From the Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri, Kansas City, Missouri 64110-2499
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ABSTRACT |
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Many aspects of the mechanism by which the GroEL/ES chaperonins mediate protein folding are still unclear, including the amount of structure present in the substrate bound to GroEL. To address this issue we have analyzed the susceptibility to limited proteolysis and to alkylation of cysteine residues of mitochondrial aspartate aminotransferase (mAAT) bound to GroEL. Several regions of the N-terminal portion of GroEL-bound mAAT are highly susceptible to proteolysis, whereas a large core of about 200 residues containing the C-terminal half of the polypeptide chain is protected in the complex. This protection does not extend to the mAAT sulfhydryl groups which in the GroEL-mAAT complex have similar reactivity as in fully unfolded mAAT. These results suggest that the mAAT species bound to GroEL represent folding intermediates with a conformation that is substantially more disorganized than that of the native state. The N-terminal half of the molecule is more flexible and lies exposed at the mouth of the central cavity of GroEL. The more compact C-terminal section of mAAT, which contains residues located at the subunit interface in the native dimer, appears to be hidden in the central cavity of GroEL. Thus, the bulk of the interactions in the GroEL·mAAT complex seems to involve residues from the more compact C-terminal section of the substrate.
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INTRODUCTION |
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Although the precursor (pmAAT)1 and mature (mAAT) forms of rat liver mitochondrial aspartate aminotransferase can spontaneously refold at low temperatures following guanidine hydrochloride (GdnHCl) denaturation (1), the yield of refolding of the chemically denatured proteins decreases dramatically at increasing temperatures, and no refolding can be observed at physiological temperatures (2). A similar temperature dependence is observed for the folding of the proteins synthesized in a cell-free extract such as rabbit reticulocyte lysate (3). However, folding of these newly translated proteins is much slower than the refolding of the chemically denatured forms in buffer. By contrast, after import into isolated mitochondria at 30 °C mAAT folds rapidly (3). The rapid and efficient folding of mAAT once it reaches the mitochondrial matrix probably results from the assistance of chaperones present in the matrix, including the mitochondrial members of the hsp60/hsp10 family of chaperones which have been reported to be involved in intramitochondrial folding of other translocated proteins (4). Indeed, the chaperonins GroEL/GroES from Escherichia coli, which are structurally and functionally homologous (5, 6) to the intramitochondrial hsp60/hsp10 chaperonin system, greatly extend the temperature range over which mAAT successfully refolds in vitro (2).
Many structurally unrelated proteins, both cytosolic and mitochondrial,
have been shown to bind GroEL with different affinities in both
in vivo and in vitro conditions. Discharge from
the complex and recovery of active dimeric (94 kDa) protein require the
presence of both MgATP and the co-chaperonin GroES, although the latter does not seem to be strictly necessary for a number of proteins with a
lower affinity for the chaperonin (7). The crystal structures of GroEL
(8, 9) and of the GroEL-GroES complex (10) have been recently reported.
The protein is a tetradecamer of 57-kDa identical subunits, arranged in
a double heptameric ring, which form a cylindrical cavity of ~45 Å in diameter. A putative polypeptide-binding site, consisting mainly of
hydrophobic residues, has been located on the inside surface of the
apical domain of each of the subunits, facing the central channel (11).
Indeed, some experimental evidence suggests that the binding of
substrate polypeptide takes place within the central cavity of the
hsp60 cylinder (12-14). However, the details of the interaction
between GroEL and its substrates in terms of sequence elements directly
involved in recognition and binding or the conformation of the
GroEL-bound polypeptides still remain unsolved. GroEL has been reported
to bind a wide variety of protein substrates, from unfolded proteins
(15-17) and various folding intermediates with different affinity
(18-23) to some late and highly structured folding intermediates and
quasi-native protein conformations (24-28), or even to further unfold
the bound proteins (23, 29). A few common structural features have been identified in many of the GroEL-bound polypeptides including a collapsed, molten globule-like conformation, with a significant amount
of native-like secondary structure, lack of stable tertiary structure
interactions, and exposed hydrophobic surfaces. Moreover, it has been
shown that a peptide, largely unstructured when free in solution, can
form an -helix upon binding to GroEL (30). On the other hand, it has
been reported that, in some cases, GroEL captures aggregation-prone or
kinetically trapped intermediates and at least locally unfolds them
upon binding, to put them back on the productive folding pathway (23,
29).
The presence of a molten globule-like intermediate has been proposed for the refolding mechanism of GdnHCl-unfolded pmAAT (1). This transient intermediate(s), which rapidly forms upon dilution of the denaturant and then slowly isomerizes to the native dimer, shows over 80% of the secondary structure found in the native protein and contains exposed hydrophobic surfaces. These properties, together with their high propensity to aggregate, make these species good potential substrates for binding to GroEL, which is able to suppress aggregation in the early stages of mAAT refolding (2).
