(Received for publication, November 11, 1994)
From the
The oligomeric form (14-mer) of the chaperonin protein, Cpn60
(GroEL) from Eschericia coli, displays restricted hydrophobic
surfaces and binds tightly one to two molecules of the fluorescent
hydrophobic reporter, 1,1`-bi(4-anilino)naphthalene-5,5`-disulfonic
acid (bisANS). The 14-mer is resistant to proteolysis by chymotrypsin,
and none of the three sulfhydryl groups/monomer react with
6-iodoacetamidofluorescein. When monomers of Cpn60 that are folded and
competent to participate in protein folding are formed by low
concentrations of urea (<2.5 M), the hydrophobic exposure
increases to accommodate approximately 14 molecules of bisANS/14-mer,
the binding affinity for bisANS decreases, and 1 sulfhydryl
group/monomer reacts with 6-iodoacetamidofluorescein. These monomers
display limited proteolysis by chymotrypsin at several points within a
hydrophobic sequence centered around residue 250 to produce a
relatively stable N-terminal fragment (26 kDa) and a partially
overlapping C-terminal fragment (
44 kDa). The exposure of
hydrophobic surfaces is facilitated by ATPMg. Ions increase hydrophobic
exposure more effectively than urea without dissociation of Cpn60. For
example, subdenaturing concentrations of guanidinium chloride (
0.75 M) or the stabilizing salt, guanidinium sulfate, as well as
NaCl or KCl are effective. The trivalent cation, spermidine, induces
maximum exposure at 5 mM. The results suggest that hydrophobic
surfaces can be involved in stabilizing the oligomer and/or in binding
proteins to be folded, and they are consistent with suggestions that
amphiphilic structures, presenting hydrophobic surfaces within a
charged context, would be particularly effective in binding to Cpn60.
Proteins termed molecular chaperones have been implicated, in vivo, in processes ranging from protein folding and processing to organelle import(1, 2) . The most widely studied of these proteins are the chaperonins encoded by the GroESL gene of Escherichia coli: Cpn60, a tetradecamer (14-mer) of 60-kDa subunits, and Cpn10, a heptamer (7-mer) of 10-kDa subunits(3) . Cpn60 appears to be the essential component, and the most widely studied effect in vitro is the ability of Cpn60 to assist the refolding of denatured proteins(4) .
A number of elegant experiments have delineated the overall requirements and the general mechanism by which the chaperonin Cpn60 can assist the refolding of proteins(5) . However, the detailed molecular mechanism is still incompletely understood. One outstanding question asks about the nature of the interactions that bind a passenger protein to Cpn60 that can account for the essential features of the interactions, including: (a) tight binding of partially folded proteins while only interacting weakly with folded proteins; (b) relatively little specificity for the sequence of the particular protein(6) ; (c) the capacity to be modulated by accessory components such as Cpn10 and ATP; and (d) the capacity to bind substantial sized proteins while being stable in solution against self-association.
All models invoke the ability of Cpn60 to stabilize folding intermediates by forming stable binary complexes with partially folded proteins. This has the effect of preventing irreversible aggregation that would compete with folding, and the passenger protein is maintained in a nonnative conformation that is competent to participate in further biological processes, which are precluded for the native protein(7, 8, 9, 10) . In vitro, under normal circumstances, the release of active protein is ATP-dependent, and, in some cases, it is also dependent on the presence of the co-chaperonin Cpn10. The requirements for ATP and/or its hydrolysis, as well as the need for Cpn10, are different for different proteins.
Since many different proteins can be bound by Cpn60(6) , interest has focused on the nature of the interactions that would be general enough to account for this diversity. One suggestion has been that hydrophobic surfaces, formed by the assembly of Cpn60 monomers, can interact with hydrophobic surfaces on loosely folded protein conformers, e.g. molten globule intermediates(7, 10) . Hydrophobic exposure has been suggested to be a general attribute of folding intermediates that are characteristic of many, if not all, proteins (11) . Additionally, it has been suggested that the ability to form amphipathic helices may represent a part of the recognition motif(12) . However, fluorescent probes of hydrophobic exposure indicate that, in the intact tetradecameric Cpn60, the accessible hydrophobic region is small and restricted(10) .
