From the Escherichia coli ClpX, a member of
the Clp family of ATPases, has ATP-dependent chaperone
activity and is required for specific ATP-dependent
proteolytic activities expressed by ClpP. Gel filtration and electron
microscopy showed that ClpX subunits (Mr
46,000) associate to form a six-membered ring
(Mr ~ 280,000) that is stabilized by binding
of ATP or nonhydrolyzable analogs of ATP. ClpP, which is composed of
two seven-membered rings stacked face-to-face, interacts with the
nucleotide-stabilized hexamer of ClpX to form a complex that could be
isolated by gel filtration. Electron micrographs of negatively stained
ClpXP preparations showed side views of 1:1 and 2:1 ClpXP complexes in
which ClpP was flanked on either one or both sides by a ring of ClpX.
Thus, as was seen for ClpAP, a symmetry mismatch exists in the bonding
interactions between the seven-membered rings of ClpP and the
six-membered rings of ClpX. Competition studies showed that ClpA may
have a slightly higher affinity (~2-fold) for binding to ClpP. Mixed
complexes of ClpA, ClpX, and ClpP with the two ATPases bound
simultaneously to opposite faces of a single ClpP molecule were seen by
electron microscopy. In the presence of ATP or nonhydrolyzable analogs of ATP, ClpXP had nearly the same activity as ClpAP against
oligopeptide substrates (>10,000 min The Clp family of ATP-dependent chaperone-linked
proteases are high molecular weight complexes composed of a protease
with limited peptidase and virtually no intrinsic proteolytic activity and an ATPase that activates proteolysis by binding and unfolding protein substrates (1, 2). Clp proteases were first described in
Escherichia coli, where ClpAP and ClpXP were shown to
consist of a common proteolytic core, ClpP, which can be activated by either of two ATPases, ClpA or ClpX (3-6). Recently another branch of
the Clp family consisting of a unique proteolytic component, ClpQ (or
HslV), and the ATPase ClpY (or HslU) was described (7-9). Despite high
degrees of amino acid sequence homology, Clp ATPases appear to fall
into two groups, the ClpA/ClpX-like proteins that have intrinsic
chaperone activity and also act as part of proteolytic complexes and
the ClpB-like proteins that appear to function solely as molecular
chaperones independent of proteolytic components (10, 11). Clp ATPases
are widespread in eukaryotes and prokaryotes indicating that, at the
least, the protein-remodeling activity of Clp ATPases is highly
conserved (1, 10).
The two Clp proteolytic components described in E. coli,
ClpP and ClpQ, are not related to each other, differing in their amino
acid sequences and in their catalytic mechanisms of peptide bond
cleavage (12-14). ClpP is representative of a family of serine proteases that is unique both in sequence and in the folding domains seen in the recently solved x-ray crystal structure (15). The ClpP
subfamily is highly conserved in prokaryotes and is found in plant
chloroplasts as well as in mammalian cell mitochondria (16). ClpQ is a
member of the proteasome family (13, 17). Proteasomes are multimeric
proteases that not only form the proteolytic core of the major
ATP-dependent protease in the eukaryotic cytosol but that
are also found in eubacteria and in Archaea (18). ClpQ has an
amino-terminal catalytically active threonine residue and a tertiary
structure similar to that of the proteasomal The Clp ATPases not only carry out the energy-dependent
steps in protein remodeling and degradation, but they also determine the selection of protein substrates for both activities. Proteins that
bind to and are remodeled by ClpX are also degraded by the corresponding holoenzyme complexes with ClpP (5, 6). The same can be
said for substrate selection in all three activities carried out by
ClpA or ClpAP (22, 23). Thus, it is likely that protein binding and
unfolding by Clp ATPases is an integral part of their ability to
promote specific protein degradation by ClpP. The structure of the
ClpAP complex as revealed by electron microscopy is consistent with
protein binding and enzymatic properties of the enzyme. ClpA binds on
the planar surface of each ring of ClpP, controlling access to the
openings of the axial channels (24). Presumably, substrates must
interact with ClpA and pass through or around the rings formed by the
two domains of ClpA to gain access to the proteolytic active sites. We
have proposed a model in which ATP-dependent protein
unfolding is coupled to translocation of segments of the substrate to
the interior of ClpP (11, 25). Because of the unequal number of
subunits in the respective rings, the subunits in ClpA will not all be
in the same register with those in ClpP. Progressive movement of different pairs of subunits into alignment during successive rounds of
ATP hydrolysis may aid in translocation of protein substrates through
the narrow channels.