In this study, we have investigated some structural aspects of refolding mAAT complexed with GroEL by examining the accessibility of the bound protein to exogenous proteases and thiol-specific reagents. In its native state, mAAT is highly resistant to proteases (31), and in the absence of substrate none of its seven cysteine residues is accessible to thiol reagents (32). By contrast, we show that the GroEL-bound protein displays susceptibility to proteolysis, particularly in its N-terminal half, and that all the cysteine residues are still highly exposed to alkylating agents. The results obtained provide valuable information with regard to the conformation of a large protein folding intermediate trapped by GroEL.
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EXPERIMENTAL PROCEDURES |
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Materials and Protein Purification-- GdnHCl was electrophoresis grade from Fisher. L-1-Tosylamido-2-phenylethyl chloromethyl ketone-treated bovine pancreatic trypsin was purchased from Worthington. Proteinase K was from IBI. Chymotrypsin, elastase, subtilisin, and soybean trypsin inhibitor were from Sigma. [14C]Iodoacetamide was from ICN. N-(1-pyrene)maleimide (NPM), 5-(((2-iodoacetyl)aminoethyl)amino)naphthalene-1-sulfonic acid (IAEDANS), and 4-acetamido-4'-((iodoacetyl)amino)stilbene-2,2'-disulfonic acid (STB) were from Molecular Probes, Inc. All other reagents were of the highest purity available.
The mature form of mAAT was obtained by trypsin cleavage of the presequence from the precursor pmAAT expressed in E. coli and purified as described previously (3, 33). This method of mature mAAT preparation renders a protein containing an additional alanine at the N-terminal end. The molecular mass of this protein, determined by ESI-MS (44599.1 ± 4.9), agrees with the molecular mass (Mr = 44598.27) calculated from the amino acid sequence of mAAT (34) plus an additional alanine residue. To facilitate the purification of radiolabeled pmAAT and the detection of the presence of an intact C terminus in the proteolytic fragments, we introduced a His6 tag at the C-terminal end of the polypeptide chain by cloning the pmAAT cDNA into pET23a (Novagen). The cDNA encoding pmAAT was altered to introduce the XhoI site necessary for subcloning into pET23a. This involved polymerase chain reaction mutagenesis (Stratagene's Quick Change kit) of the pBSKS-4 plasmid (3) to insert the XhoI recognition site immediately upstream of the stop codon. The NdeI, XhoI fragment from the resulting plasmid was subcloned into pET23a to produce pET23a-2. Radiolabeled pmAAT-HT was prepared by expressing the pET23a-2 plasmid in E. coli BL21(DE3)pLysS growing in minimum media and adding 1 mCi of Tran35S-label (ICN Biomedicals Inc.) per 100 ml of culture 45 min after induction with isopropyl-Formation of the GroEL·mAAT Complex and Limited Proteolysis-- The native protein was denatured by incubation in 4 M GdnHCl, 100 mM HEPES, pH 7.5, 10 mM dithiothreitol, and 0.1 mM EDTA for 30 min at room temperature (1) at a protein concentration of 1.5 mg/ml. Complexes between the refolding transaminase and GroEL were formed by diluting the denatured protein 40-fold to 40 µg/ml (or 0.89 µM monomer) into the appropriate volume of ice-cold refolding buffer (100 mM HEPES, pH 7.5, 0.1 mM EDTA, 1 mM dithiothreitol, with 50 mM KCl) containing 1.08 µM GroEL 14-mer (1.2-fold molar excess over mAAT) in a polypropylene microtube while vortexing (2). After incubating the complex on ice for 5 min, proteolysis was begun by adding the appropriate amount of one of five different proteases, and the sample was incubated at 10 °C. The protease concentrations used were as follows: trypsin, 2.5 or 20 µg/ml; chymotrypsin, 2.5 or 0.8 µg/ml; proteinase K, subtilisin, and elastase, 0.8 µg/ml. At various incubation times, an aliquot was withdrawn, and the hydrolysis was quenched by addition of a 2:1, w/w, excess of soybean trypsin inhibitor to reactions containing trypsin or 2 mM phenylmethylsulfonyl fluoride (PMSF) to the other reactions. After incubation on ice for at least 10 min, an equal volume of 2 × SDS-PAGE sample buffer was added, and the samples were analyzed by SDS-PAGE on 12% acrylamide gels (35). The proteolytic fragments were detected either by silver staining or by autoradiography using a Molecular Dynamics PhosphorImager. When pmAAT-HT was used, the fragments containing an intact C-terminal end could be visualized by chemiluminescence using a nickel-loaded carboxymethyllysine (CM-lys) horseradish peroxidase2 and the ECL kit from Amersham Corp. after blotting on a nitrocellulose membrane.
