(Received for publication, December 20, 1994; and in revised form, May 19, 1995)
From the
The heat shock protein GroEL from Escherichia coli is a
tetradecameric oligomer that facilitates the refolding of nonnative
polypeptides in an ATP-hydrolysis dependent reaction. A mutant in GroEL
was prepared in which lysine 3 was substituted with glutamate, which
destabilizes the oligomeric structure of GroEL (Horovitz, A.,
Bochkareva, E.S., and Girshovich, A.S.(1993) J. Biol. Chem.
268, 9957-9959). The highly expressed and purified GroEL was judged to be monomeric by sedimentation equilibrium, yielding
a molecular weight of 54,500, despite a weak tendency of the mutant to
reversibly form higher order aggregates above 4 mg
ml
. The monomeric variant appears to be folded based
on the far UV circular dichroism spectrum, which shows significant
-helical content, but with slight differences in conformation
relative to wild-type GroEL. The increase in exposed hydrophobic
surface of the monomer was probed with the dye
4,4`-bis-1-anilino-3-naphthalenesulfonate (bis-ANS). The fluorescence
of bis-ANS increases approximately 150-fold in the presence of the
mutant, and about 4 mol of bis-ANS bind per mol of monomer, with a
binding constant of 1.6 µM. Adenosine nucleotide binding
to monomeric GroEL
resulted in considerable quenching of
bis-ANS fluorescence, correlating with significant structural changes
as seen in the far UV circular dichroism, and permitted the measurement
of binding isotherms for ATP and ADP. Hyperbolic ATP binding isotherms
yield a dissociation constant of 82 µM, about 4-fold
weaker than the K
for ATP seen in steady-state
kinetics assays of the wild-type GroEL ATPase. A similar difference was
seen for ADP binding. These results suggest that the mutation disrupts
the native tetradecameric quaternary structure through conformational
changes that may also weaken nucleotide binding. The monomeric mutant
exhibited no chaperone activity as evidenced by a failure to inhibit or
facilitate the refolding of chemically denatured enolase, an inability
to refold denatured rhodanese above spontaneous levels, and a lack of
binding to
-casein, a competitor in many chaperonin-promoted
refolding reactions. Thus, the formation of assembly incompetent
monomers by the lysine 3 to glutamate mutation results in a dramatic
decrease in the affinity for nonnative polypeptide chains and suggests
that the oligomeric nature of GroEL is crucial for its molecular
chaperone function.
The Escherichia coli chaperonin GroEL, a member of the highly conserved Hsp60 family of heat shock proteins, is assembled from 14 copies of identical chains into an oligomer with 7-fold symmetry (1, 2, 3) and a large, central cavity that has been implicated in binding nonnative polypeptide chain substrates(1, 4, 5) . The oligomeric nature of GroEL appears to be strongly linked to its function as a molecular chaperone since it binds only 1-2 polypeptides/oligomer(6, 7) , binds 1-2 mol of the co-chaperonin GroES(1, 8, 9) , and hydrolyzes ATP in a cooperative manner(10, 11) . There have been numerous reports that have described the surprising relative ease of dissociation of the tetradecameric structure of GroEL into monomers either by limited chemical denaturation, proteolysis, or by mutagenesis(5, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21) . A basis for these results can be suggested by a consideration of the recently determined x-ray crystal structure of GroEL, which has revealed that only a fraction of the accessible surface area is buried at subunit interfaces upon assembly of the oligomer (3) relative to that estimated for similarly sized proteins(22) .
Motivated by mutagenesis studies, which
suggested that amino acid residues in the N-terminal region of the
equatorial domain of GroEL were essential for its proper
assembly(14, 15) , a mutant in GroEL was constructed
in which lysine 3 was replaced with glutamate (14) and the
variant was purified and characterized to compare with, and yield
insight into, the molecular properties of wild-type GroEL. The K3E
mutant of GroEL exists as an extensively folded monomer that binds more
bis-ANS ()than wild-type GroEL(21, 23) ,
reflecting an increase in exposed hydrophobic surface for the mutant.
The monomer is an inactive ATPase, yet it maintains reasonable affinity
for adenosine nucleotides. The weaker binding of nucleotides to the
monomer relative to that seen for wild-type GroEL suggests that
although it is significantly folded, its conformation may be
significantly nonnative. Furthermore, the monomeric GroEL
mutant is unable to arrest, or refold, either enolase or
rhodanese, which may be attributable to a marked reduction in its
affinity for nonnative polypeptide chains. These results suggest that
the oligomeric nature of GroEL is crucial for its biological activity.
