(Received for publication, September 22, 1995; and in revised form, November 2, 1995)
From the
Although the role of nucleotides in the catalytic cycle of the
GroESL chaperonin system has been extensively studied, the molecular
effects of nucleotides in modulating exposure of sites on GroEL has not
been thoroughly investigated. We report here that nucleotides (ATP,
ADP, or adenosine 5`-(,
-imino)triphosphate) in the presence
of Mg
make the oligomer selectively sensitive to
trypsin proteolysis in a fashion suggesting conformational changes in
the monomers of one heptameric ring. The site of proteolysis in the
monomer that is exposed upon nucleotide binding by the oligomer is in
the apical domain (Arg-268). Further, complexes of GroEL with GroES or
rhodanese display the same sensitivity to proteolysis, unlike the
GroEL-GroES-rhodanese complex, which is protected from proteolysis. The
influence of various cations on trypsin proteolysis is investigated to
elucidate the differential effects that monovalent and divalent cations
have on the oligomeric structure of the chaperonin. These results are
discussed in relation to the molecular basis for the chaperonin
activity.
The molecular chaperones are a class of proteins that have been
shown to facilitate the in vivo folding and transport of
nascent polypeptides(1, 2) . The chaperonins, one
class of molecular chaperones, have been found in prokaryotes,
mitochondria, and chloroplasts(3) . One widely studied
chaperonin from Escherichia coli, GroEL, has been demonstrated
to promote the in vitro refolding and assembly of a variety of
chemically denatured proteins, including
rhodanese(4, 5) , ribulose-bisphosphate
carboxylase/oxygenase (Rubisco)(6, 7) , and glutamine
synthetase(8) . The promiscuity of GroEL in polypeptide
recognition and binding suggests that the hydrophobic interactions
accounting for the complex formation vary from substrate to
substrate(9, 10, 11) . The fact that some
proteins require only K and ATP-Mg (or a
non-hydrolyzable analog) for release of functional enzyme from the
complex, whereas others also require the co-chaperonin GroES, supports
this idea that complexes with some non-native proteins are stronger
than others(12, 13) .
GroEL is a homotetradecamer
(14-mer) of 57.2-kDa subunits, arranged as two stacked heptameric
rings, with a central cavity at each end(14) . The 2.8-Å
x-ray crystal structure reveals that each monomer is organized into an
equatorial, intermediate, and apical domain, with interactions between
equatorial domains exclusively defining the heptamer-heptamer
interface(15) . Monomer-monomer interactions within each ring
are mediated by the equatorial domains and by conserved interactions at
the intermediate-apical domain interface(15, 16) . The
apical domains line the opening to the central cavity at either end of
the oligomer and contain a region(200-263) that has been
implicated by site-directed mutagenesis and photoincorporation of the
hydrophobic probe bis-ANS ()as the site of polypeptide
binding(17, 18) .
Studies of the effects of nucleotides on GroEL have focused on regulation of the ATPase activity of the chaperonin(19, 20) , including the elements of positive and negative cooperativity in nucleotide binding/hydrolysis, and the debate over complex formation(21, 22, 23, 24, 25, 26) . Several studies have shown that two GroES molecules can bind to a single GroEL oligomer in the presence of ATP to form symmetric complexes, while others have demonstrated asymmetric complexes (1:1 GroEL:GroES ratio) to be the functional, physiological unit. The few studies that have evaluated the conformational changes in GroEL due to ATP binding or hydrolysis have demonstrated quaternary structural changes consisting of monomer pivoting and apical domain reorientation upon ATP binding(27, 28, 29) . Unfortunately, the results do not provide much information about the exposure of specific sites believed to mediate protein-protein interactions. Finally, nucleotides have also been shown to affect the affinity of GroEL for substrate, with the ADP-Mg complex displaying tight binding and the ATP-Mg complex displaying weak binding(30) .
There have been several reports on ions (monovalent, divalent, and polyvalent) affecting structural changes in GroEL(31, 32, 33) . Monovalent and polyvalent cations are reported to increase the exposure of hydrophobic surfaces on GroEL without disrupting the oligomeric structure(33) . Divalent, but not monovalent, cations stimulate the ATPase activity of GroEL and induce structural changes that allow preferential cross-linking of the heptameric rings(31) . These results suggest that cations of various charges may work at several different levels to produce structural changes in the GroEL oligomer.