Purification and Identification of Proteolytic Fragments-- Scaled up reactions (1.5 ml) of the GroEL·pmAAT or pmAAT-HT complex treated with different proteases were used to purify several of the major intermediate products for subsequent sequencing and ESI-MS analysis. Conditions for the formation of the complex and treatment with proteases were as described above. Different concentrations of proteases and incubation times were used to accumulate a selected fragment as indicated in the legends to figures and under "Results." After quenching proteolysis by addition of the appropriate inhibitor, the reactions were analyzed by SDS-PAGE and autoradiography and fractionated by HPLC on a RP-304 analytical column (4.6 × 250 mm, Bio-Rad) to separate the mAAT fragments from GroEL. The chromatography was performed at a flow rate of 1 ml/min using a 95% acetonitrile/water gradient (20-45% over 25 min, followed by an increase from 45 to 55% over 20 min) containing 0.1% trifluoroacetic acid. Each fraction was analyzed by SDS-PAGE to determine the distribution of the proteolytic fragments observed in the electrophoretic pattern of the unfractionated reaction. Fractions collected from the RP-HPLC column were directly analyzed by electrospray mass spectrometry (ESI-MS) using a VG PLATFORM (VG Biotech) single quadrupole instrument or a LCQ ion trap system (Finnigan MAT). Samples were injected directly into the ion source via a loop injection at a flow rate of 2-10 µl/min, and data were acquired and elaborated using the MASS-LINK program (VG Biotech) or the Navigator software (Finnigan), respectively. All masses are reported as average values. The identification of the origin of the fragments within the primary structure of mAAT was based on the accurate molecular weight measured and the presence of a His-tagged C-terminal end, according to the known amino acid sequence of rat liver mAAT (34). To confirm that the large intermediate fragments arising from the proteolysis of the GroEL·mAAT complex remain associated with the chaperonin, reaction samples treated as described above were centrifuged at 10,000 rpm for 5 min to remove a small amount of protein aggregate, and the supernatant was loaded on a 2-ml, 10-30% glycerol gradient in refolding buffer. Following ultracentrifugation at 55,000 rpm for 4 h at 4 °C (Optima TLX ultracentrifuge, Beckman; TLS-55 swinging bucket rotor), the gradient was fractionated from the bottom of the tube. The identity of the proteins present in each fraction was established by SDS-PAGE analysis and silver staining or chemiluminescence detection as described above.
Chemical Modification-- For the labeling with radioactive iodoacetamide ([14C]IAM), the GroEL·mAAT complex was formed as described previously at a mAAT concentration of 0.89 µM and a 1.2:1 molar excess of GroEL. Samples were incubated at 20 °C with a 20:1 molar excess of [14C]IAM over free thiol groups in the transaminase. Aliquots were withdrawn at different times; the reaction was quenched with an excess of dithiothreitol, and the proteins were precipitated with 10% trichloroacetic acid. The pellet, containing GroEL and the labeled mAAT, was analyzed by SDS-PAGE, followed by Coomassie staining and PhosphorImager analysis. As a control, the same procedure was followed to label unfolded mAAT, except that the reaction was performed in buffer containing 4 M GdnHCl. The amount of mAAT present in each sample was estimated by densitometric analysis of the Coomassie-stained gel using a Personal Densitometer Scanner (Molecular Dynamics). A standard curve, obtained by running on the same gel different known amounts of unlabeled mAAT, was used to calculate the protein concentration from the band intensity. The amounts of protein loaded on the SDS-PAGE gels (0.5-2.0 µg) were well within the linear range for both stain binding and instrument response (at least 0.1-2.5 µg). The amount of radioactivity incorporated at each time point was determined by PhosphorImager analysis of the intensity of the corresponding radioactive band using the Molecular Dynamics ImageQuant program.