The molecular weight of the K3E mutant of GroEL was determined from the concentration distribution of the protein at sedimentation equilibrium at 25 °C using the partial specific volume of 0.732 ml/g determined from the amino acid composition(29) . Data obtained under different rotor speeds or initial solute concentrations were analyzed using an extension of the basic equation for sedimentation equilibrium that includes terms for a monomeric component in the presence of aggregates (32) . This equation is
where C is the concentration at a
given radial position, C
is the
concentration of a particular species, i, (monomer or
aggregate of i monomers) at a reference position, e.g. the meniscus, M
, is the molecular weight of
the monomer, v is the partial specific volume,
is the
solvent density,
is the angular velocity, r is the
radial position in centimeters from the center of rotation, r
is the distance in centimeters from the
center of rotation to the reference position, R is the gas
constant, T is the absolute (Kelvin) temperature, and B is a correction term for a non-zero base line.
One of our
first goals after purifying the mutant was to reproduce the results
that indicated the protein was unable to assemble into its native
quaternary structure. Thus, sedimentation equilibrium was employed to
assess the oligomeric state of GroEL. As can be seen in Fig. 1, the concentration distribution at sedimentation
equilibrium of GroEL
in neutral buffer can be described
by a single homogeneous species of molecular weight 54,500, which shows
a slight tendency to associate into higher order aggregates above
approximately 4 mg ml
(
1.0 A
), possibly due to an increase in exposed
hydrophobic surface. This monomeric molecular weight is in reasonable
agreement with that expected from the nucleotide sequence (29) and confirms the previous results of Horovitz et al.(14) who first reported the effect of this mutation on the
quaternary structure of GroEL. Since a chemically denatured form of
wild-type GroEL requires ATP to promote its
reassembly(12, 35) , additional sedimentation
equilibrium experiments were performed in the presence of ATP
(concentration monitored at 295 nm). These experiments showed no effect
of the nucleotide to promote assembly of monomeric GroEL
to a higher order oligomer (data not shown).
Figure 1:
Sedimentation equilibrium of
GroEL. Sedimentation equilibrium was performed in 50
mM Tris-Cl, pH 7.5, in the presence of 0.1 mM EDTA
and 0.1 mM DTT. A, lowerpanel,
representative trace of absorbance at 280 nm versus radial
distance in centimeters at 13,000 rpm. B, upperpanel, the residuals (A
- A
) for a single species of
molecular weight 54,500 (55,550 - 53,450) that shows a slight
tendency to aggregate above about 4 mg
ml
.
Because
GroEL is monomeric, even in the presence of nucleotides,
it was of interest to analyze whether the mutant possessed similar
functional attributes to the tetradecameric wild-type chaperonin,
especially since monomers prepared either by proteolysis or by
renaturation of chemically denatured oligomers have been reported to
possess chaperone activity(19) . Furthermore, the far UV
circular dichroism spectrum for GroEL
shows some
similarity to wild-type GroEL, suggesting that the monomer has a folded
conformation, in spite of its inability to assemble (see below). Thus,
we first measured the ATPase activity of the monomer relative to that
for wild-type GroEL. Wild-type GroEL displayed a turnover number of
4.5-5.7 min
in ATPase assays, similar to
previous estimates(11) , whereas the mutant showed no ability
to hydrolyze ATP above background. Thus, monomeric GroEL
is an inactive ATPase.
Figure 2:
The effect of GroEL in the
absence and presence of MgATP on the fluorescence of bis-ANS. The
fluorescence spectrum of bis-ANS (10 µM) was obtained at
25 °C in 0.1 M Tris-Cl, pH 7.5, containing 10 mM KCl, 10 mM MgCl
, 0.1 mM DTT, and 0.1
mM EDTA. The upper spectrum is bis-ANS in the presence of 2
µM GroEL
, the middle spectrum is bis-ANS in
the presence of 2 µM GroEL
containing 7.5
mM MgATP, and the lower spectrum is bis-ANS fluorescence in
the absence of protein.
