it cannot be
excluded that as yet unidentified motifs in one or more of
the P-ring subunits might confer ClpC association, the presence
of the ClpT proteins on the P-ring is also likely to interfere
with a stable ClpC interaction. As such, it would appear that
substrate translocation into the chloroplast Clp proteolytic core
is unidirectional, a phenomenon so far unique among known Clp
proteases. Although the Hsp100 partners of other Clp proteases
including the cyanobacterial ClpP3/R can potentially bind to
either end of the proteolytic core, it remains uncertain if more
than one substrate protein is translocated in vivo into the core at
any given time.
AUTHOR CONTRIBUTION
Anders Tryggvesson and Frida Ståhlberg substantially contributed to data acquisition
(Figures 1–4) and to drafting of the paper; Axel Mogk substantially contributed to data
acquisition (Figure 4) and to drafting of the paper; Kornelius Zeth substantially contributed
to data acquisition (Figure 5) and to drafting and revising of the paper; Adrian Clarke
substantially contributed to the conception and design of the study, interpretation of the
data, drafting the paper and revising it for important intellectual content.
c The Authors Journal compilation c 2012 Biochemical Society
320
A. Tryggvesson and others
ACKNOWLEDGEMENTS
We would like to thank Kerstin Lindgren at Biovitrum (Göteborg, Sweden) for allowing
access to their FluoSTAR optima for assays.
FUNDING
This work was supported by the Swedish Research Council (VR).
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Biochem. J. (2012) 446, 311–320 (Printed in Great Britain)
doi:10.1042/BJ20120649
SUPPLEMENTARY ONLINE DATA
Interaction specificity between the chaperone and proteolytic components
of the cyanobacterial Clp protease
Anders TRYGGVESSON*1 , Frida M. STÅHLBERG*1 , Axel MOGK†, Kornelius ZETH‡ and Adrian K. CLARKE*2
*Department of Biological and Environmental Sciences, Gothenburg University, Box 461, S-405 30 Göteborg, Sweden, †Zentrum fur Molekulare Biologie der Universität Heidelberg,
Im Neuenheimer Feld 282, D-69120 Heidelberg, Germany, and ‡Department of Protein Evolution, Max-Planck-Institute for Developmental Biology, 72076 Tübingen, Germany.
Supplementary Figure S1 is on the next page.
1
2
These authors contributed equally to this work.
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2012 Biochemical Society
A. Tryggvesson and others
Figure S1
Sequence alignment of SyClpR and SyClpP against the current PDB database
For both sequences the same PDB models 3P21 and 1YG6 with high similarity were chosen. The sequences were aligned and the similarity between individual residue positions is shown for identical
(*), highly conserved (:) and less conserved (.) positions. Residues not present in either model or template sequences are marked ( − ). SyClpR (ClpR SE) shows additional residues compared with
the protein models in the N-terminal and C-terminal part as well as insertions within the sequence. SyClpP3 (ClpP SE) is more similar to the models used with a short N-terminal extension.
Received 18 April 2012/29 May 2012; accepted 1 June 2012
Published as BJ Immediate Publication 1 June 2012, doi:10.1042/BJ20120649
c The Authors Journal compilation c 2012 Biochemical Society