In this
report we show that proteolysis by trypsin can be used as a probe for
conformational changes in the tetradecameric structure of GroEL. In a
native state with Mg as the only ligand, GroEL is not
susceptible to proteolysis by trypsin, whereas unliganded GroEL is
rapidly digested, leaving only 10% of intact monomers within 30 min.
Upon addition of nucleotides (ADP, ATP, or the ATP analog AMP-PNP) and
Mg
, exposure of one predominant cleavage site in the
apical domain occurs. When ADP-Mg is liganded to GroEL, producing a
state with high affinity for non-native protein, approximately half of
the monomers become proteolyzed without loss of the oligomeric
structure. Complexes of GroEL-ADP-Mg with GroES or denatured rhodanese
are proteolytically sensitive in a manner similar to the
nucleotide-liganded state. In contrast, a GroES-GroEL-rhodanese complex
formed in the presence of ADP-Mg is significantly protected from
proteolysis (
90% protected). Monovalent and polyvalent cations
also affect trypsin proteolysis of GroEL, protecting approximately half
of the sites from cleavage compared with an unliganded state. These
results suggest that nucleotide binding induces asymmetry in the GroEL
tetradecamer, exposing specific sites in the apical domain of only one
heptameric ring such that they are accessible to other proteins in
solution, either protease or substrate proteins.
Rhodanese was prepared as described
previously (34) and stored at -70 °C as a crystalline
suspension in 1.8 M ammonium sulfate. Rhodanese concentrations
were determined using A = 1.75 (35) and a molecular mass of 33 kDa(36) . The
chaperonin, GroEL, was purified from lysates of E. coli cells
bearing the multicopy plasmid pGroESL(37) . The purification
was by a modified version of published protocols(20) ,
excluding the Mono Q and hydroxyapatite columns. After purification,
GroEL was dialyzed against 50 mM Tris-HCl, pH 7.5, and 0.1
mM dithiothreitol and then made 10% (v/v) in glycerol, rapidly
frozen, and stored at -70 °C. The co-chaperonin, GroES, was
purified from lysates of E. coli cells bearing the multicopy
plasmid pND5, using a previously published protocol(38) . After
purification, GroES was stored at 4 °C in 70% ammonium sulfate.
Prior to use, GroES was centrifuged in a microcentrifuge, and the
pellet was resuspended in 50 mM triethanolamine hydrochloride,
pH 8.0, and dialyzed overnight against the same buffer. The protomer
concentration of GroEL was measured under denaturing conditions, and
GroES was measured under non-denaturing conditions using the
bicinchoninic acid assay (Pierce) according to the procedure
recommended by the manufacturer.
For the experiments involving GroEL-rhodanese protein complexes, rhodanese was unfolded in 8 M urea as described previously (40) and diluted into a solution containing GroEL and all the other components of the buffered solution as indicated in the figure legends. In forming ternary complexes of GroEL-GroES-rhodanese, GroES was added to a solution already containing GroEL, nucleotide, and unfolded rhodanese. In each case, the final solutions were incubated at room temperature for at least 10 min to allow complex formation prior to proteolysis.
Figure 1:
Time course of trypsin proteolysis of a
GroEL-ADP-Mg complex. A, denaturing gel showing the time
course of trypsin digestion. GroEL (10.4 µM monomer) in 50
mM Tris-HCl, pH 7.8, 10 mM ADP, 20 mM
MgCl, and 10 mM 2-mercaptoethanol was digested
with 5% (w/w) trypsin at room temperature. Aliquots were removed at
increasing times, and the reaction was stopped as described under
``Materials and Methods'' prior to electrophoresis of samples
on a 12% denaturing polyacrylamide gel. The leftmost lane contains molecular mass standards, from the top: 97.4 kDa,
phosphorylase b; 66.2 kDa, bovine serum albumin; 42.7 kDa,
ovalbumin; 31 kDa, carbonic anhydrase; 21.5 kDa, soybean trypsin
inhibitor; and 14.4 kDa, hen egg lysozyme. Lanes 1-9 correspond to GroEL standard (untreated) and 0, 1, 3, 6, 9, 12,
15, and 18 min of digestion, respectively. B, semi-log plot
showing percent of undigested monomers remaining at increasing times of
trypsin proteolysis. Data points (
) represent an average of four
separate experiments, all performed as outlined in A. In each
experiment the ``0 min digestion'' or ``untreated GroEL
standard'' was taken as the 100% point for calculating the
undigested monomer percentages. The line is the best fit
exponential curve determined as outlined under ``Materials and
Methods,'' with parameters as stated in the
text.