Materials--
Unless noted, chemicals were purchased from
Sigma. Nucleotides were obtained from Sigma and Boehringer Mannheim.
Clp proteins were prepared by R. Grimaud and M. R. Maurizi. ClpX Purification--
The clpX coding region was
amplified by polymerase chain reaction and inserted in plasmid pET11a
(Novagen) so that expression was under control of the T7 promoter.
Details of the construct will be published
elsewhere.1 BL21(DE3) cells
containing the plasmids pLysS and pET11a/clpX were grown at
room temperature (25 °C) for 17 h in Luria broth containing 100 µg/ml ampicillin, 30 µg/ml chloramphenicol, and 0.25 mM
isopropyl-1-thio- Enzymatic Assays--
ATPase activity was assayed by liberation
of inorganic phosphate from ATP after incubating ClpX in 50 µl of 50 mM Tris/HCl, pH 7.5, 10 mM MgCl2, 1 mM dithiothreitol, and 1 mM ATP for 25 min at
37 °C. The inorganic phosphate was measured by the procedure described by Lanzetta et al. (26), except that the Sterox
was omitted from the malachite green/ammonium molybdate solution, and
16 µl of 10% Tween 20 was added to the solution after the addition
of the color reagent. Assay conditions and methods for casein and the
propeptide, FAPHMALVPV, degradation were published previously (27).
Assays were performed with limiting ClpP (0.2-0.5 µg) and 2-5 µg
of ClpX. For degradation of Isolation of ClpX Oligomers and the ClpXP Complex--
ClpX and
various complexes of ClpX were separated on a 0.3 × 20-cm
Superdex 200 column in either 50 mM Tris/HCl or 50 mM Hepes/KOH, pH 7.5, with 0.2 M KCl and 2 mM Electron Microscopy--
A 4-µl drop of the sample at a
concentration of 20 µg/ml was placed on a glow-discharged,
carbon-stabilized, collodion-coated grid for 30 s. The sample
droplet was blotted away, and the grid was negatively stained with
several drops of 1% aqueous uranyl acetate. Specimens were viewed in a
Zeiss 902 or a Phillips CM120 transmission electron microscope, and
micrographs were recorded on Kodak SO-163 emulsion at a nominal
magnification of 50,000. Magnification of the electron micrographs was
calibrated using the 40.6-Å spacing of T4 bacteriophage tail
striations (28).
Laboratory of Cell Biology,
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
1/tetradecamer of
ClpP). Thus, ClpX and ClpA interactions with ClpP result in
structurally analogous complexes and induce similar conformational
changes that affect the accessibility and the catalytic efficiency of
ClpP active sites.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-subunits (17).
Surprisingly, ClpQ subunits assemble into rings with only six subunits
(8, 19), unlike the proteasome, which has seven subunits per ring. For
both ClpP and ClpQ, the active sites are buried within an aqueous
cavity formed by the joining of the two rings, and access to the active
sites is limited to a narrow axial channel through the center of each
ring. It has been proposed that binding of the chaperone component and
cycles of ATP hydrolysis may alter the size and properties of the
channel and increase substrate access (20, 21).
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
O
protein was a gift from Sue Wickner, Laboratory of Molecular Biology,
NCI, NIH, Bethesda, MD. Polyclonal rabbit antibodies against ClpX were described previously (5).
-D-galactopyranoside. Cells (6.5 g) were suspended in 26 ml of buffer D (50 mM Tris, pH 7.5, 10 mM MgCl2, 5 mM dithiothreitol and
10% (v/v) glycerol) and broken by a single pass through an ice-chilled
French pressure cell (Aminco) at 20,000 psi. The crude extract was
centrifuged at 30,000 × g for 30 min at 4 °C.
Polyethyleneimine was added to the supernatant to a concentration of
0.1%. Precipitated material was removed by centrifugation at
30,000 × g for 30 min at 4 °C. The supernatant solution was brought to 0.1 M KCl and loaded on a 2.5 × 20-cm Q-Sepharose (Amersham Pharmacia Biotech) column equilibrated
with buffer D plus 0.1 M KCl. Proteins were eluted by a
100-ml gradient from 0.1 to 0.4 M KCl, and the ClpX was
detected by SDS-polyacrylamide gel electrophoresis and Western
blotting. The ClpX-containing fractions, which eluted at about 0.3 M KCl, were pooled, and ClpX was precipitated with 55%
saturation of ammonium sulfate. The precipitated protein was collected
by centrifugation at 30,000 × g for 30 min at 4 °C
and dissolved in buffer D plus 0.1 M KCl. The protein
solution was clarified by centrifugation at 30,000 × g
for 10 min at 4 °C and loaded onto a size exclusion TSK 250 column
(Bio-Rad) equilibrated with buffer D plus 0.1 M KCl. The ClpX was further purified by chromatography on a 1 × 10 cm MonoQ (Amersham) column using a 40-ml gradient from 0.1 to 0.4 M
KCl gradient in Buffer D. ClpX fractions were stored separately at 0-4 °C.