For the labeling with either NPM, IAEDANS, or STB, the GroEL·mAAT complex was prepared as before and incubated with different concentrations of the alkylating agent ranging from 6 µM to 2 mM. After incubation at room temperature for different times, the reaction was quenched with an excess of dithiothreitol. The extent of mAAT modification was estimated from the absorbance associated with the mAAT peak at either 336 nm (IAEDANS and STB) or 339 nm (NPM) following removal of free label and separation of mAAT from GroEL by HPLC on a RP-304 column, using the same acetonitrile/water gradient described before. The area under the 336 nm or 339 nm peak was normalized by the absorbance at 214 nm recorded simultaneously using a photodiode array detector attached to the Waters chromatographer (Waters 626 pump, 600S controller and 996 photodiode array detector, equipped with Millenium Software) to correct for differences in the total protein present in the sample. Samples of mAAT unfolded in 4 M GdnHCl were analyzed in parallel following identical labeling and analytical procedures. To determine the extent of labeling of each individual cysteine residue, a peptide digest was prepared for samples of mAAT labeled either in 4 M GdnHCl or while in complex with GroEL. After purification of the labeled protein on a RP-304 column as described previously, the mAAT peak was dried under vacuum, resuspended in 50 mM ammonium bicarbonate, pH 8.5, and incubated with L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin, approximately 1:20, w/w, ratio, overnight at 37 °C. The peptide mixture was fractionated by RP-HPLC on a Vydac C18 analytical column (4.6 × 250 mm, The Separation Group, Hesperia, CA) using a flow rate of 1 ml/min and an acetonitrile/water gradient (5-60% over 55 min) containing 0.1% trifluoroacetic acid. Elution was monitored at both 214 nm and 336 or 339 nm (IAEDANS, ![]() |
RESULTS |
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Limited Proteolysis of GroEL-bound mAAT-- A complex between mAAT and GroEL is formed when the GdnHCl-unfolded transaminase is diluted into refolding buffer containing the chaperonin (2). On the other hand, fully folded native mAAT does not bind to GroEL (data not shown). GroEL shows a high affinity for refolding mAAT and forms a stable complex with a stoichiometry of one subunit (46 kDa) of mAAT bound per GroEL 14-mer (2). This complex can be isolated by HPLC-size exclusion chromatography and rate zonal centrifugation in a 10-30% glycerol gradient (data not shown). Moreover, the transaminase is bound in a folding competent state, for GroEL alone completely inhibits the spontaneous refolding of the protein, but the process is resumed upon addition of GroES and MgATP (2). For the experiments described in this work, it was not necessary to reisolate the complex before studying its protease susceptibility because under the experimental conditions used (1.2-fold molar excess of GroEL over mAAT), essentially all mAAT was complexed with the chaperonin. Furthermore, any mAAT that may have escaped trapping by GroEL and continued folding to its native state would be fully resistant to proteolysis. Much harsher conditions (equimolar amounts of trypsin and hours of incubation at 37 °C) than those used in these experiments are needed to hydrolyze just two peptide bonds in the N-terminal region of native mAAT (31). Thus, limited hydrolysis with a variety of proteases under controlled conditions appeared as a suitable approach to examine whether folding intermediates trapped by GroEL resembled the compact native state.
The GroEL-transaminase complex was treated with five different proteases as follows: trypsin, chymotrypsin, elastase, proteinase K, and subtilisin. The conditions for proteolysis were designed so that the bound protein was cleaved, whereas GroEL remained essentially intact. As previously reported (12), we could also cleave from GroEL a 16-residue C-terminal peptide with proteinase K; however, the chaperonin maintained its oligomeric structure and was fully active (12). The concentration of proteases used ranged from 20 µg/ml for trypsin to 2.5 µg/ml chymotrypsin and 0.8 µg/ml for the others. Although different time aliquots were analyzed, Fig. 1 shows only a representative time point for each proteolytic reaction (15 min for trypsin and 10 min for the other enzymes, at 10 °C), at which all of the predominant intermediate fragments are present. It can be seen that, in contrast to the stability of the native enzyme, incubation of the mAAT folding intermediate bound to GroEL with low concentrations of proteases under mild conditions (with regard to temperature and incubation time) produces a reproducible pattern of a limited number of proteolytic fragments. The predominant peptides range in molecular mass from 42 to 23 kDa. Smaller size fragments were sometimes observed in very small amounts but never accumulated. As observed by comparing Fig. 1A (silver-stained gel) and Fig. 1B (autoradiogram), peptides arising from hydrolysis of GroEL can contribute to the electrophoretic pattern when visualized by silver staining. Although only a very small fraction of GroEL is digested (~10%), their absolute amounts are comparable with those of the mAAT fragments due to the large excess of GroEL monomer in the reaction (14:1 relative to mAAT monomer). Nevertheless, since we can specifically detect mAAT-derived species by their radiolabel, the presence of small amounts of GroEL peptides was of little consequence to this analysis.
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Characterization of mAAT Proteolytic Fragments-- To localize the cleavage sites for the different proteases within the amino acid sequence of mAAT, selected proteolytic fragments were partially purified by RP-HPLC and analyzed by electrospray-mass spectrometry (ESI-MS). Before injection in the HPLC, the samples were dried and resuspended in 0.1% trifluoroacetic acid (chromatography buffer A) which denatures the proteins and thereby dissociates the complex. A representative RP-HPLC chromatogram of a tryptic digest (incubation with 20 µg/ml trypsin for 20 min at 10 °C) includes a large peak eluting at ~45 min, which was identified as GroEL by analysis of an aliquot of purified GroEL (Fig. 3A). An additional broad peak appeared with a retention time (35.3 ± 0.1 min) only slightly different from that of intact transaminase (35.7 min), run as a control. Similar chromatograms were obtained with the digests prepared using the other proteases. SDS-PAGE analysis of fractions from the 35.3 peak using radioactive mAAT showed that it contained all of the large molecular weight tryptic fragments, which are not well resolved in the reverse phase C4 column used for this separation. The lower retention time of the peptides eluting earlier (at ~24 min) was consistent with a much smaller size and probably more hydrophilic nature than the large fragments bound to GroEL. Mass spectrometric analysis of several of these free peptides originating from treatment of GroEL·mAAT with trypsin, chymotrypsin, and elastase led to their unequivocal localization within the primary structure of rat liver mAAT. As summarized in Table I, all of them were indeed relatively small peptide fragments, arising from either the N-terminal end of the protein (see peptides T1c, T2c, and E2c and the peptides labeled as CT2c in Table I) or from internal sequences (CT4c or E3c). In all cases they represent pieces of the polypeptide chain that are complementary to the large fragments detected by SDS-PAGE analysis of the digested samples. Those small peptide fragments were recovered in the filtrate upon ultrafiltration of the digest in a Microcon-100, which indicates that upon nicking of the peptidic bond that connected them to the core of the polypeptide they were readily released into solution.
