(Received for publication, December 19, 1994)
From the
ClpA is the ATPase component of the ATP-dependent protease Ti
(Clp) in Escherichia coli and contains two ATP-binding sites.
A ClpA variant (referred to as ClpAT) carrying threonine in place of
the 169th methionine has recently been shown to be highly soluble but
indistinguishable from the wild-type, 84-kDa ClpA in its ability to
hydrolyze ATP and to support the casein-degrading activity of ClpP.
Therefore, site-directed mutagenesis was performed to generate
mutations in either of the two ATP-binding sites of ClpAT (i.e. to replace the Lys or Lys
with Thr).
ClpAT/K220T hydrolyzed ATP and supported the ClpP-mediated proteolysis
10-50% as well as ClpAT depending on ATP concentration, while
ClpAT/K501T was unable to cleave ATP or to support the proteolysis.
Without ATP, ClpAT and both of its mutant forms behaved as trimeric
molecules as analyzed by gel filtration on a Sephacryl S-300 column.
With 0.5 mM ATP, ClpAT and ClpAT/K501T became hexamers, but
ClpAT/K220T remained trimeric. With 2 mM ATP, however,
ClpAT/K220T also behaved as a hexamer. These results suggest that the
first ATP-binding site of ClpA is responsible for hexamer formation,
while the second is essential for ATP hydrolysis. When trimeric
ClpAT/K220T was incubated with the same amount of hexameric ClpAT/K501T (i.e. at 0.5 mM ATP) and then subjected to gel
filtration as above, a majority of ClpAT/K220T ran together with
ClpAT/K501T as hexameric molecules. Furthermore, ClpAT/K501T in the
mixture strongly inhibited the ability of ClpAT/K220T to cleave ATP and
to support the ClpP-mediated proteolysis. Similar results were obtained
in the presence of 2 mM ATP and also with the mixture with
ClpAT. On the other hand, the ATPase activity of the mixture of ClpAT
and ClpAT/K220T was significantly higher than the sum of that of each
protein, particularly in the presence of 2 mM ATP, although
its ability to support the proteolysis by ClpP remained unchanged.
These results suggest that a rapid exchange of the subunits, possibly
as a trimeric unit, occurs between the ClpAT proteins in the presence
of ATP and leads to the formation of mixed hexameric molecules.
Protease Ti, also called Clp, consists of two different multimeric components, both of which are required for ATP-dependent proteolysis in Escherichia coli(1, 2, 3, 4) . While component A (ClpA) contains the ATP-hydrolyzing sites, component P (ClpP), which is a heat shock protein(5) , contains the serine active sites for proteolysis. The isolated ClpA shows protein-activated ATPase activity, which in the reconstituted enzyme is linked to protein breakdown.
The clpA gene has recently been shown to contain dual initiation sites for translation and therefore to encode two polypeptides with different sizes (i.e. 84- and 65-kDa subunits), of which the smaller polypeptide is derived from the internal start site(6) . Accordingly, mutagenesis of the 5`-end AUG codon results in an exclusive synthesis of the 65-kDa protein, while mutation at the internal 169th AUG codon (Met) to ACG (Thr) produces only the 84-kDa protein (henceforth referred to as ClpAT). In addition, the purified ClpAT has been found to be highly soluble and to show little or no nonspecific interaction with gel filtration matrices, unlike the wild-type, 84-kDa ClpA(7, 8) . Nevertheless, ClpAT is indistinguishable from the 84-kDa ClpA in its ability to cleave ATP and to support the ClpP-mediated protein degradation(6) . The isolated ClpAT behaves as a trimer in the absence of ATP but as a hexameric complex in its presence(6, 9) .
