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 N-terminus 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.
- Clp protein
Proteolysis performs a vital homoeostatic role in all organisms, within which energy-dependent proteases are major contributors . 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 . In the case of the bacterial Clp proteases, the proteolytic core commonly has a single ClpP subunit organized in two heptameric rings . Compartmentalized within are the proteolytic active sites, with each ClpP subunit having a catalytic triad of serine, histidine and aspartic acid residues . 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 . 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 .
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 so-called 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 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 .
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 . 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 . 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 .
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 . These subunits are arranged to two distinct rings, the P-ring containing ClpP3–6 and the R-ring with ClpP1 and ClpR1–4 . Also associated with the outer surface of the P-ring are two accessory proteins, ClpT1 and ClpT2, that appear to regulate the assembly of the core complex . 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 . 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.
Purification of Synechococcus ClpP3/R complexes
SyClpP3 and SyClpR proteins were co-expressed in E. coli as described previously . 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 (14000 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 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 . 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 . 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.
The ATPase activity of the different Hsp100 proteins was measured by the release of inorganic phosphate using a Malachite Green assay . 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 K2HPO4 in buffer E and diluting to a range of 5–50 nmol of inorganic phosphate.
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. . Separation of purified SyClpP3/R under non-denaturing conditions was done using a Tris Borate gel system . 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. 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 side-chain clashes were removed through energy minimization using the CNS program package [27c].
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 N-terminus of EcClpP , 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 degraded almost all of the α-casein by 12 min (Figure 1B). In comparison, the core complex containing the chimaeric R-N1 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 α-casein degradation, whereas the core with the least modified chimaeric R-N3 had a degradation rate twice that of the wild-type (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 . As shown in Figure 1(C), the addition of wild-type SyClpP3/R stimulated SyClpC ATPase activity approximately 3-fold, consistent with previous observations . 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 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, 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 α-casein (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. . 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 non-denaturing PAGE. As shown in Figure 3(B), the wild-type core separated to a size of 270 kDa, consistent with previous size determinations . The R-N2, R-N3 and P3-N3 chimaerics all formed stable cores of approximately the same size as the 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 wild-type 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 . 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 . 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 . 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 . 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 , 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 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 wild-type 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 ), 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 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 14-meric 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 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 three-dimensional 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 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.
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 . 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 . 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 .
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 . 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 . Interestingly, all of the cyanobacterial ClpP3 orthologues have a matching arginine residue (position 11 in SyClpP3) to that in EcClpP, but the ClpR 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 N-terminal 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 . They also appear important for the actual proteolytic activity, helping to stabilize the acyl-enzyme intermediate . A more recent study proposes that charged amino acids within the N-terminus of ClpP which line the channel determine the maximal rate of degradation . Modelling of SyClpP3/R revealed that the axial pore is narrower than that of EcClpP and is essentially closed . 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 . 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 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 , 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 . Each of the six P-loops within the ClpX hexamer is important for both tight ClpX binding and efficient ClpXP proteolytic activity . 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 . 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 pore-2 loop was mutated . 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 ATP-dependent 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 cyano-bacterial ClpCP3/R association also has direct relevance for the plastid Clp protease in plants. According to the endosymbiotic theory, cyanobacteria are the progenitors of plant chloroplasts, and the ClpCP3/R protease is clearly the ancestor of the chloroplast Clp protease . 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 . 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 con-served 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 .
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.
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.
This work was supported by the Swedish Research Council (VR).
We would like to thank Kerstin Lindgren at Biovitrum (Göteborg, Sweden) for allowing access to their FluoSTAR optima for assays.
Abbreviations: AAA+, ATPases associated with various cellular activities; BsClpC, B. subtilis ClpC; DTT, dithiothreitol; EcClp, E. coli Clp; FtClpP, Francisella tularensis ClpP; SyClp, Synechococcus Clp
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