(Received for publication, June 20, 1995; and in revised form, August 22, 1995)
From the
The urea-induced dissociation and subsequent conformational
transitions of the nucleotide-bound form of GroEL were studied by light
scattering, 4,4`-bis(1-anilino-8-naphthalenesulfonic acid) binding, and
intrinsic tyrosine fluorescence. Magnesium ion alone (10 mM)
stabilizes GroEL and leads to coordination of the structural
transitions monitored by the different parameters. The midpoint of the
light-scattering transition that monitored dissociation of the 14-mer
with bound magnesium was raised to 3 M, which is
considerably higher than the ligand-free form of the protein, which
exhibits a transition with a midpoint at
2 M urea.
Binding of ADP results in destabilization of the GroEL oligomeric
structure, and complete dissociation of the 14-mer in the presence of 5
mM ADP occurs at about 2 M urea with the midpoint of
the transition at
1 M urea. The same destabilization by
ADP and stabilization by Mg
were seen when the
conformation was followed by the intrinsic fluorescence. Complexation
with the nonhydrolyzable ATP analog, 5`-adenylimidodiphosphate gave an
apparent stability of the quaternary structure that was between that
observed with Mg
and that with ADP. The ADP-bound
form of the protein demonstrated increased hydrophobic exposure at
lower urea concentrations than the uncomplexed GroEL. In addition, the
GroEL-ADP complex is more accessible for proteolytic digestion by
chymotrypsin than the uncomplexed protein, consistent with a more open,
flexible form of the protein. The implication of the conformational
changes to the mechanism of the GroEL function is discussed.
Molecular chaperones are proteins that can assist in the folding of other proteins (Hartl et al., 1994). One well studied example is GroEL that is found in Escherichia coli and that is homologous to Hsp60 found in the mitochondrial matrix. GroEL is a large, multisubunit, oligomeric protein consisting of two stacked rings. Each 7-fold rotationally symmetric ring contains seven identical 60-kDa subunits. Each subunit is folded into three domains: 1) an equatorial domain holding the two rings together, 2) an apical domain forming the ends of the cylinder, and 3) a small intermediate domain that connects the equatorial and apical domains (Braig et al., 1994).
GroEL participates in protein folding by first forming a
tight, noncovalent complex with the partially folded target protein,
which is thereby prevented from misfolding or precipitating, two
activities that commonly compete with successful folding. During its
function, GroEL requires the presence of adenine nucleotides and the
cations, K and Mg
. Release of a
bound target protein is accompanied by GroEL-mediated ATP hydrolysis
(Hartl et al., 1994), and some proteins require a second
protein, the co-chaperonin GroES, which is a seven-subunit
homooligomeric protein. For example, the most efficient folding of
proteins such as Rubisco (Goloubinoff et al., 1989), rhodanese
(Mendoza et al., 1991), 6-hydroxy-D-nicotine oxidase
(Brandsch et al., 1992), and ornithine transcarbamylase (Zheng et al., 1993) require the complete chaperonin system,
including GroES and ATP. On the other hand, ornithine transcarbamylase
monomers that cannot assemble can be released from the GroEL-ornithine
transcarbamylase complex by the sole addition of ATP or a
nonhydrolyzable ATP analog (Zheng et al., 1993). Refolding of
some other target proteins from their GroEL-polypeptide complexes can
be induced just by addition of nucleotides. Proteins of this type
include dihydrofolate reductase (Viitanen et al., 1991),
tryptophanase (Mizobata et al., 1992), and the Fab fragment of
a monoclonal antibody (Schmidt et al., 1992). GroEL-protein
complexes of various proteins have been shown to be capable of
dissociation just by the addition of ATP, ADP, ATP
S, (
)or AMP-PNP. In some more complicated cases, the addition
of nucleotide can initiate renaturation of a protein, but the rate of
release is increased in the presence of GroES (Fisher, 1992, 1994;
Höll-Neugebauer et al., 1991).