Fig. 1B is a semi-log plot representing
the percentage of parent band (monomeric GroEL) remaining at various
times during the trypsin digestion of the GroEL-ADP-Mg complex. The
curve was best fit by a single exponential with the following
parameters: k = 0.186 s, A
= 0.423, and A
= 0.516, where A
represents the
fraction of undigested protein remaining at long proteolysis times. The
results indicate that within the first 15 min of proteolysis
approximately half of the monomers in the tetradecamer were cleaved at
Arg-268, while the other half of the monomer population remained
intact. Longer periods of digestion (up to 60 min) did not produce
significantly more than 50% digestion (data not shown).
Fig. 2A shows a similar plot for the proteolysis of
GroEL under different conditions. The upper curve (solid
circles) represents trypsin proteolysis of the GroEL oligomer in
the presence of 20 mM Mg. Very little
digestion occurred (10% at most) without production of a doublet such
as that seen in Fig. 1A. The lower curve (open
squares) demonstrates the significant digestion of the unliganded
GroEL oligomer. These data (open squares) were best fit by a
single exponential with the following parameters: k =
0.067 s
, A
= 0.852, and A
= 0.094. The digestion of unliganded
GroEL also produced the doublet shown in Fig. 1A, but
the percentage of the parent band converted to doublet was less, and
many other weakly staining bands appeared (data not shown). These
observations are consistent with the presence of a broader distribution
of GroEL structures in the absence of ligand. Finally, in the absence
of ligand, higher trypsin concentrations (10-20%) completely
digested the chaperonin, producing fragments that were too small (or
too weakly staining) to be visualized on the 12% denaturing gels used
(data not shown).
Figure 2:
Trypsin proteolysis of GroEL with or
without Mg. A, semi-log plot showing the
time course of trypsin digestion of GroEL, represented as percent of
undigested monomers remaining at increasing times of proteolysis. GroEL
(10.4 µM monomer) in 50 mM Tris-HCl, pH 7.8, 10
mM 2-mercaptoethanol, with 20 mM MgCl
(
) or without MgCl
(
) was digested with
5% trypsin (w/w) as in Fig. 1A. Data points represent
averages of either two (+MgCl
,
) or three
(unliganded,
) separate experiments. The data were fit to a
straight line (
) or an exponential curve (
) as described
under ``Materials and Methods.'' B, effect of
increasing Mg
concentration on trypsin digestion of
GroEL. Separate samples of GroEL (10.4 µM monomer) in 50
mM Tris-HCl, pH 7.8, 10 mM 2-mercaptoethanol and
increasing concentrations of MgCl
, as indicated on the x axis, were digested with 5% trypsin (w/w) for 30 min and
then treated as in Fig. 1A. Data points (
)
represent averages of two separate denaturing gels. Since this
phenomenon is more complex than a simple tight binding
ligand(31) , the dashed line is not a fitted line but
is simply drawn to guide the eye.
To investigate the differences in proteolysis
between unliganded and magnesium-liganded GroEL an experiment was
conducted where the degree of proteolysis was measured at fixed times
(15 or 30 min) at increasing concentrations of Mg.
The results, graphed in Fig. 2B, show that an
increasing percentage of the monomers were protected from trypsin
proteolysis as the Mg
concentration was increased
from 0 to 20 mM, with maximum protection afforded by
10-12 mM. This protection from trypsin proteolysis at
increasing Mg
concentrations is strikingly similar to
the effect of increasing Mg
concentrations on the
cross-linking of heptameric rings in GroEL (31) .
Trypsin proteolysis of GroEL in the presence of ATP-Mg or AMP-PNP-Mg produced results similar to those seen with ADP-Mg. Fig. 3is a semi-log plot comparing the results with the three nucleotides, showing that all three produced an oligomeric structure that was sensitive to trypsin proteolysis. It is clear that nucleotide binding alone and not hydrolysis produced the conformational change necessary to expose the clip site in the apical domain. In each of the three cases the same digestion products appeared on denaturing gels, with the bands of the doublet described above representing the predominant species (data not shown).