A titration of bis-ANS was performed with
GroEL to assess the average affinity of the fluorophore
for the protein. The fluorescence increase of bis-ANS measured at 490
nm was saturable and displayed a hyperbolic dependence on the mutant
protein concentration (Fig. 3). Assuming simple binding, the
average dissociation constant for protein-fluorophore binding was
determined to be 1.6 ± 0.2 µM. These data, and
results from a titration of the K3E variant with bis-ANS, yielded an
estimate for a lower limit of about 4 mol of bis-ANS bound per mol of
monomer. The strong affinity of bis-ANS for GroEL
, and
the large decrease in bis-ANS fluorescence promoted by ATP binding,
enabled precise binding isotherms for nucleotides to be obtained for
the monomeric mutant.
Figure 3:
Saturable binding of bis-ANS to
GroEL. GroEL
(0.2-30 µM)
was added to bis-ANS (0.5 µM) in 0.1 M Tris-Cl,
pH 7.5, containing 0.1 mM DTT, 0.1 mM EDTA, 10 mM KCl, and 10 mM MgCl
at 25 °C. The
excitation wavelength was 382 nm, and the change in bis-ANS emission
was measured at 490 nm after equilibration. The theoretical curve
corresponds to simple, hyperbolic binding with a K
of 1.6 µM.
Figure 4:
Effect of ATP on the
GroEL-promoted increase in bis-ANS fluorescence.
GroEL
(20 µM) was added to bis-ANS (0.5
µM) and equilibrated for 25 min at 25 °C in 0.1 M Tris-Cl, pH 7.5, containing 0.1 mM DTT and 0.1 mM EDTA. The decrease in bis-ANS fluorescence at 490 nm was measured
after the addition of MgATP (0.01-2 mM) followed by a
15-min equilibration and excitation at 382 nm. The theoretical curve
corresponds to simple, hyperbolic binding of ATP to GroEL
with a K
of 82
µM.
Circular dichroism spectroscopy was also used as a probe for
nucleotide binding to GroEL for two reasons. First, it
was of interest to compare the spectrum for the monomeric mutant with
wild-type GroEL to assess the extent to which the mutation disrupted
secondary structure. Secondly, it was of interest to correlate
nucleotide binding with potential conformational changes in GroEL. As
can be seen in Fig. 5, the far UV spectrum for GroEL
resembles that seen for the wild-type chaperonin. The spectrum is
characteristic of highly helical proteins (38) and is
consistent with the secondary structural composition of the chaperonin
from x-ray crystallography(3) . This similarity suggests that
the mutant is in a significantly folded conformation, although it is
not identical with the wild-type. In addition, there was a significant
effect of ATP on the conformation of monomeric GroEL
as
can be seen in Fig. 5. The spectroscopic changes in the region
of the signature bands for helical structure at 191, 208, and 222 nm
are enhanced significantly, suggesting a modest increase in the extent
of helical structure in the monomer upon binding nucleotide. These
changes were in sharp contrast to the effects of ATP on wild-type
GroEL, which displays almost no spectroscopic changes upon nucleotide
binding.
Figure 5:
Effect of ATP on the circular dichroism of
wild-type GroEL and GroEL. Mean residue ellipticity versus wavelength was measured at 25 °C in 0.1 M Tris-Cl, pH 7.5, containing 10 mM MgCl
, 1.0
mM KCl, 0.1 mM EDTA, and 0.1 mM DTT. There
is some similarity in the spectrum obtained for GroEL
(A, thinline) with that seen for
wild-type GroEL (B, thinline). A
significant change in the spectrum of GroEL
(34.9
µM) was seen upon the addition of 0.5 mM ATP (A, thickline) relative for wild-type GroEL
(0.75 µM) in the presence of ATP (B, thickline).