It has
been suggested that during the GroEL-GroES refolding cycle the GroEL
molecule undergoes several conformational changes (Todd et
al., 1993, 1994; Staniforth et al., 1994a). For example,
the affinity of GroEL for the unfolded polypeptide is decreased upon
binding of GroES and MgATP (Todd et al., 1994; Staniforth et al., 1994b). The binding of ATP leads to conformational
changes in the GroEL-ATP complex such that the ATP initially forms a
weak collision complex with GroEL (K = 4 mM), which isomerizes to a strongly
binding state at a rate of 180 s
(Jackson et
al., 1993). The GroEL conformation formed in the presence of ATP
was proposed to be distinct from the conformation formed in the
presence of ADP (Mizobata et al., 1992). Other investigators
proposed the existence of two interconvertable forms of GroEL, one,
stabilized by MgATP, which associates weakly with unfolded polypeptide,
and a second destabilized by MgATP, which associates strongly with the
target protein (Badcoe et al., 1991).
In the present work,
we have directly monitored conformational consequences of the binding
of Mg and the Mg
complexes of ADP
and AMP-PNP. We show that the stability of the protein is greatly
decreased in the GroEL-ADP complex, and it is significantly less
affected upon binding of AMP-PNP. In contrast, the addition of
Mg
stabilizes the GroEL oligomeric structure.
Possible implications of these phenomena to the mechanism of GroEL
function are discussed.
The data describing
the structural transitions in GroEL were analyzed using a nonlinear
least squares fit to the equation described by Pace (1990). Values were
extracted for the free energy, G, and the urea
concentration at the midpoint of the transition, U
. Computation was done using the PS-Plot
version 1.1 (PolySoft Ltd.).
G
The dissociation of the 14-mer, monitored by the decrease in the intensity of the scattered light, is shown for GroEL as a function of urea concentration in Fig. 1(open circles). The protein was incubated in the standard Buffer A (see ``Materials and Methods''), containing 50 mM Tris, pH 7.8. No species with intermediate degrees of oligomerization were detected in the present study. Thus, the GroEL dissociation can be well described as a two-state transition. The midpoint of the transition monitored by the light-scattering assay was estimated to be at 2.06 M urea, and dissociation was complete at 2.6 M urea in the standard Buffer A (see ``Materials and Methods''). These results are consistent with previous reports that, under similar conditions, the dissociation to monomers is complete at 2.5-2.6 M (Mendoza et al., 1994).
Figure 1: The dissociation transition of GroEL monitored by light scattering. Separate samples were prepared with 380 µg/ml of GroEL in the corresponding standard buffer (see ``Materials and Methods''). Samples were preincubated for 90-120 min at the indicated concentration of urea and then measured at 323 nm. Signals were corrected for the corresponding blank values. The lines correspond to 1) GroEL-Mg complex in standard Buffer B (see ``Materials and Methods'') (closed circles), 2) free GroEL in standard Buffer A (see ``Materials and Methods'') (open circles), 3) GroEL-ADP complex in standard Buffer B (see ``Materials and Methods'') (open squares). The lines are plotted according to nonlinear least squares fit to the data as described under ``Materials and Methods.'' The fit for the GroEL-Mg complex uses two transitions, and the major one was used for calculation and comparison. All other transitions were fit to single transitions.
Figure 2:
Sedimentation velocity analysis
presented as the integral distribution of s values for GroEL. The y axis measures the fraction of the material with s
value less than or equal
to the value given on the abscissa. The samples shown are
GroEL (5.1 µM protomer) in buffer containing 50 mM Tris and 10 mM MgCl
, pH 7.8, that was
preincubated for 120 min at 25 °C in the presence of 2.5 M urea (solid squares), 3 M urea (open
triangles), or 3.6 M urea (open
circles).