Electron microscopic and crystallographic structures of Cpn60 have been used to speculate about the nature and location of the interactions with partially folded protein, and it has been suggested that at least part of the partially folded proteins bind in the central cavity of the chaperonin oligomer(4, 13, 14) . Recent crystal structures at a resolution of 2.8 Å are compatible with electron micrographs, and they provide additional detailed structural information. The monomers in the isolated Cpn60 14-mer are arranged in two seven-member rings associated in a double doughnut structure with the two rings stacked back to back so that the external faces on both ends of the cylindrical 14-mer are the same. This cylindrical oligomer is approximately 14 nm in diameter and 15 nm in height with a central channel that is approximately 45 Å in diameter in the crystal structure. The monomers appear to be structurally equivalent(4, 14, 15, 16) .
Each
Cpn60 monomer consists of a linear sequence of 547 amino acids that is
folded into three domains in the form reported in the x-ray
structure(14) . The first or equatorial domain, is formed by
residues from both ends of the polypeptide chain (residues 6-133
together with residues 409-523). This domain has a high content
of helix and provides the ATP binding site and all of the
contacts between the rings. Residues at the extreme N and C termini are
disordered and not resolved. The second or intermediate domain is
composed of residues 134-190 plus residues 377-408. The
third or apical domain is comprised of residues 191-376, and the
apical domains collectively form the opening of the central,
cylindrical channel. The apical domain contains segments with the
highest temperature factors in the structure, and most of the
disorder/flexibility of the protein appear to be in segments facing the
channel and forming the top surface of the cylinder. Furthermore,
mutations in this region disrupt polypeptide binding(17) .
It has recently been demonstrated that Cpn60 monomers alone are able
to exert the essential interactions that permit the stabilization of
folding intermediates and the subsequent release of proteins that are
able to fold(18, 19) . The rhodanese-Cpn60 complex
could be dissociated by moderate concentrations of urea to allow
folding of rhodanese to proceed, thereby removing the obligatory
requirement for Cpn10 and ATP that had formerly been considered
requisite for the in vitro folding of rhodanese. Thus,
perturbation of the Cpn60 quaternary structure, alone, was capable of
permitting a complete refolding cycle. In addition, unfolded rhodanese
could induce the reassembly of tetradecameric Cpn60 from monomers, and
the quaternary structure of Cpn60 was stabilized by bound rhodanese.
Subsequent studies demonstrated that monomeric Cpn60, prepared at urea
concentrations 2.5 M urea, were not unfolded, and they
could spontaneously reassociate on urea removal (10) . (
)
In the present study we report that assembly competent monomers, compared with their properties in the tetradecamer, have increased hydrophobic exposure and flexibility. These results are compatible with recent electron micrographs and the crystal structure, and they are consistent with the hypothesis that sterically restricted hydrophobic surfaces are formed by the juxtaposition of monomers in the oligomeric structure(10) . In addition, it is demonstrated that hydrophobic exposure can be modulated by ionic interactions without dissociation of Cpn60, thus demonstrating that the hydrophobic regions that become exposed are not required for stabilizing the Cpn60 tetradecamer.
The chaperonins,
Cpn60 and Cpn10, were purified from lysates of E. coli cells
bearing the multicopy plasmid pGroESL(20) . The purifications
were modified versions of published
protocols(21, 22) . After purification, the
chaperonins were dialyzed against 50 mM Tris-HCl, pH 7.5, and
1 mM dithiothreitol and then made 10% (v/v) in glycerol,
rapidly frozen in liquid nitrogen, and stored at -70 °C. The
protomer concentrations of Cpn10 and Cpn60 were measured at 280 nm with
extinction coefficients of 3,440 M cm
for Cpn10 (23) and 12,200 M
cm
for Cpn60 as
reported by Fisher (24) and assuming molecular masses of 10 and
60 kDa, respectively. Protein concentrations of Cpn60 stock solutions
were also measured using the bicinchoninic acid protein assay (Pierce)
using the procedure recommended by the manufacturer. Protein
concentrations measured in this way were within 10% of those determined
using A
. Stoichiometries for bisANS binding were
based on measurements of A
.