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Accessibility of mAAT Cysteine Residues to Chemical Modification-- In its native conformation, the seven cysteines found in the mAAT structure (32, 34, 36) are not accessible to modification by thiol reagents. In fact, only when incubated with high concentrations of alkylating agents and for extended periods, one of its cysteine residues, Cys166, may be labeled, and this only in the closed structure of the enzyme which is induced by binding substrate or substrate analogs (32). To determine how much of the compact three-dimensional structure of native mAAT (36) is already present in the GroEL-bound protein, we analyzed the accessibility of its seven cysteine residues to alkylating agents such as iodoacetamide (radioactively labeled, [14C]IAM), a small and neutral molecule; NPM, a large hydrophobic molecule; IAEDANS also quite bulky but a hydrophilic and charged reagent; and STB, also large and carrying two negative charges. NPM, STB, and IAEDANS were chosen also because their strong absorbance in the near UV (with maximum at 339 nm for NPM and 336 nm for the others) allowed the detection of labeled cysteine-containing tryptic fragments separated by RP-HPLC. In contrast to the native protein, the cysteines in mAAT bound to GroEL showed high reactivity toward all the alkylating agents mentioned above. By using different concentrations of [14C]IAM, we followed the kinetics of incorporation of radioactive carboxyamidomethyl groups in both GroEL-bound and free mAAT unfolded in 4 M GdnHCl but were unable to detect significant differences in the kinetics of alkylation between the two states of the protein. The alkylation reaction per se was unaffected by the high ionic strength of the unfolding solution, at least when a relatively high concentration of IAM (20 mM) was used. After 1 h incubation under these conditions both GroEL-bound and free unfolded transaminase were completely labeled, as evidenced by the absence of any sulfhydryl groups that could be titrated with Ellman's reagent (5,5'-dithiobis(2-nitrobenzoic acid)) (data not shown).
Given the small size and neutral character of iodoacetamide, we reasoned that perhaps more bulky and/or charged reagents could be more discriminatory as to their accessibility to the region of mAAT where the cysteines are located and therefore provide better probes of the conformational state of mAAT in the complex. The degree of labeling was estimated from the absorbance at 336 nm (STB and IAEDANS) or 339 nm (NPM) of mAAT eluting from an RP-304 column chromatography performed to remove the excess free reagents. Simultaneous detection at 214 nm was used to normalize the absorbance at 336 or 339 nm for the total mAAT present. Surprisingly, whereas GroEL-bound mAAT was completely alkylated after incubation with 60 µM STB for 60 min at room temperature, the unfolded protein (in 4 M GdnHCl) was only partially modified (data not shown). Nevertheless, full modification of the unfolded protein could be achieved in the presence of higher concentrations of reagent (3 mM for 60 min). Apparently, the high ionic strength of the unfolding buffer interferes with the reaction with these probes. The labeling of GroEL-bound mAAT was always performed in refolding buffer containing a residual 0.1 M GdnHCl and the incorporation of the three probes followed kinetics very similar to that observed with IAM (data not shown). We took advantage of the spectroscopic properties of IAEDANS, STB, and NPM to analyze the kinetics of modification of individual cysteine residues in mAAT bound to GroEL. The experimental approach used involved the peptide mapping of the labeled enzyme using simultaneous detection at 214 nm and 336 or 339 nm to identify the peptides containing modified residues. mAAT was completely labeled in 4 M GdnHCl using a large excess of STB (3 mM, 120 min incubation). After removing the denaturant by dialysis, which causes the precipitation of the protein, the labeled mAAT was extensively digested with trypsin (see "Experimental Procedures"). The tryptic map, as shown in Fig. 6A for STB, displays seven major peaks at 336 nm. According to the distribution of tryptic cleavage sites in the mAAT sequence, only six tryptic peptides can be expected, with one of them containing two cysteine residues, Cys251 and Cys253, which are only one residue apart (34). The seven labeled peptides were identified on the basis of their molecular mass, determined by ESI-MS, and taking into account the presence of one (or two) alkylated cysteine residue. The peak eluting at 21.2 min corresponds to the tryptic peptide spanning residues Val267-Lys275 and containing labeled Cys274; the 24.2-min peak corresponds to the His242-Lys258 peptide that includes two cysteines, Cys251 and 253; the 26.7-, 28.8-, and 31.7-min peaks correspond to peptides containing Cys191 (Ile180-Lys206), Cys166 (Thr165-Lys179), and Cys361 (Glu344-Arg372), respectively. The two peaks eluting at 29.3 and 30.1 min correspond to peptides arising from alternative cleavage by trypsin at either Lys68 (29.3-min peak, Glu69-Lys81 peptide) or Lys63 (30.1-min peak, Asn64-Lys81 peptide), and both contain Cys80.