Fig. 3demonstrates the ability of
Mg to reassemble monomeric GroEL into 14-mers. In
this case, all samples are in 2.5 M urea and, in the absence
of Mg
, the GroEL is mainly monomeric. The increase of
the light-scattering signal, upon addition of MgCl
(Fig. 3), indicates an increase in the average molecular
weight of the protein. As noted above, the sample at 10 mM Mg
and 2.5 M of the denaturant is
almost entirely 14-mer as determined by ultracentrifugation. These
results are consistent with previous studies showing that the stability
of GroEL, analyzed by gel electrophoresis, increases upon addition of
divalent cations such as Mg
, Mn
,
Ca
, or Zn
(Azem et al.,
1994). The present results extend those previously reported, since the
nondenaturing gel electrophoresis used to detect intact oligomers, as
normally performed, removes denaturant, and, therefore, it detects only
irreversible dissociation (Mendoza, et al., 1995).
Figure 3:
Changes in light scattering of GroEL as a
function of MgCl concentration. Aliquots of MgCl
were added to a sample of 280 µg/ml GroEL in Buffer B that
was 2.5 M in urea (see ``Materials and Methods'').
Light scattering was monitored at 323 nm. Signals were corrected for
the corresponding blank values. The line shown is to guide the
eye.
Several distinct
species were detected for the GroEL-MgADP complex at 0.8 and 0.896 M urea by native polyacrylamide gel electrophoresis, where the
gel contained MgCl (10 mM), ADP (10 mM),
and the corresponding amount of urea (data not shown). This can reflect
the fact that 7-mers and other forms of GroEL, produced as a result of
the protein dissociation, have increased relative stability in the
presence of MgADP. We cannot exclude the possibility that GroEL
molecules with different mobilities could reflect different numbers of
bound ADP molecules.
Figure 4: The urea concentration dependence of the tyrosine-intrinsic fluorescence of GroEL. Separate samples for each point were made with 1.0-1.7 µM GroEL in the corresponding standard buffer (see ``Materials and Methods''). Samples were incubated for 90-120 min. prior to measurements. Fluorescence was excited at 280 nm, and it was detected at 300-340 nm. The lines correspond to 1) GroEL-Mg complex in Buffer B (closed squares), and 2) GroEL-ADP complex in Buffer B with 5 mM ADP (open squares). The lines are plotted according to nonlinear least square fits to the data as described under ``Materials and Methods.''
Figure 6: Fluorescence intensity of bis-ANS bound to GroEL as a function of the urea concentration. Individual samples at the indicated urea concentrations were prepared as under ``Materials and Methods'' with 1 µM GroEL and 10 µM bis-ANS. Curves represent 1) GroEL in Buffer B containing 10 mM ADP (open squares), and 2) GroEL in Buffer B (closed squares) without ADP. Fluorescence was excited at 399 and detected at 500 nm.
Fig. 5shows the results of an experiment in which GroEL was
treated with chymotrypsin (1%, w/w) in the absence of urea at 37 °C
in the absence (lanes 4 and 5) or the presence of 10
mM ADP (lanes 2 and 3). Proteolysis was
stopped after either 60 (lanes 2 and 4) or 120 (lanes 3 and 5) min. The appearance of a distinct
26-kDa band in the case of the sample that contained nucleotide is
consistent with the idea that the protein is more open in the GroEL-ADP
complex (Fig. 5, arrow). Similar species have been
observed before (Horowitz et al., 1995; Seale et al.,
1995) for the GroEL digest, and it was shown that this band represents
the N-terminal portion of the protein (approximately up to 250 amino
acids) and contains parts of the equatorial and apical domains.
Figure 5: Chymotrypsin digest of GroEL in the presence of ADP. Protein (250 µg/ml), dissolved in Buffer B was subjected to the proteolysis by chymotrypsin (1%, w/w) at 37 °C for 60 min (lanes 2 and 4) and 120 min (lanes 3 and 5). Samples contained either no ADP (lanes 4 and 5) or 10 mM ADP (lanes 2 and 3). Lane 1 shows undigested GroEL as a control. The arrow marks a band at 26 kDa. The reaction was stopped by adding phenylmethylsulfonyl fluoride to 3 mM and analyzed by SDS-polyacrylamide gel electrophoresis. The gels were stained by Coomassie R-250.