Figure 1: bisANS binding to Cpn60 at 0 and 2.5 M Urea. Solutions containing Cpn60 (1 uM protomer) in 50 mM Tris-HCl, pH 7.8, were titrated with bisANS to the concentrations shown. Fluorescence emission spectra with excitation at 394 nm were recorded after the addition of bisANS, and the maximum fluorescence intensities were measured. The curves shown were generated by a nonlinear least squares procedure as described under ``Materials and Methods.'' The curve through the 0 M urea data (solidcircles) corresponds to a dissociation constant of 0.6 uM, and a maximum number of bisANS molecules/14-mer = 1.4. The data through the 2.5 M urea (solidsquares) has been fit with a curve corresponding to a dissociation constant for bisANS = 55.4 uM, and a maximum number of bound bisANS/14-mer = 13.9.
Figure 2: a, Fluorescence intensity of bisANS bound to Cpn60 as a function of the urea concentration. Individual samples at each of the indicated urea concentrations were prepared as in Fig. 1with a concentration of bisANS = 10 uM. Fluorescence spectra were recorded, and the intensities at the maxima are plotted as a function of the urea concentration. Closedtriangles, no ATPMg; closedcircles, 5 mM ATPMg. The excitation wavelength was at 394 nm. b, wavelength maxima of the bisANS fluorescence in the presence of Cpn60 as a function of the urea concentration. The wavelength of the maximum fluorescence of the bisANS in the presence of Cpn60 was determined for each urea concentration. The experimental conditions were the same as for a.
Fig. 2b shows the response of the wavelength maximum of the fluorescence of bisANS to changes in the urea concentration. There is a shift toward shorter wavelengths as the urea concentration increases in the region (2.0-2.75 M), leading to dissociation of the 14-mer to monomers that can reassemble. It corresponds to the increase in intensity in Fig. 2a. This change is in a direction that would be associated with an increased hydrophobicity of the bisANS binding site. Beyond 2.75 M urea, the wavelength maximum shifts toward longer wavelengths to reach approximately 525 nm at 4 M urea. This latter effect would be that expected for a decreasing hydrophobic character of the binding site. Therefore, not only are the number of binding sites changing in this region, but the hydrophobic character provided for the bisANS is changing. The shift to slightly longer wavelengths at low urea concentrations (0-0.5 M) may indicate that Cpn60 contains some very sensitive regions of structure. This latter effect was not considered further in the present study.
Figure 3: Gel fluorometric determination of the binding of 6-iodoacetamidofluorescein to Cpn60 as a function of the concentration of urea. Panel A, photograph of the fluorescence of an SDS gel for 6-IAF labeled Cpn60 at different urea concentrations. Lanes1-8 correspond to the increasing urea concentrations: 0, 1, 2, 2.25, 2.5, 2.75, 3, and 4 M urea respectively. Fluorescein, unconjugated to the protein, moved at the dye front, which is not shown in this photograph. Panel B, areas under fluorescent bands determined by video densitometry. Details of the procedure are presented under ``Materials and Methods.''
Samples of 6-IAF labeled protein, prepared at 2.5 M urea, that were fluorescent completely lost any fluorescence in the 60 kDa band when the samples were first treated with Proteinase K (data not shown) under conditions that have previously been shown to remove approximately 50 residues from the C-terminal end of intact Cpn60 monomers(32) . Cys-519 is the only cysteine residue within the last 50 residues of the Cpn60 sequence, which suggests that the sulfhydryl labeling occurs in the equatorial domain in the region of subunit contacts(14) .
Figure 4: a, urea concentration dependence of the chymotryptic digestion of Cpn60. Cpn60 (0.8 mg/ml) was treated with 2% chymotrypsin (w/w) in 50 mM Tris-HCl, pH 7.5, at the indicated urea concentrations. The urea concentrations were from lanes1-9: 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, and 4 M urea, respectively. Digestion was allowed to proceed at room temperature for 1 h before the samples were treated with 1 µl of 10 M phenylmethylsulfonyl fluoride to stop proteolysis. Arrows on the left mark the molecular mass standards which are, from the top: 97 kDa, phosphorylase B; 66 kDa, bovine serum albumin; 45 kDa, ovalbumin; 31 kDa, carbonic anhydrase; 21.5 kDa, soybean trypsin inhibitor; and 14.4 kDa, lysozyme. b, time course of chymotryptic digestion of Cpn60 at 2.5 M urea. SDS gel showing time course of the chymotryptic digestion. Samples of Cpn60 (0.8 mg/ml) were digested as in Fig. 7at 2.5 M urea with 0.1% (w/w) chymotrypsin. The parent band is denoted as P and bands A-D are the digestion products selected for quantification. Lanes1-6 correspond to: 0, 15, 30, 60, 90, and 120 min, respectively. The leftmost lane contains molecular weight standards as in a.