O protein and other proteins, the
substrates (5-10 µg) were incubated at 37 °C with 0.5 µg of
ClpP and 4 µg of ClpX in 50 mM Tris/HCl, pH 8, with 10 mM MgCl2 and 4 mM ATP for 30-60
min. Reactions were quenched with 5% trichloroacetic acid, and the
precipitated protein was dissolved in SDS-containing buffer and heated
at 95 °C for 5 min before loading on 12% acrylamide, SDS gels. The
protein remaining was estimated by inspection after staining with
Coomassie Blue.
-mercaptoethanol with or without 10% (v/v) glycerol.
Columns were run at room temperature and at a flow rate of 0.1 ml/min.
For stabilization of high molecular weight oligomers, samples of ClpX
alone or ClpX and ClpP were mixed with 25 mM
MgCl2 and 1-2 mM
ATP
S2 before running the
column, and these ligands were included in the running buffer.
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RESULTS |
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ClpX Purification--
When ClpX was expressed under the strong T7
promoter, only 10-20% was found in the soluble portion of cell
extracts, and the remainder was found in the low speed pellet. The
soluble material was used for purification by the procedure outlined
under "Experimental Procedures." After the last purification step,
anion exchange chromatography on MonoQ, several fractions contained
ClpX that was 95% pure, estimated by inspection of Coomassie-stained
SDS-polyacrylamide electrophoresis gels (Fig.
1). These fractions were stored
separately and used for the studies presented here.
|
ATPase Activity--
During purification the activity of ClpX was
monitored by measuring the ATPase activity. The ATPase activity eluted
from the MonoQ column coincided with the major protein band (Fig. 1),
which also cross-reacted with anti-ClpX antibodies (data not shown). The ATPase activity of purified ClpX was ~0.3 µmol/min/mg (turnover number 15 min1), which is consistent with what was
previously reported by Wawrzynow et al. (23).
Protein and Peptide Degradation in the Presence of ClpX--
In
the presence of MgATP and ClpP, the ClpX in our preparation promoted
the degradation of purified O protein (data not shown), which has
been shown to be a specific substrate for ClpX and ClpP in
vivo and in vitro (5, 6) but, as has been reported (6), had virtually no activity against
-[3H]casein (see
Fig. 4, below).
|
Oligomerization State of ClpX--
In the absence of nucleotides,
ClpX tended to run as a broad peak on gel filtration columns. The
fastest migrating fraction had an apparent Mr of
~200,000, and smaller species were also evident (data not shown).
Electron microscopy of this protein showed mostly heterogeneously sized
particles with no obvious regularity (data not shown). The addition of
ATPS to ClpX produced a more uniform, higher molecular weight peak
that eluted from a Superdex 200 column with an apparent
Mr of ~300 ± 20 × 103
(Fig. 2, dotted line).
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|
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The ClpXP Complex--
When a mixture of ClpP and ClpX was run
over Superdex 200 in the presence of ATPS, both proteins eluted
together in a peak with an apparent molecular weight >600,000 (Fig. 2,
solid line). In the experiment shown, the amount of ClpX
protein in the complex was almost twice the amount of ClpP (estimated
from the stained bands in Fig. 2) as would be expected if two hexamers
of ClpX combined with a tetradecamer of ClpP. In other experiments with ClpP added in excess of ClpX, the new peak had slightly lower molecular
weight and an obviously lower ratio of ClpX to ClpP (data not shown).
When ClpXP complexes were examined by electron microscopy after
negative staining, several different projections were observed. The
most informative of these were projections composed of three or four
strong parallel striations (Fig. 3b). After selection, the
respective projections were rotationally and translationally aligned
and then averaged to result in the final reconstructed images (Fig.