ClpA is a member of a family of
highly conserved proteins that have two regions of particularly high
homology, each of which contains a consensus sequence for an adenine
nucleotide binding(10, 11) . Both of the ATP-binding
regions are characterized by the presence of
Gly-X-Gly-X-Gly-Lys-Thr elements, of
which the lysine residue interacts with one of the phosphoryl group of
the bound nucleotide(12, 13) . hsp104 is also a member
of the highly conserved protein family and contains the two consensus
ATP-binding sites(14) . The isolated hsp104 exists
predominantly as monomer or dimer in the absence of ATP but
oligomerizes to form a hexameric complex in its presence(15) .
In addition, mutational analysis has revealed that both of the
ATP-binding sites of hsp104 are necessary for its in vivo function, such as tolerance to high temperatures and high
concentrations of ethanol(14) . It has also been demonstrated
that the second ATP-binding site is primarily responsible for the
oligomerization of hsp104(15) .
In an attempt to determine
the structural and functional relationship of ClpA, we generated two
mutant forms of ClpAT, in which the Lys or Lys
residue in the ATP-binding sites was replaced with Thr, and
examined the effects of the mutations on the ATPase activity of ClpAT
and on its ability to support the ATP-dependent proteolysis by ClpP. We
also examined whether any of the mutations affected the hexamer
formation of ClpAT in the presence of ATP. In addition, we examined the
effects of the ClpAT mutant proteins on the structure and function of
ClpAT.
Figure 1:
Polyacrylamide gel electrophoresis of
the purified mutant forms of ClpAT. ClpAT (lanea)
and its mutant forms, ClpAT/K220T (lane b) and ClpAT/K501T (lanec), were purified as described under
``Experimental Procedures.'' 10 µg of each were then
electrophoresed on a 10% slab gel containing SDS(22) . After
electrophoresis, the gel was stained with Coomassie Blue R-250. Lanem indicates the size markers (from top to bottom): myosin heavy chain, -galactosidase,
phosphorylase b, BSA, ovalbumin, and carbonic
anhydrase.
To
determine the effects of the mutations on ATP hydrolysis, the purified
proteins were incubated in the presence of increasing concentrations of
ATP. As shown in Fig. 2, A and B, ClpAT/K220T
hydrolyzed ATP 10-50% as well as ClpAT, depending on ATP
concentration. However, casein was still capable of stimulating the
ATPase activity of ClpAT/K220T by about 2-fold, similar to that of
ClpAT, indicating that the mutation shows little or no effect on the
interaction of casein with the mutant protein. Upon double-reciprocal
plot of the data obtained in the presence of casein, the K for ATP was estimated to be 0.21 and 1.1 mM for ClpAT and ClpAT/K220T, respectively. In addition, the V
for ATP cleavage by ClpAT was estimated to be
0.51 nmol/min, and that by ClpAT/K220T was 0.26 nmol/min. In the
absence of casein, the V
for each of the
proteins was reduced to about one-half of the values seen in its
presence while the K
values remained unchanged.
Therefore, the mutation at the first of the two ATP-binding sites
reduces not only the affinity of ClpAT to ATP but also its V
. On the other hand, ClpAT/K501T showed little
or no ATPase activity regardless of whether casein was present (Fig. 2C). Thus, it appears that the ATPase activity of
ClpA depends more strictly on the second ATP-binding site than the
first.
Figure 2: ATP hydrolysis by ClpAT and its mutant forms. The ATPase activities of ClpAT (A), ClpAT/K220T (B), and ClpAT/K501T (C) were assayed in the absence (opensymbols) and presence of 10 µg of casein (closedsymbols). Reaction mixtures containing 0.2 µg of the purified proteins and various amounts of ATP were incubated at 37 °C for 30 min, and the inorganic phosphates released were determined.
Figure 3:
Ability of ClpAT and its mutant forms to
support the ClpP-mediated proteolysis. Casein hydrolysis was measured
by incubating 0.2 µg of ClpAT (), ClpAT/K220T (
), or
ClpAT/K501T (
), 0.1 µg of ClpP, and 10 µg of
[
H]casein in the presence of increasing amounts
of ATP. Incubations were performed for 1 h at 37 °C, and the
radioactivities released into acid-soluble products were determined
using a scintillation counter.