Figure 7:
Binding
of bisANS to Cpn60 in the presence of different salts. bisANS
titrations were carried out as described under ``Materials and
Methods'' on samples of Cpn60 pretreated with 0.5 M GdmCl (opensquares), 1 M (Gdm)SO
(filledtriangles), 20 mM spermidine (opentriangles), 0.5 M NaCl (filledsquares), or no pretreatment (filledcircles). Symbols indicate actual data points. Lines are best-fit curves derived as outlined under
``Materials and Methods.''
Densitometry was used to follow the time course of the appearance of bands marked A-D in Fig. 4b. Bands B and D appear early in the digestion, and band D is quite stable throughout. Blotting and amino acid sequencing (see ``Materials and Methods'') indicate that band A begins at residue 203 and is approximately 44 kDa. Band C starts at residue 268. Band D begins at the N terminus of the mature protein and has a molecular mass of 26 kDa and therefore contains approximately 236 residues.
Figure 5: Fluorescence intensity of bisANS bound to Cpn60 as a function of the guanidinium chloride concentration. Cpn60 (1 uM protomer) was treated with the indicated concentrations of GdmCl in the presence of 10 uM bisANS. The spectra were collected, and the intensities at the maxima are plotted. All other conditions were the same as in Fig. 2.
Figure 6:
a, effect of ions on the fluorescence of
bisANS bound to Cpn60. Separate samples of 1 µM Cpn60 in
50 mM Tris-HCl, pH 7.8, were treated with pH 8-adjusted salts
(GdmCl, filledcircles,
(Gdm)SO
, opensquares, or
NaCl, filleddiamonds) to final concentrations as
shown. bisANS was added to salt-treated protein (final concentration,
10 µM). Excitation wavelength for bisANS fluorescence was
360 nm; emission wavelength was 500 nm. b, The effect of
spermidine on bisANS fluorescence. pH 8-adjusted spermidine was added
to samples prepared as in a, and the bisANS fluorescence was
measured at 500 nm, after excitation at 360
nm.
Fig. 6b, shows that the trivalent cation, spermidine, was particularly effective at exposing hydrophobic surfaces on Cpn60. With spermidine, as with the other cations used, there was no cooperative transition, and the maximum fluorescence occurred at about 5 mM spermidine.
It has been widely supposed that hydrophobic interactions are important for binding proteins to Cpn60. One reason for this conjecture is that hydrophobic exposure appears to be characteristic of folding intermediates, and this could account for the generality of the interactions and explain how Cpn60 could compete with aggregation in assisting refolding of a diverse group of proteins. This idea suggests that hydrophobic surfaces should be present on Cpn60 for initial binding and that the interactions should be capable of being modulated to permit release and folding of the bound protein. A difficulty with this proposal, in its simplest form, is that Cpn60 binds only a few small hydrophobic probe molecules that might be presumed to have ready access to any extensive hydrophobic surfaces that would be available for binding to large polypeptide chains. Similarly, it is not clear how the central cavity in a rigid Cpn60 14-mer cylinder could bind and completely enclose proteins up to 90 kDA using hydrophobic interactions and yet have the accessibility of those surfaces not lead to self association.
The present work shows that the restricted hydrophobic surfaces on native Cpn60 can be increased in two ways: (a) by relatively mild structural perturbations that produce assembly competent monomers that are folded and functional (16) and (b) by ionic interactions, especially with cations, that are especially effective and do not dissociate the 14-mer. Therefore, the hydrophobic surfaces can be modulated by the detailed assembly of the quaternary structure of the 14-mer, and the exposed surfaces are not those that are required for maintaining the quaternary structure of the Cpn60 oligomer. Furthermore, the results also suggest that at least portions of the monomers are flexible so that the exposure of hydrophobic surfaces could, in part, be determined by the interactions with the bound proteins. These considerations suggest that structural malleability can participate in the functions of Cpn60.