Figure 6:
The effect of wild-type and GroEL on chemically-denatured enolase and rhodanese refolding. A, the reconstitution of enolase activity was measured at 25
°C in refolding buffer consisting of 50 mM Tris-Cl, pH
7.8, containing 10 mM MgCl
, 20 mM KCl, 2
mM DTT. Denaturation of enolase was achieved by dilution to a
final concentration of 17.1 µM dimer (80 units/mg) in 4.0 M guanidinium chloride for 1 h. Reconstitution was initiated
by rapid dilution into refolding buffer to give a final concentration
of 0.257 µM dimer either in presence of 0.407 µM wild-type GroEL (squares) or 75.3 µM
GroEL
(circles). Activity of 40-µl aliquots
was assayed at the indicated time after dilution into 1 ml of assay
buffer and compared with the effect of 2 mM ATP added at the
eighth minute (filledsymbols) and with a control in
the absence of the chaperonins (filledtriangles). B, rhodanese was denatured in 8 M urea for 2 h at a
concentration of 36.4 µM. Refolding was initiated at 37
°C in the absence or presence of various chaperone components by
dilution to 364 nM in buffer containing 50 mM Tris-Cl, pH 7.5, 50 mM KCl, 10 mM MgCl
, 10 mM DTT, 0.5 mM EDTA, and 50
mM Na
S
O
. At various times
following refolding, rhodanese activity was measured at a concentration
of 14.6 nM by diluting 40 µl of the refolding mixture into
1 ml of assay buffer. Chaperone-assisted refolding was performed either
in the presence of 0.75 µM wild-type GroEL (openbars) or 7.3 µM GroEL
(filledbars). Companion experiments of
rhodanese refolding were performed (from left to right) in the absence (lightlyhatchedbar) or presence (heavilyhatchedbar) of 7.2 µM bovine serum albumin, with no
additions but chaperones, or with chaperones in the presence of 5
mM ATP, with ATP and 1.1 µM GroES, with ATP,
GroES, and 7.2 µM casein and, finally, with ATP, GroES,
and 7.2 µM subtilisin BPN`
PJ9.
The sedimentation, the enzyme kinetics, and the spectroscopic
results presented here indicate that GroEL exists in
solution as a folded monomer that, although inactive as an ATPase,
binds nucleotides with an affinity that is comparable with wild-type.
The limits of the sedimentation analysis (Fig. 1) suggest that
whereas the mutant is predominantly monomeric in the absence or
presence of nucleotides, confirming the observations of Horovitz et
al.(14) , and about 4-8 chains aggregate at high
concentration, there is no evidence of a stable tetradecameric
structure. However, since the monomers appear to be significantly
folded from circular dichroism spectroscopy (Fig. 4), and the
limited, nonspecific aggregation detected in sedimentation experiments
appears to be reversible, we cannot exclude the possibility that the
monomers weakly assemble into a specific, higher order quaternary
structure at high concentration, and this structure spontaneously
dissociates upon dilution.
The surprising ability to construct
``stable,'' folded monomers of GroEL by a variety of
means(12, 13, 14, 16, 17, 19, 21) is
consistent with the recent crystal structure determination that
indicates only a small fraction of the accessible surface area of the
individual monomers is buried at subunit interfaces(3) . The
exposed surfaces of GroEL were therefore probed with
bis-ANS, a fluorophore that binds to hydrophobic regions in
proteins(36, 37) . Monomeric GroEL
promotes a 150-fold increase in the fluorescence of bis-ANS (Fig. 2). This is consistent with the ``folded''
character of the mutant since there is no effect on bis-ANS
fluorescence by the addition of wild-type GroEL that has been unfolded
in guanidinium chloride(35) , as well as with the binding of
bis-ANS to urea-dissociated, assembly-competent monomers(21) .
About 4 mol of bis-ANS bind tightly to the GroEL
monomer,
with a dissociation constant of approximately 1.6 µM (Fig. 3). Although this binding constant is comparable with the
value of 1.2 µM determined for the two to three molecules
of bis-ANS bound to the wild-type GroEL tetradecamer under similar
conditions(23) , the stoichiometry of dye binding to
GroEL
is higher. This in part reflects the increase in
exposed hydrophobic surface in the mutant monomer relative to the
wild-type, but it is somewhat larger than the value seen for GroEL
monomers generated in the presence of 2.5 M urea (21) , suggesting a conformational difference between
assembly-competent monomers and the K3E mutational variant.