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DISCUSSION |
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Where along the folding pathway a protein is recognized by GroEL and how much structure is present in the bound substrates are questions that remain unsolved. Some studies have suggested that GroEL-bound polypeptides are fully unfolded, whereas others show that the substrate is partially structured and may contain different degrees of secondary and possibly unstable tertiary interactions (for a recent review, see Ref. 7). In addition, there are claims that the folding intermediate is fully trapped in the GroEL cavity which may act as a folding cage (14). The present work clarifies some of the issues under debate by showing an overall view of the conformation of mAAT stoichiometrically bound to GroEL in a stable complex.
The susceptibility of a polypeptide to exogenous proteases as a probe of its conformation either free in solution (37, 38) as well as in a complex with DNA (39) or other proteins (40, 41) has been used in the past with encouraging results. The rationale behind the method is that amino acid residues that are accessible to protease recognition under controlled conditions must be located within regions of the polypeptide chain exposed to the solvent. However, surface exposure is not a sufficient criteria for proteolysis to occur; chain flexibility is also required for binding of a polypeptide segment to the active site of the protease where the hydrolytic reaction takes place (37). Thus, by using the limited proteolysis approach, structural information about both the accessibility to the solvent of specific residues and the local flexibility of the polypeptide chain can be obtained.
Native mAAT can be cleaved only in a very limited number of sites,
regardless of the specific protease used and only at high temperatures
(37 °C) and enzyme:substrate
ratios.3 In its crystal
structure, each of the two identical subunits of mAAT is composed of a
large domain (residues 49-325), a small domain (residues 15-48 and
326-410), and an N-terminal extended region forming a bridge between
the two subunits (residues 3-14) (36). In the folded protein the few
protease-accessible sites are confined to the N-terminal region, with
the cleavable peptide bonds located in the -helix 1 (Pro16-Arg26) of the small domain and the
contiguous
-turn (Asn29-Lys32). For
instance, trypsin cleaves after Arg26 and Lys31
of chicken mAAT (42). The large domain and the C-terminal portion of
the small domain in the native transaminase are very resistant to
proteolysis (31), even after the initial cleavage of the N-terminal
peptide, indicating that the overall three-dimensional structure of the
dimeric protein is very rigid and stable. At the other extreme of
protease susceptibility is the unfolded protein, where all potential
tryptic sites are accessible.
In the case of a binary complex such as the one under investigation here, additional factors that may contribute to the observed pattern of proteolysis need to be considered. In fact, protection against proteolysis could be conferred not only by the existence of a rigid structure within the polypeptide chain bound to GroEL but also by direct protein-protein interaction between the substrate and the chaperonin in the regions of tighter binding, as well as by steric hindrance, due to the blocked location of the scissile substrate within the complex. In the case of the mAAT folding intermediate(s) bound to GroEL, our limited proteolysis study shows two main characteristics of the structure of the protein as part of the binary complex. First, the most accessible cleavage sites are all located at residues within the N-terminal half of the transaminase polypeptide chain, regardless of the specificity of the protease used and despite the presence of numerous other potential cleavage sites along the chain (42 cleavage sites, evenly distributed along the amino acid sequence of mAAT, for trypsin alone, Fig. 5). Second, there appears to be a specific pattern of proteolytic events, starting with cleavage in the stretch from Arg26 to Lys31 (region 1, Fig. 5), and continuing in a sequential fashion with hydrolysis occurring in regions 2-5. Time course experiments, performed to determine the order of formation and the relative stability of each intermediate fragment (data for trypsin and proteinase K are shown in Fig. 4, A and B, respectively), revealed that the region most exposed to proteolysis that produces relatively stable intermediates is located between Tyr48 (fragments CT2 and E2) and Arg54 (T2) (see Fig. 5). Those are the first high molecular weight fragments to accumulate significantly. The species T1, produced by cleavage at position 26, is indeed observed, and it reaches a maximum amount slightly ahead of T2,4 but it does not accumulate as much as T2. No fragments of comparable size are detected with other proteases. Moreover, even though after 15 min incubation with trypsin the most prominent small peptide is the one complementary to T1 (T1c, eluting at 24.2 min, Fig. 3), at earlier time points we also detected the presence of the peptide complementary to T2 (T2c, residues 3-54), together with the two smaller fragments 3-26 and 27-54. These data indicate that the regions 1 and 2 are almost equally accessible, with slightly faster cleavage at region 1. Cleavage at region 2 does not require, however, previous cleavage at region 1, as the presence of both T1c and T2c complementary fragments at the early stage of proteolysis suggests. Further cleavage of T2c by trypsin (at Arg26) following its release from GroEL might explain the predominance of the 3-26 peptide (T1c) at later time points (Fig. 3A).