3b, inset). The two shorter contiguous striations
had the same lengths previously seen for ClpP in the ClpAP complex
(24). The third striation, by analogy with previous images of ClpA and
ClpAP, was interpreted as a side projection of a ClpX ring. The fourth
striation, which flanked the central ClpP molecule, appeared to be a
side projection of ClpX that had undergone some flattening or other
disruption of structure.
ClpX and ClpA Have Similar Affinities for ClpP-- The relative affinities of ClpX and ClpA for binding to ClpP were compared by a competition assay using casein degradation as a measure of ClpAP. The addition of ClpX in amounts sufficient to prevent ClpA binding to ClpP should inhibit casein degradation because ClpXP cannot degrade casein. Fig. 4 shows that the addition of increasing amounts of ClpX to assay mixtures with a fixed amount of ClpA resulted in a decrease in casein degradation by ClpP. A 3-5-fold excess of ClpX6 over ClpA6 was required to inhibit 50% of the activity measured in the absence of ClpX. The initial aliquots of ClpX produced no inhibition (or even a slight increase in casein degrading activity), suggesting that displacement of a single ClpA6 by ClpX6 was not sufficient to block casein degradation. In control experiments, when the ClpAP complex, which dissociates very slowly under assay conditions, was allowed to form before the addition of ClpX, a substantial lag in the kinetics of inhibition was observed. Thus, inhibition by ClpX was dependent on its ability to bind to ClpP and not on competition with the substrate, casein, or excessive hydrolysis of the ATP (data not shown). Thus, hexameric ClpX appears to bind ClpP with an affinity similar to that shown by hexameric ClpA.
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ClpX and ClpA Can Bind Simultaneously to ClpP-- Efforts to observe the relative amounts of ClpX and ClpA bound to ClpP by electron microscopy were hampered by the tendency of complexes to stand on end on the grid. Nevertheless, these experiments did yield the novel observation of a mixed complex of ClpA and ClpX with ClpP. Fig. 5a shows a field of negatively stained particles in which projections having five striations are clearly distinguishable along with the expected ClpAP and ClpXP complexes. The particles with five striations were averaged to give the image shown in the Fig. 5b. The dimensions of the subdomains strongly suggest that the particles are composed of a double ring of ClpP flanked on one side by the doubly striated ClpA and on the other by the single-striated ClpX. The average side views for ClpXP and ClpAP are shown in Figs. 5, c and d for comparison. These data are the first to demonstrate that a mixed ClpAPX complex can be formed and indicate that binding of ClpX or ClpA to one end of a ClpP tetradecamer does not preclude the other ATPase from binding at the other end. Whether such hybrid complexes form in vivo (for example, under circumstances in which the supply of ClpP is limiting) and their possible physiological significance remain to be determined.
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DISCUSSION |
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ATP-dependent proteases from a variety of sources and from different evolutionary families have complex, multimeric structures. Modular assemblies (as in ClpAP and ClpXP or the complex of the proteasome with either the ATP-dependent 19 S or ATP-independent 11 S activators) and alternative subunit compositions (as in the isoforms of eukaryotic proteasomes) are now among the well established features of these proteases (14, 18, 31). The variety of ways in which the different components can be combined allows the specificity, the activity, and the response to regulatory signals to be fine-tuned. One goal of our studies is to distinguish the structural features that underlie the activities common to all the proteases and those that reflect unique functions of particular proteases.
We had shown previously that ClpA formed a complex with ClpP in which a hexameric ring of ClpA was bound to each of the two heptameric rings of ClpP (24). One surprising aspect of the ClpAP complex was the symmetry mismatch between the six-membered and seven-membered rings of the two components. Although intriguing speculations regarding a function of the symmetry mismatch in allowing ratcheting or rotation of the ClpA and ClpP rings about each other during catalysis were attractive, it remained possible that this structural feature was peculiar to ClpAP. Such reservations were of heightened concern because much more was known from genetic and biochemical studies about the physiological activities of a putative complex between ClpX and ClpP, two proteins that are encoded in an operon and subject to co-regulation in vivo (5). It was thus imperative to determine the oligomeric structure of ClpX and to establish the nature of the ClpX and ClpP interaction in forming the active proteolytic complex.
Our results demonstrate that ClpX, like ClpA, is composed of six subunits arranged in a symmetrical ring. Like ClpA, ClpX binds tightly to ClpP only in the oligomeric state stabilized by nucleotide binding. ClpX can form 1:1 and 2:1 complexes with ClpP, and the ClpXP complexes, like ClpAP, have a symmetry mismatch between the ATPase and proteolytic components. The ability to generalize this structural feature of the ATP-dependent Clp proteases suggests that, although the details are still not understood, the symmetry mismatch must be a fundamental determinant of the mechanism of chaperone-mediated proteolysis by ClpAP and ClpXP.