The profile shown here for GroEL in the absence of ADP (Fig. 6, closed squares) demonstrates a sharp increase in the bis-ANS fluorescence between 2 and 3 M urea. This considerable increase in the bis-ANS fluorescence demonstrates that a large amount of hydrophobic surface is hidden in the unperturbed conformation of GroEL. Addition of ADP to the GroEL leads to a large shift in the bis-ANS fluorescence profile to the lower concentrations of urea (Fig. 6, open squares). The high level of bis-ANS fluorescence at about 1.5 M urea for protein that is mainly monomeric (Fig. 1, open squares) supports the conclusion that the oligomeric form of GroEL is less stable in the presence of ADP.
Based on these transitions, the
values of free energy of stabilization of GroEL were derived from the
parameters of the nonlinear, least squares fitting procedure described
under ``Materials and Methods'' (Table 1). The analysis
shows that the structure of GroEL monitored by intrinsic fluorescence
is slightly stabilized by binding of Mg (
G = -5.10 kcal/mol), while the
quaternary structure monitored by light scattering is stabilized to a
greater extent (
G = - 11.99 kcal/mol).
Binding ADP in the presence of Mg
produces a large
destabilization (
G = -14.12 kcal/mol by
intrinsic fluorescence) of the quaternary structure. Binding of AMP-PNP
under these conditions produces a smaller change (
G =
-10.5 kcal/mol).
Two features of GroEL that have been suggested to be important for binding target proteins are 1) coordination of the multiple sites in the oligomer, and 2) conformational changes that could lead to exposure of interactive surfaces. For example, studies with fluorescent probes suggested that conformational changes in GroEL are required for extensive hydrophobic exposure, so that important functional aspects of chaperonin-assisted refolding reside in the ease with which ligand interactions can trigger conformational changes (Horowitz et al., 1995). Previous studies have demonstrated that relevant conformational changes are related to changes in subunit interactions that can be monitored by the facility with which chaotropes such as urea can perturb the quaternary structure of GroEL and expose hydrophobic surfaces (Horowitz et al., 1995; Gibbons and Horowitz, 1995).
The present work has demonstrated major
changes in the stability of GroEL when it binds functionally relevant
ligands such as Mg or the Mg
complex of ADP at concentrations common to in vivo conditions (Diamant, et al., 1995). In general,
Mg
increased the stability of GroEL to produce a
state in which conformational changes are correlated. Nucleotide
binding destabilized this state, with ADP giving the greatest change.
These results are consistent with previous observations that nucleotide
binding can influence cooperativity among subunits (Diamant et
al., 1995). The present results provide further support since they
show that ligand binding can couple conformational changes in the
monomer to quaternary structure and, therefore, increase cooperativity
involved in binding folding intermediates.
In the absence of added ligands, the structural changes in GroEL are uncorrelated. The light-scattering transition occurred at lower concentrations of urea than the transition monitored by the intrinsic fluorescence. This is in keeping with the previous results showing that monomers could be formed at lower concentrations of urea than those causing denaturation of the protein (Mendoza et al., 1994). The transition monitored by the bis-ANS fluorescence increase starts before significant changes in the intrinsic fluorescence, and the maximum of the hydrophobic exposure occurs at the end of the scattering transition and before the end of the changes in the intrinsic fluorescence. In short, the transitions followed by the three parameters are not coincident.