Figure 4:
Size estimation of ClpAT and its mutant
forms using a Sephacryl S-300 column. Aliquots (0.2 mg each) of the
purified ClpAT (), ClpAT/K220T (
), and ClpAT/K501T
(
) were incubated with 0.5 (rightpanel) or 2
mM ATP (leftpanel) for 15 min at 4 °C.
Each was then loaded onto the gel filtration column (1
40 cm)
equilibrated with 50 mM Tris-HCl buffer (pH 8) containing 5
mM MgCl
, 0.5 mM EDTA, 1 mM dithiothreitol, 20% glycerol, 0.1 M NaCl, and 0.5 or 2
mM ATP. Opensymbols are for the proteins
incubated and chromatographed without ATP. Fractions of 0.5 ml were
collected at a flow rate of 7 ml/min, and aliquots of them were assayed
for protein concentration. The dottedlines indicate
the positions where the protein peaks eluted. The arrows show
the size markers: lanea, thyroglobulin (669 kDa); laneb, apoferritin (443 kDa); lanec, alcohol dehydrogenase (150 kDa); laned, BSA (66 kDa).
Figure 5:
Effects of increasing concentrations of
ClpP on the ATPase activities of ClpAT and ClpAT/K220T. The ATPase
activities of ClpAT () and ClpAT/K220T (
) were assayed
without (A) and with 10 µg of casein (B) as in Table 1but in the presence of increasing amounts of ClpP. The
assays were also performed with increasing amounts of BSA (dottedlines) instead of ClpP. However, the amounts of BSA added
were in 50-fold excess of the indicated amounts of ClpP. The activities
seen in the absence of ClpP are expressed as 1.0, and the others are
expressed as their relative values.
Figure 6:
Elution of the mixture of ClpAT/K220T and
ClpAT/K501T from a Sephacryl S-300 column. The mixture of 0.2 mg each
of ClpAT/220T and ClpAT/K501T (total 0.4 mg) containing 0.5 mM ATP () was incubated for 15 min at 4 °C and subjected to
gel filtration on a Sephacryl S-300 column as described in the legend
to Fig. 4but in the presence of 0.5 mM ATP only. The
same experiments were performed with 0.2 mg of ClpAT/K220T (
) or
ClpAT/K501T (
) alone. The size markers (arrows) are
the same as shown in Fig. 4.
We then examined the effect of ClpAT/K501T on the casein-activated ATPase activity of ClpAT or ClpAT/K220T in the presence of ClpP. As shown in Table 2(experiment A), ClpAT/K501T strongly inhibited the ATPase activities of the latter proteins. Similar inhibitory effects were observed in the presence of 2 mM ATP, at which concentration all of the ClpAT proteins by themselves behave as hexameric molecules. On the other hand, the ATPase activity of the mixture of ClpAT and ClpAT/K220T was higher than the sum of that of each protein. Particularly in the presence of 2 mM ATP, ATP hydrolysis by the mixture of ClpAT and ClpAT/K220T was enhanced to an extent that could be seen by the doubled amount of ClpAT alone. Similar stimulatory or inhibitory effects of ClpAT/K220T or ClpAT/K501T, respectively, on the ATPase activity of ClpAT were observed when the same experiments were performed in the absence of casein, ClpP, or both (data not shown). Furthermore, the mixture of any of the two ClpAT proteins eluted in the fractions corresponding to a hexameric size from the Sephacryl S-300 column (data not shown). These results clearly suggest that the hexameric ClpAT proteins can also interact with each other and form hybrid molecules.