The competition assays (Fig. 4) indicate that the affinities of ClpA and ClpX for ClpP are within a factor of three of each other, and thus, ClpA and ClpX should compete for ClpP in vivo. Estimates of the intracellular concentrations of ClpA, ClpX, and ClpP indicate that, in exponentially growing cells on rich media, ClpA is in slight excess of ClpX, and ClpP is limiting compared with ClpA and ClpX combined.1 Thus, the distribution of ClpP between ClpA and ClpX could affect the selection of substrates under different physiological conditions. ClpA is degraded in cells with a half-life of 1 h, but the degradation appears to be autocatalytic (3) and is not dependent on ClpX.3 Further studies are required to understand the relative amounts of ClpAP and ClpXP and their significance in vivo.
A unique finding of this study is the formation of a mixed complex between ClpA, ClpX, and ClpP. Because so few side views were available, we were prevented from obtaining accurate quantitation of the relative numbers of the three complexes, ClpAP, ClpXP, and ClpAPX. Experiments are under way to determine the frequency with which the mixed complexes occur. It would appear, however, that there is no bias against such complexes, i.e. no negative cooperativity between ClpA and ClpX, and thus we think it highly probable that mixed complexes exist in vivo as well. Activity measurements with ClpAP had suggested that 1:1 and 2:1 complexes of ClpAP had nearly the same specific activity for casein degradation, indicating that ClpA might not translocate substrates from both sides of ClpP simultaneously (32). Whether ClpA and ClpX can activate degradation of different substrates simultaneously cannot be determined from our data. It is interesting, however, that the inhibition of ClpAP activity by ClpX appeared to be cooperative, suggesting that displacing a single ClpA failed to inhibit casein degrading activity and implying that ClpAPX has casein degrading activity comparable to that of the 1:1 and 2:1 ClpAP complexes.
The 6-fold symmetry of ClpX has been observed in all micrographs of
native ClpX that we have studied. In contrast, the homologous ATPase,
ClpY (HslU), formed rings with 6- or 7-fold symmetry (8, 19). Sequence
homology apparently does not dictate that proteins will form rings with
the same numbers of subunits. Another example of such structural
deviation is the difference in symmetry between E. coli ClpQ
(HslV) (6-fold) and the homologous -subunits of proteasomes from
Archaea and eukaryotic cells (7-fold) (14, 18). The circular alignment
of active sites or binding sites produced by the ring-like structure
appears to be the critical structural element rather than the exact
number of such sites. In the case of ClpYQ, it is interesting to note
that, if the predominant form of ClpY has seven subunits per ring (8,
19), there would be an inside out (with respect to ClpAP or ClpXP)
symmetry mismatch between the ATPase and the proteolytic component
(ClpQ).
It is not yet possible to generalize about the occurrence of symmetry mismatches in other ATP-dependent proteases. Symmetry mismatch cannot be essential, because homooligomeric proteases such as Lon and FtsH, which have the ATPase and proteolytic sites in the same polypeptide, are perforce symmetrical. The structure of the ATPases in the 26 S proteasome has not been defined, but in yeast there appears to be only six such ATPases (33), which suggests at least a nonstoichiometric interaction between ATPase and proteasome subunits. A symmetry mismatch between the 11 S activator (PA28) and the 20 S proteasome may exist, although there is controversy regarding the number of subunits in the rings of PA28 (six or seven) (34, 35). With the uncertainty regarding the numbers of subunits in ClpY and in the 11 S, it is reasonable to consider the possibility that some of these proteins may exist in both forms and that the degradative activities of the resulting complexes may vary. We would like to note that preliminary studies of ClpX structure using a His-tagged ClpX protein (kindly supplied by T. Baker, MIT) had shown rings with 7-fold symmetry.1 Structural or chemical perturbations may have significant effects on the assembly of Clp ATPases.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: NCI, National Institutes of Health, Bldg. 37, Rm. 1B07, 37 Convent Dr., MSC 4255, Bethesda, MD 20892-2755. Tel.: 301-496-7961; Fax: 301-402-0450.
1 R. Grimaud and M. R. Maurizi, unpublished observations.
2
The abbreviation used is: ATPS, adenosine
5'-O-(thiotriphosphate).
3 M. R. Maurizi, unpublished observations.
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REFERENCES |
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