Figure 3:
Trypsin proteolysis of GroEL in complex
with ATP, ADP, or AMP-PNP. The semi-log plot shows the time course of
trypsin digestion of GroEL (percent undigested monomers remaining versus time of proteolysis) complexed with various
nucleotides. GroEL (10.4 µM monomer) in 50 mM Tris-HCl, pH 7.8, 10 mM 2-mercaptoethanol, 20 mM
MgCl, and 10 mM ADP (
), AMP-PNP (
),
or ATP (
) was digested with 5% trypsin (w/w) as in Fig. 1A. Data points for ADP are the same as Fig. 1B and are simply replotted here for ease of
comparison. Data for each nucleotide were fit to an exponential curve
as described under ``Materials and
Methods.''
Figure 4:
Effect of trypsin proteolysis on the
tetradecameric structure of GroEL. Non-denaturing gel showing the time
course of trypsin digestion of a GroEL-ADP-Mg complex. Trypsin
proteolysis of GroEL (10.4 µM monomer) in 50 mM Tris-HCl, pH 7.8, 10 mM ADP, 20 mM MgCl, and 10 mM 2-mercaptoethanol was
performed as in Fig. 1A, and the samples were
electrophoresed on a non-denaturing gel as described under
``Materials and Methods.'' Lanes 1-7 correspond to: GroEL standard (untreated) and 1, 3, 5, 10, 20, and
30 min of digestion.
There was a question of whether the non-denaturing gels were visualizing tetradecamers that had not fallen apart or tetradecamers that had reassembled during electrophoresis. This question was addressed by running samples treated in the same manner as above on a gel permeation column (TSK-4000), a system that resolves monomers from tetradecamers. The samples of ADP-Mg-liganded GroEL treated with trypsin for 30 min eluted in the same volumes as controls with native, unperturbed GroEL, or ADP-Mg-liganded oligomers that had not been proteolyzed, indicating that the tetradecamers remained intact after proteolysis (data not shown). Two-dimensional polyacrylamide gel electrophoresis was also performed. The bands from non-denaturing gels were excised, dissolved in SDS sample buffer, and electrophoresed on denaturing gels. The results were exactly the same as when samples were run directly on denaturing gels, as shown in Fig. 1A. These results confirmed that the bands visualized on the non-denaturing gels consisted of cut and uncut monomers, which remained associated as oligomers after proteolysis.
Figure 5:
Trypsin proteolysis of GroEL in complex
with GroES and/or rhodanese. The semi-log plot shows the time course of
trypsin digestion of GroEL in a binary complex with either GroES or
rhodanese or in a ternary complex with GroES and rhodanese. GroEL (0.78
µM oligomer) in 50 mM Tris-HCl, pH 7.8, 10 mM ADP, 20 mM MgCl, and 10 mM 2-mercaptoethanol was incubated with GroES (0.78 µM oligomer) (filled circles), unfolded rhodanese (1.96
µM) (open diamonds), or GroES and unfolded
rhodanese (GroEL:GroES:Rhod, 1:2:2) (asterisks) for 10 min at
room temperature to form a complex. The complexes were then treated
with 5% trypsin (w/w) as in Fig. 1A. The data points
represent averages of two (ternary complex, asterisks; and
GroEL-rhodanese complex, open diamonds) or three (GroEL-GroES
complex, filled circles) separate experiments. The lines represent the best fit to a straight line (asterisks) or
exponential curve (filled circles and open diamonds)
as outlined under ``Materials and
Methods.''
Figure 6:
Effect of monovalent or polyvalent cations
on the trypsin proteolysis of GroEL. The semi-log plot shows the
percent of undigested monomers remaining at increasing times of trypsin
proteolysis in the presence of KCl or spermidine. GroEL (10.4
µM monomer) in 50 mM Tris-HCl, pH 7.8, 10 mM 2-mercaptoethanol, and 25 mM KCl () or 5
mM spermidine (
) was digested with 5% trypsin (w/w) as
in Fig. 1A. The lines represent the data fit
with exponential curves as outlined under ``Materials and
Methods.''
An understanding of how various ligands induce conformational
changes in GroEL and the specific sites which become exposed is crucial
to deciphering the molecular mechanism of chaperonin activity. We were
able to follow such conformational changes by using the differential
trypsin proteolysis of GroEL under various conditions. Initially, an
important distinction arises, which should be carefully considered.