To determine whether the interaction between the ClpAT proteins may also influence their ability to support the ClpP-mediated proteolysis, casein hydrolysis was assayed by incubation of their mixtures in the presence of 0.5 and 2 mM ATP. Table 2(experiment B) shows that ClpAT/K501T strongly inhibited the ability of ClpAT or ClpAT/K220T to support the proteolytic activity of ClpP, similar to its effect on ATP cleavage. Furthermore, the inhibitory effects of ClpAT/K501T occurred in a dose-dependent manner on the ability of ClpAT or ClpAT/K220T in supporting the ClpP-mediated proteolysis as well as in ATP hydrolysis (Fig. 7), again indicating that ATP hydrolysis is tightly coupled to protein breakdown by protease Ti. On the other hand, ClpAT/K220T showed little or no effect on the ability of ClpAT in supporting the proteolysis by ClpP at both of the ATP concentrations (i.e. the casein-degrading activity of ClpP in the presence of their mixture was nearly identical to the sum of that seen with each protein) (Table 2, experiment B). To clarify further the differential effect of ClpAT/K220T on casein hydrolysis from that on ATP cleavage, the same assays were performed as in Table 2(experiment B) but by varying the incubation period. As shown in Fig. 8, nearly the same results were obtained at all time points for ATP hydrolysis and for casein degradation. These results suggest that the mutation in the first ATP-binding site may indeed influence the interaction of ClpA with ClpP, and therefore the enhanced ATP hydrolysis by the mixture of ClpAT and ClpAT/K220T could not increase its ability to support the ClpP-mediated casein degradation.
Figure 7:
Effects of increasing concentrations of
ClpAT/K501T on the ability of ClpAT or ClpAT/K220T in ATP hydrolysis (A) and in supporting the casein hydrolysis by ClpP (B). ATP hydrolysis was assayed by incubating 10 µg of
casein and 0.2 µg of ClpAT () or ClpAT/K220T (
) for 30
min at 37 °C in the presence of various amounts of ClpAT/K501T. The
incubation mixtures also contained ClpP with one-half the amount of the
ClpAT proteins. For casein hydrolysis, incubations were performed as
above but in the presence of twice the amount of the ClpAT
proteins.
Figure 8:
Effects of ClpAT/K220T on the ability of
ClpAT in ATP hydrolysis (A) and in supporting the casein
hydrolysis by ClpP (B). ATP hydrolysis was assayed by
incubating 10 µg of casein and 0.2 µg of ClpAT (), 0.2
µg of ClpAT/K220T (
), 0.2 µg each of both (
), or
0.4 µg of ClpAT (
) for various periods at 37 °C in the
presence of various amounts of ClpAT/K501T. The incubation mixtures
also contained ClpP with one-half the amount of the ClpAT proteins. For
casein hydrolysis, incubations were performed as above but in the
presence of twice the amount of the Clp
proteins.
The present studies have demonstrated that the first of the
two ATP-binding sites in ClpA is responsible for the ATP-mediated
hexamer formation, while the second site is critical for the ATPase
function. The mutation in the first site (K220T) prevents hexamer
formation of ClpAT at 0.5 mM ATP but not at 2 mM. Of
interest is the finding that the mutation results in the reduction of
not only the affinity of ClpAT to ATP but also the V value for ATP hydrolysis and hence also decreases the ability of
ClpAT to support the proteolytic activity of ClpP. It has been reported
that the kinetics of proteolysis by protease Ti shows positive
cooperativity with respect to ATP with a Hill coefficient of
1.6(9) . Therefore, it appears that binding of ATP to the first
site influences the efficiency of its binding to the second site and
thus the ability of ClpA to support the ClpP-mediated proteolysis.
Noteworthy was the finding that the mode of interaction of ClpP with ClpAT/K220T appears to differ from that with ClpAT. We have previously shown that ClpP reduces the rate of ATP hydrolysis by ClpA in the absence of casein but increases the ATPase activity in its presence (8) . However, the ATPase activity of the mutant form of ClpAT was found to be increased by ClpP whether or not casein was present. Furthermore, the extent of the increase in ATP hydrolysis by ClpAT/K220T was significantly greater than that by ClpAT. Therefore, it appears that the reduced ability of the mutant form of ClpAT in supporting the ClpP-mediated proteolysis may also be attributed to the change in the mode of its interaction with ClpP.