There are two
major effects when Mg is added to solutions of GroEL:
1) the protein becomes more stable relative to the unliganded state;
and 2) the transitions become closely correlated, in that they occur
over the same range of urea concentrations. The most obvious change is
that the light-scattering transition is moved to higher urea
concentrations. Thus, the Mg
binding stabilizes the
quaternary structure of the protein (Table 1). These results are
compatible with previous studies using chemical cross-linking of GroEL,
which showed that Mg
increased the interactions among
the monomers of GroEL, particularly the contacts between monomers
within the heptameric rings (Azem et al., 1994).
The most
surprising result is that the quaternary structure of GroEL is very
markedly destabilized by MgADP. The transitions become closer to being
coincident, and it is much easier to expose hydrophobic surfaces in the
presence of MgADP. The G was 14.12 kcal/mol relative
to the Mg
bound state for the transition monitored by
intrinsic fluorescence.
When AMP-PNP was used, the structure of
GroEL was destabilized relative to the magnesium-bound state as
assessed by light scattering, although to a smaller extent than with
MgADP. For the transition monitored by intrinsic
fluorescence, the
G was 10.5 kcal/mol relative to
the Mg
bound state. A comparison of these results
with those reported previously in which ATP appeared to destabilize
GroEL suggests that at least part of the reported effect might have
been due to the spontaneous hydrolysis of ATP (Horowitz et
al., 1995).
An interesting feature of GroEL is that many
diverse proteins can interact with this chaperonin and there are
different requirements for their release (Lorimer et al.,
1993). ATP binding is sufficient for release of several target
proteins, so there is no obligatory requirement for ATP hydrolysis or
for GroES. Thus, nonhydrolyzable analogs of ATP can release glutamine
synthase (Fisher, 1994), ornithine transcarbamylase (Zheng et
al., 1993), barnase (Gray and Fersht, 1993), lactate dehydrogenase
(Badcoe et al., 1991), and dehydrofolate reductase (Viitanen et al., 1991). In a number of cases, target proteins are bound
more tightly in the presence of MgADP. Thus, lactate dehydrogenase and
dehydrofolate reductase remain bound to GroEL in the presence of ADP
under conditions that would lead to release if ATP were used (Badcoe et al., 1991; Staniforth et al., 1994a; Viitanen et al., 1991). Specifically, in the presence of GroES, the
exchange of ADP for ATP at one of the sets of nucleotide binding sites
on GroEL results in considerably tighter binding of lactate
dehydrogenase with bound ADP (K = 34
nM) compared with bound ATP (K
=
440 nM) (Staniforth et al., 1994a). Even GroES
itself, whose binding sites on GroEL overlap the binding sites for
bound polypeptides, binds tightly in the presence of MgADP (Todd et
al., 1994, Schmidt et al., 1994). These results are
reminiscent of nucleotide effects on the binding of proteins to the
chaperonin, Hsp70. In that system, oscillation between a loose binding
ATP state and a tight binding ADP state are involved in the alternate
binding and release of target proteins during the cycle of this
chaperonin (Palleros et al., 1994).
It has been suggested that hydrophobic exposure on unperturbed GroEL is less important than the ease with which hydrophobic surfaces can be induced when the chaperonin interacts with targets or ligands (Horowitz et al., 1995; Gibbons et al., 1995). Therefore, the effects of nucleotides on the ease of exposure of hydrophobic surfaces may have important consequences. If hydrophobic binding involving GroEL were too strong and uncoordinated, two possibilities are suggested: 1) GroEL might aggregate or the 14-mer might bury those surfaces internally so they would not be available for interactions with target polypeptides, and/or 2) Hydrophobic interactions with the target would be so strong that subsequent release would be difficult. Therefore, facile and reversible binding and release of polypeptide chains requires a balance between the exposure and coordination of hydrophobic surfaces on the chaperonin. Thus, nucleotide and ion binding to GroEL can provide a number of functions, and they can 1) shift the energy for exposure of hydrophobic surfaces into a biologically relevant range, 2) help coordinate the changes among the subunits in the 14 mer; and 3) permit structural differences induced by ATP and ADP to participate in the binding and release of peptide segments required for productive folding of proteins.