Previous
studies of the effect of ligands on the interaction energy between
subunits in GroEL indicate that nucleotide binding in the presence of
2.5-3.5 M urea facilitates the dissociation of oligomers
to monomers(15, 21) . Since ligand binding and subunit
assembly are coupled in allosteric systems(40) , this suggests
that nucleotides should bind more strongly to monomers than the
assembled tetradecamer. It was therefore of interest to compare the
nucleotide binding affinity to the GroEL monomer with the
average affinity estimated from cooperative ATP hydrolysis by wild-type
GroEL. Assuming that the sigmoidal dependence of the initial velocity
of nucleotide hydrolysis by GroEL is directly proportional to the
fractional saturation, the average affinity for nucleotides can be
estimated from the K
values obtained under
conditions similar to the binding experiments. Steady-state kinetics of
ATP hydrolysis by GroEL under similar conditions to the nucleotide
binding experiments performed on the monomeric mutant yields a K
for ATP of about 20 µM,
consistent with previous estimates(11) . The value for the
dissociation constant for ATP binding to the monomer is 82
µM, about 4-fold weaker. This difference suggests that ATP
strengthens the interaction between subunits in GroEL since it binds
more tightly to the oligomer relative to the monomer (40) . A
similar difference was seen for the effect of ADP using binding data
from calorimetry experiments with wild-type GroEL. (
)
The
difference seen here for the predicted ligand binding affinity to
monomeric GroEL may reflect merely the difference in the
reference conditions employed in these investigations or a difference
between the monomeric mutant and assembly-competent monomers generated
by dissociation with urea. Several functional properties of GroEL are
quite sensitive to mutational alterations in the intermediate,
``allosteric'' domain in the subunit(3, 5) ,
and the integrity of this domain, or of the equatorial domain that
provides the nucleotide binding site, may be altered in the presence of
denaturants(17, 35) . Alternatively, the binding of
adenine nucleotides to the mutant might be affected by the
nonconservative substitution of lysine 3 to glutamate. Since the first
five residues were disordered in the recent crystal structure of
GroEL(3) , it is probable that the mutation disrupts the
quaternary structure indirectly, through conformational changes.
Although the monomeric mutant showed considerable secondary structure
as evidenced by circular dichroism spectroscopy, similar to previous
observations on chemically prepared
monomers(12, 13, 35) , there are detectable
differences in the spectrum relative to wild-type GroEL. Moreover, the
large changes in the far UV CD spectrum (Fig. 5) suggest that
ATP binding promotes a significant increase in the amount of structure
in the mutant monomer, which was not seen for the wild-type oligomer.
Thus, the conformational changes promoted by the K3E mutation may lead
not only to a disruption of the wild-type tetradecameric quaternary
structure, but may also lead to a decrease in ATP binding affinity
relative to that expected for putative native monomers.
The
inability of GroEL to refold either yeast enolase or
mitochondrial rhodanese argues that it is inactive as a molecular
chaperone. Additionally, in contrast to the effect of either casein or
subtilisin BPN` PJ9 on reducing the yield of refolded rhodanese in the
presence of wild-type GroEL, there is no effect by these competitors
seen on the small yield of spontaneously refolded rhodanese in the
presence of GroEL
. These results and the observation that
GroEL
failed to bind casein as detected by gel filtration
indicate that the affinity of monomeric GroEL
for
nonnative chains is significantly less that of wild-type GroEL. These
conclusions, however, are in contrast with recent reports of an
intrinsic, but modified chaperone-like activity of GroEL monomers
constructed by chemical denaturation and proteolysis of the
tetradecamer(16, 19) . A small level of wild-type
contamination (e.g. only 1%) would be sufficient to explain
the chaperone activity seen at the high monomer concentrations that
were required for proteolytically-generated monomers to facilitate
refolding(19) . On the other hand, it is possible that monomers
generated by proteolysis may show residual ability to assemble, as do
urea-denatured monomers(17, 21) . The results
presented here on the lack of detectable chaperone activity for
monomeric GroEL
are in accordance with mutational studies
of the function of GroEL(5, 20) . Mutants in GroEL,
including the single-amino acid substitutions G119E, G282E, E408D, the
double mutants A377D/V378E and E386A/K390A, and carboxyl-terminal
deletions of 28 or more residues do not form 20 S particles, indicating
that they fail to assemble into stable tetradecameric quaternary
structures. Furthermore, these assembly-defective mutants either failed
to rescue the conditionally GroEL-impaired E. coli strain LG6,
which provides a sensitive in vivo assay for the chaperone
function of GroEL(41) , or were nonviable. These observations
do not rule out the role of intermediate quaternary structures of GroEL
for its molecular chaperone activity(17) , particularly since a
functional mitochondrial Hsp60 has been shown to possess a heptameric
quaternary structure(42) . However, when taken together, these
observations suggest that the capacity of GroEL to function as a
molecular chaperone is strongly linked to its ability to assemble into
an oligomeric form and that assembly defective monomeric variants of
the enzyme-like GroEL
are insufficient to actively
facilitate the refolding of nonnative substrate proteins.