As shown in Fig. 7 and Table
III, in the folded protein the cleavage
site in region 1 which produces T1 lies roughly in the middle of the
N-terminal portion of the small domain, at the end of an -helix. In
the folded protein this is the only region that is susceptible to
trypsin (42) and only in harsh conditions. Evidently, this is also the
region of greater accessibility and conformational flexibility in the
folding intermediate bound to GroEL. Its unrestricted access to trypsin
also suggests that region 1 is not involved in extensive interactions
with the chaperonin. The residues comprising region 2, which is
completely resistant to proteolysis in the native conformation, are
located near the beginning of the large domain in what is a 12-residue
-helix in the native protein (Fig. 7). This region also includes
residues at the intersubunit and domain interface in the folded protein (Table III), including a short segment in an extended conformation that
crosses over between the two domains (Pro47 and
Tyr48). It is obvious that this region is considerably more
flexible in the folding intermediate bound to GroEL than in the native protein and does not represent a binding site for the chaperonin. The
type 2 intermediates are relatively stable, and only a limited number
of its potential cleavage sites are accessible for further proteolysis.
Among these, regions 3 and 4 seem to have comparable accessibility as
both types of fragments start to appear almost simultaneously (Fig. 4).
Yet, the type 3 fragments do not accumulate much, probably because they
are subsequently converted to type 4 species. The latter are the most
stable species observed. Regions 3 and 4 are both located at the
outside edge of a pack of
-helices and
-strands that form the
core of the compact large domain in the native structure (Fig. 7),
partly exposed but still resistant to proteolysis. Those two regions
therefore appear to be less structured in the folding intermediate
trapped by GroEL than in the native protein.
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Type 4 fragments are slowly degraded, but the major products detected
(type 5) rapidly disappear. The only type 5 fragment we characterized
(K5a) arises from cleavage at Asp199, at the domain
interface (36). This region appears to become accessible only after
removal of the first 100 N-terminal residues. A possible explanation
for the rapid degradation of the K5a fragment could be the loss of the
protection afforded by GroEL if it were released from the complex.
However, the K5a fragment remains bound to GroEL (data not shown). More
likely, the increased instability of K5a is due to further unfolding or
greater exposure of the C-terminal core (~200 residues) following
cleavage of the 106-199 segment from the relatively stable K4
fragment. Interestingly, no stable intermediates were detected
originating from cleavage within this ~100-residue region (106-190)
that seems to stabilize K4. In the native dimer, this section is
organized in alternating -helix and
-strand (3 of each) elements
in the middle of the compact
/
core of the large domain, with
many of the residues contributing to the subunit or domain interfaces
and even to the active site (36). By contrast, the 194-199 sequence
forms a loop connecting the last
-strand of the previous region
(strand A5, 184-190) with the next
-helix (helix 8, 202-215) (Fig.
7). A similar arrangement of secondary structure elements in the
GroEL-bound intermediate would explain the preferential cleavage after
Asp199 in K4.
Taken together our results suggest that the folding domains revealed by
the pattern of protease-susceptible and -resistant regions observed in
the mAAT polypeptide bound to GroEL might be related to the structural
organization of the native protein. The regions with greatest
accessibility and conformational flexibility are located either at the
N-terminal peptide, which forms an exposed bridge between the two
subunits in the native structure (region 1), or at the N-terminal edge
of the /
ensemble that forms the compact large domain (regions
2-4, Fig. 7). The sections forming the central core of the large
domain and the C-terminal portion of the small domain in the native
protein, together comprising over 70% of the polypeptide, are largely
resistant to proteolysis and probably much more structured. The fact
that the only other cleavage site identified (region 5) is in a loop at
the domain interface (Fig. 7) might indicate that the docking of the
two domains in the intermediate is still incomplete.