Flexibility or deformability within Cpn60 is consistent with previous proteolysis experiments as well as with x-ray studies that observed local changes in the crystal packing of Cpn60 that were suggested to reflect a plasticity of the Cpn60 molecule that would allow for large domain or subunit movements(37) . This type of structural malleability would be consistent with the ability of urea to both induce the biphasic disassembly/unfolding of the 14-mer that gives rise to a maximum in the hydrophobic exposure and to induce the differential accessibility of sulfhydryl groups.
The recent 2.8-Å crystal structure of Cpn60 provides an important interpretive context for the present work. The main areas of flexibility are in the apical domain between residues 220 and 360. Based on site directed mutagenesis, the putative polypeptide binding site has been suggested to be in the apical domain and to involve hydrophobic residues (Tyr-199, Tyr-203, Phe-204, Leu-234, Leu-237, Leu-259, Val-263, and Val-264)(17) . This region is the least well resolved part of the structure. A plot of the side chain hydrophobicities using a sliding window of 50 residues with the values tabulated by Kyte and Doolittle (38) is shown in Fig. 8, and it provides a measure that is sensitive to long stretches of hydrophobic residues. The apical domain in such a plot contains by far the highest maximum centered around residue 250.
Figure 8: Hydrophobicity and charge distribution along the sequence of Cpn60. The figure shows the net charge (lowercurve) or the sum of the side-chain hydrophobicities (uppercurve) placed in the center of a sliding window of 50 residues. These measures are sensitive to long stretches of like charged amino acids or long stretches of hydrophobic side chains. The charge was determined for neutral pH, and the sum of the charges within a 50 residue window is plotted at the center of the window. The sums of the side-chain hydrophobicities are analogously presented using the hydrophobicity values tabulated by Kyte and Doolittle(43) .
Even though the hydrophobic exposure in the unperturbed Cpn60 tetradecamer is relatively low, the structural malleability within the apical domain would permit the protein to expose additional hydrophobic surfaces when the 14-mers bind the target protein. Thus, the increased hydrophobic surfaces that have been detected when partially folded proteins interact with Cpn60 may be partly from Cpn60 itself and not entirely from the bound protein(7) . The results presented here indicate that hydrophobic exposure is very sensitive to the ionic environment and can be modulated without disrupting the quaternary structure of Cpn60, even though subunit interactions may be involved in the modulation. It is interesting that the reported Cpn60 structure was derived with crystals grown between 0.86 and 1.72 M ammonium sulfate and stabilized for analysis in 1.8 M ammonium sulfate. These are conditions that the present results indicate give rise to increased flexibility and increased hydrophobic exposure in the molecule. This is consistent with ionic triggering of a conformational change that leads to increased flexibility and hydrophobic exposure that is expressed in the apical domain. This suggestion is supported by the finding that perturbation of the quaternary structure of Cpn60 greatly enhances proteolytic susceptibility in the apical domain between residues 203 and 268, a region that is not cleaved in the absence of perturbation.
The considerations above suggest that
potential binding interactions between target proteins and Cpn60 would
be favorable if hydrophobic surfaces are presented to Cpn60 in a
charged context to take advantage of ionic triggering of the exposure
of hydrophobic surfaces. This would be in keeping with the well
documented finding that charged hydrophobic structures such as
amphiphilic helices are preferentially bound to
Cpn60(12, 39) . It has been shown, for example, that
Cpn60 shows strong interactions with the signal sequences of
-lactamase (40) and the N-terminal sequence of
rhodanese(12) . Both sequences are positively charged, and they
can form helices with hydrophobic faces. The implied binding of the
helical form has been shown directly for the rhodanese N-terminal
sequence(12) . It has also been shown that Cpn60 binding to
granulocyte ribonuclease depends on a sequence of 18 residues
containing four positively charged arginine residues, no negatively
charged residues, and 9 hydrophobic residues(41) . The
importance of charge interactions can also be seen with reduced
apo-
-lactalbumin, which only binds to Cpn60 when the salt
concentration is increased (e.g. 50 mM KCl or NaCl)
under conditions where it was shown that the effect was not on the
structure of the lactalbumin(42) .
In summary, based on the results presented here, it is tempting to suggest two important aspects of the mechanism for binding of partially folded proteins to Cpn60: (a) binding to Cpn60 can organize amphipathic structures; and (b) interactions with the target proteins can trigger changes in Cpn60 that favor binding.