Mg alone shifts the conformation of the chaperonin
from a state that is 100% susceptible to proteolysis to one that is
completely protected from trypsin. This is consistent with published
studies showing that 50% of the protein is cross-linked as a heptamer
at Mg
concentrations of 10 mM or
higher(31) . Apparently, conditions that cause association of
the monomers in one heptameric ring (such that cross-linking is
facilitated) result in an oligomeric structure that is fully protected
from trypsin proteolysis. Considering the concentration range of this
effect and the fact that Mg
concentrations in E.
coli may vary between 20 and 40 mM(47) , it is
more reasonable that the magnesium-liganded state of GroEL is the form
that should be thought of as the physiological state (control) rather
than the unliganded form.
Previous reports have suggested that nucleotides bind asymmetrically to the tetradecamer occupying only seven sites, presumably of a single heptameric ring(29, 48) . The results presented here (Fig. 1, A and B) support those earlier studies by showing that 50% of the monomers become proteolytically sensitive upon nucleotide binding to the magnesium-liganded tetradecamer and extend those reports by identifying the specific site of exposure within the monomeric structure (Arg-268). Proteolysis at other sites in this region (203, 237, and 268) has also been demonstrated when GroEL is perturbed by 2.5 M urea(33) . Significantly, this site (Arg-268) is in the region that has been identified by site-directed mutagenesis and bis-ANS incorporation as the putative protein binding region in the apical domain(17, 18) . This suggests that nucleotide binding to the magnesium-liganded tetradecamer exposes the regions of the apical domains of one heptamer to allow for interaction with substrate protein.
The results presented here of the effects of
Mg and ADP-Mg on the structure of GroEL are perfectly
consistent with a recent study evaluating the stability of GroEL to
urea-induced dissociation(49) . The report showed that 10
mM Mg
alone stabilized GroEL against
urea-induced dissociation, as measured by light scattering, bis-ANS
binding, or intrinsic tyrosine fluorescence. The ADP-Mg complexed form
of GroEL was more easily dissociated by urea, and exposure of
hydrophobic surfaces occurred more readily. Finally, the GroEL-ADP-Mg
complex was susceptible to limited chymotrypsin proteolysis at an
apparent cleavage point in the apical domain. Taken all together, it
seems clear that ligands such as Mg
and nucleotides
can coordinate and induce conformational changes in functionally
important regions of the GroEL structure so that interactions with
other proteins can be regulated.
Proteolytic analysis of complexes reveals no significant differences between nucleotide-bound GroEL and GroEL in a binary complex with GroES or rhodanese. These findings suggest that any additional conformational changes in GroEL upon binding protein (either co-chaperonin or non-native substrate) are local, confined to the specific site of interaction, rather than global. By contrast, formation of a ternary complex of GroEL-GroES-rhodanese effectively blocks proteolysis of GroEL, suggesting either that association of the two proteins with the chaperonin occurs at opposite ends of the tetradecamer or that binding of the two to one end of the oligomer results in global conformational changes that prevent proteolysis at the opposite end of the oligomer.
Local flexibility in the chaperonin has been shown to be an important characteristic that is frozen upon binding to non-native proteins, producing a complex which stabilizes the tetradecamer(50, 51) . Although nucleotide binding induces interdomain and quaternary structural rearrangements in GroEL(27, 28) , the local flexibility of the protein is not affected by formation of a GroEL-ATP complex(50) . This suggests that structural regulation in the chaperonin exists at two different levels: 1) exposure of the reactive sites in one heptameric ring by nucleotide binding and/or hydrolysis; and 2) structural rearrangements upon protein-protein interaction that stabilize regions of local flexibility.
The effects of ions on the GroEL structure
also suggest this multilevel basis for structural regulation. Divalent
cations (i.e. Mg and Mn
)
stimulate the GroEL ATPase activity, stimulate rapid cross-linking of
monomers within a heptameric ring by glutaraldehyde, and completely
prevent trypsin proteolysis (31, 32) (Fig. 2A). Monovalent and
polyvalent cations increase the exposure of hydrophobic surfaces that
bind bis-ANS and prevent trypsin proteolysis of the chaperonin by only
50% (31, 33) (Fig. 6). With the exception of
K
, monovalent and polyvalent cations do not affect
ATPase activity. Taken all together, divalent cations appear to mediate
intermonomer and heptamer/heptamer interactions, whereas monovalent and
polyvalent cations appear to affect the local structure of monomers in
a fashion that may possibly be localized to the apical domain.