Some of the protease protection observed in mAAT bound to GroEL could be due to the binding to the chaperonin or by the intrinsic structural rigidity of the resistant fragments. However, the trypsinolysis pattern of mAAT at the early stages of refolding in the absence of GroEL is qualitatively identical to that of the complex with the chaperonin.4 Thus, the structural organization of the protein bound to GroEL appears to be an intrinsic property of the folding intermediate and not the result of its binding to the chaperonin. Binding to GroEL, however, confers some retardation to proteolysis either by limited steric interference to access of the protease or by locking the fluctuating conformation of the folding intermediate into one of the more protease-resistant conformations. Although these data do not support a clear model for the binding of mAAT to GroEL, they are most compatible with the location of the more compact C-terminal part in the central cavity of GroEL and the N-terminal portion exposed at the mouth of the cavity or perhaps extending even beyond the external opening of the cavity. The significant amount of exposure to proteases nevertheless precludes a model in which the protein is fully immersed into the GroEL central cavity. The size of the cylindrical cavity in resting GroEL (~45 Å) (8) is unlikely to accommodate a folding intermediate the size of (partially unfolded) mAAT but could probably accept the compact C-terminal half. The more flexible N-terminal region could engage in interactions with residues near the opening of the cavity and between the apical domains (11, 43, 44). The observation that mAAT-HT, having a His6-tag at the C-terminal end, is not retained by a Ni2+-NTA column when bound to GroEL (free native protein binds tightly, data not shown) supports the proposed location of the C-terminal section buried inside the complex. The sudden increase in protease accessibility of 200-residue C-terminal fragments (type 5) as time of proteolysis increases supports the perception that the N-terminal region originally forms a protective barrier between the exogenous protease and the C-terminal half of the bound protein. Changes in the intrinsic structure of the fragment brought about by the previous successive cleavages may also contribute to the instability of the type 5 fragments.
Despite the highly restricted access of the polypeptide bound to GroEL to proteases, the reactivity of the seven cysteine residues in GroEL-bound mAAT is very similar to that observed in the unfolded state, with only two of them (Cys residues 274 and 361) showing slightly slower reactivity than in the unfolded state. Cysteine residues are distributed along the sequence but are mostly located in its C-terminal half (Fig. 5), the region with greatest resistance to proteolysis in the complex with GroEL. Thus, this folding intermediate with restricted access to relatively large proteolytic enzymes may have a conformation with considerable amounts of secondary structure and limited conformational flexibility in much of its structure, but still lacks at least some of the tertiary interactions that contribute to the highly compact nature of the native folded state. All of the observed properties in fact resemble the main characteristics of the molten-globule state proposed for the conformation of other proteins when bound to GroEL (7). Furthermore, with mAAT, binding to GroEL seems to stabilize the structure of the intermediate without inducing significant further unfolding or folding of the bound protein.
In conclusion, the central cavity of GroEL seems to host the C-terminal half of mAAT. This segment probably is highly ordered, with much of secondary and partial elements of tertiary structure already present as they cannot be hydrolyzed even in the absence of chaperonin,4 and there is rapid formation of secondary structure earlier in the folding sequence (1). The compact organization of this segment from helix 11 to the end is not yet completed as alkylating agents still can reach the cysteines in the region. Since this half of the protein contains many of the hydrophobic patches that interact with its companion subunit to form the native dimer, these patches may be contact sites with residues within the GroEL cavity. On the other hand, the structure of the N-terminal half, although probably containing many of the protein's secondary structural elements, has a looser and more flexible organization. The access to the peptide region up to residue 105 is not dramatically hindered by GroEL, and therefore, it most likely rests exposed outside the GroEL cavity, hence its susceptibility to added proteases. Contact sites between some parts of this N-terminal segment and GroEL nevertheless may exist as once trapped by GroEL folding fails to progress. This section of the protein contains the critical residues 5 and 6 that interlock with the companion subunit in the native dimeric protein as well as other subunit interface contact sites spanning residues 47-70. As the dimerization step is very fast and precedes the final structural rearrangements leading to the native protein (45), it is likely that the final organization of this N-terminal segment is achieved, at least in part, through intersubunit interactions once the monomers are released from GroEL and the dimer is formed.
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ACKNOWLEDGEMENTS |
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We thank Dr. Darrel L. Peterson for the gift of Ni2+-loaded CM-lys horseradish peroxidase; Drs. Marilyn Yoder and Xin Chen for help with the molecular graphics; and John Bollin for the purification of GroEL, GroES, and wild type pmAAT. The mass spectrometric analyses performed with the VG PLATFORM were done at the International Mass Spectrometry Facility Center, CNR-Universitá di Napoli Federico II, Napoli, Italy.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants HL-38412 and GM-38341.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 816-235--5246;
Fax: 816-235-5158.
1
The abbreviations used are: pmAAT and mAAT,
precursor and mature forms of rat liver mitochondrial aspartate
aminotransferase, respectively; mAAT-HT, aspartate aminotransferase
with a (His)6tag fused at the C-terminal end; GdnHCl,
guanidine hydrochloride; PMSF, phenylmethylsulfonyl fluoride; CM-lys,
N,N
-bis(carboxymethyl) lysine; RP-HPLC,
reverse phase-high performance liquid chromatography; ESI-MS,
electrospray-mass spectrometry; NPM, N-(1-pyrene)maleimide; IAEDANS,
5-(((2-iodoacetyl)aminoethyl)amino)naphthalene-1-sulfonic acid; STB,
4-acetamido-4'-[(iodoacetyl)amino]stilbene-2,2'-disulfonic acid.
2 D. L. Peterson, personal communication.
3 Y. H. Fu, unpublished data.
4 J. R. Mattingly, Jr., A. Iriarte, and M. Martinez-Carrion, manuscript submitted for publication.
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REFERENCES |
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