On the other hand, the mutation in the second site (K501T) almost completely eliminated the ATPase activity of ClpAT but with little or no effect on the hexamer formation in the presence of 0.5 or 2 mM ATP. Furthermore, ClpAT/K501T can form a hexameric complex even in the presence of ADP, indicating that hexamer formation requires the binding but not the hydrolysis of the adenine nucleotides. Therefore, the mutation in the second ATP-binding site does not seem to exert any influence on the binding of ATP or ADP to the first site.
Of particular interest was the finding that the hexameric ClpAT or ClpAT/K501T can interact with the trimeric form of ClpAT/K220T and generate a mixed hexameric complex. Furthermore, ClpAT/K501T inhibited the ability of ClpAT or ClpAT/K220T in ATP hydrolysis and in supporting the ClpP-mediated proteolysis. These inhibitory effects of ClpAT/K501T could also be seen in the presence of 2 mM ATP, at which concentration all of the ClpAT proteins by themselves and the mixtures of any two of them behave as hexameric molecules. In addition, the ATPase activity of the mixture of ClpAT and ClpAT/K220T was found to be significantly higher than the sum of that of each protein, although its ability to support the proteolysis by ClpP remained unchanged. Therefore, it appears likely that a rapid exchange of the subunits occurs between the ClpAT proteins in the presence of ATP and results in the formation of hybrid molecules.
However, it is presently unclear whether the exchange of the subunits between the ClpAT proteins occurs at the level of a trimeric unit or otherwise. Upon the gel filtration analysis of hexameric ClpAT/K501T incubated with increasing amounts of trimeric ClpAT/K220T (i.e. at 0.5 mM ATP as in Fig. 6), we found that the right side shoulder of the protein peak increases much more significantly than the peak height itself (data not shown). These results suggest a possibility that each trimeric unit of hexameric ClpAT/K501T in the incubation mixture is interacting with a trimeric ClpAT/K220T to form a hybrid hexameric complex until all of the trimeric units of ClpAT/K501T are replaced and occupied by trimeric ClpAT/K220T molecules, and therefore the excess of ClpAT/K220T remains trimeric. This possibility is in part supported by the finding that the inhibitory effect of ClpAT/K501T on the ATPase activity of ClpAT or ClpAT/K220T does not reach completion even in the presence of a 2-6-fold excess of ClpAT/K501T (see Fig. 7A and data not shown). However, this possibility is based on the assumption that the hybrid molecule containing one of each trimeric unit of ClpAT and ClpAT/K501T should be partially active in ATP hydrolysis and in supporting the ClpP-mediated proteolysis. In addition another possibility, that the exchange of the subunits occurs at the level of a monomeric or dimeric unit and results in the formation of various kinds of hybrid molecules, cannot be totally excluded.
In contrast to the present findings that the first
ATP-binding site is responsible for hexamer formation of ClpA and the
second is for ATPase activity, Lindquist and co-workers (15) have recently demonstrated that a single amino acid
substitution in the second ATP-binding site eliminates hexamer
formation of hsp104 with only a slight defect in ATP hydrolysis.
Furthermore, a mutation in the first site completely eliminates the
ATPase activity of hsp104 without much influence on oligomerization.
These reversed functions of the two ATP-binding sites are rather
striking, because the sequences of each ATP-binding region containing
the Gly-X-Gly-X-Gly-Lys-Thr elements in
ClpA and hsp104 are known to be highly homologous with each other (14) and because in both studies the same lysine residue that
interacts with one of the phosphoryl group of the bound nucleotide (12, 13) was substituted with threonine. Therefore, it
appears that the distinctive functions (i.e. ATP hydrolysis
and oligomerization) of the two ATP-binding sites may be determined by
the tertiary structures of ClpA and hsp104, such as the proximity of
any one of the two sites to the catalytic site for ATP hydrolysis or to
certain unknown sequence(s) that is involved in oligomerization, but
not by the sequences of the homologous ATP-binding regions themselves.