Recognition of Protein Substrates by Protein-disulfide Isomerase

Refolding of partially folded mitochondrial malate dehydrogenase (mMDH) is assisted by protein-disulfide isomerase (PDI). The addition of a 20-fold molar excess of PDI over denatured protein (0.1 μm) accelerates the recovery of catalytic activity. PDI fluorescence measurements show that 1 mol of PDI binds 1 mol of denatured mMDH when their concentrations approach 1 μm. The binding of PDI, derivatized with the fluorescence probe iodoacetamide fluorescein, to partially folded mMDH is characterized by a dissociation constant of 0.2 μm.It is shown that the fluorescence probe is covalently attached to a SH residue located in the b′ domain. Based on the fluorescence measurements of native and derivatized PDI, it is suggested that recognition of the unfolded substrate involves conformational changes propagated to several domains of PDI. Refolding of partially folded mitochondrial malate dehydrogenase (mMDH) is assisted by protein-disulfide isomerase (PDI). The addition of a 20-fold molar excess of PDI over denatured protein (0.1 μm) accelerates the recovery of catalytic activity. PDI fluorescence measurements show that 1 mol of PDI binds 1 mol of denatured mMDH when their concentrations approach 1 μm. The binding of PDI, derivatized with the fluorescence probe iodoacetamide fluorescein, to partially folded mMDH is characterized by a dissociation constant of 0.2 μm. It is shown that the fluorescence probe is covalently attached to a SH residue located in the b′ domain. Based on the fluorescence measurements of native and derivatized PDI, it is suggested that recognition of the unfolded substrate involves conformational changes propagated to several domains of PDI. protein-disulfide isomerase 5-(iodoacetamide)-fluorescein-labeled PDI guanidine hydrochloride malate dehydrogenase mitochondrial MDH Protein-disulfide isomerase (PDI)1 is a multifunctional enzyme that both catalyzes the formation of disulfide bonds (1De Lorenzo F. Goldenberg R.F. Steers Jr., E. Givol D. Anfinsen C.B. J. Biol. Chem. 1996; 241: 1562-1567Abstract Full Text PDF Google Scholar, 2Lambert N. Freedman R.B. Biochem. J. 1983; 213: 225-234Crossref PubMed Scopus (124) Google Scholar) and acts as a subunit of prolyl-4-hydroxylase (3John D.C.A. Grant M.E. Bulleid N.J. EMBO J. 1983; 12: 1587-1595Crossref Scopus (95) Google Scholar). PDI has been proposed to function as a molecular chaperone by binding to unfolded protein species, thereby preventing aggregation and misfolding (4Song J.L. Wang C.C. Eur. J. Biochem. 1995; 231: 312-316Crossref PubMed Scopus (136) Google Scholar, 5Song J.L. Ouan H. Wang C.C. Biochem. J. 1997; 273: 9637-9643Google Scholar). Despite these interesting studies, the situation is complicated because PDI, unlike other chaperones, catalyzes disulfide bond formation and reduction. Several laboratories have attempted to show the site of binding of small molecular weight polypeptides that compete with misfolded protein substrates. Mutated PDI with the carboxyl terminus deleted shows neither peptide binding nor chaperone activity in assisting the refolding of denatured d-glyceraldehyde-3-phosphate dehydrogenase (6Dai Y. Wang C.C. J. Biol. Chem. 1997; 272 (27576): 27572Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). On the other hand, it has been reported that deletion of the carboxyl-terminal domain (C domain) has no inhibitory effect on the assembly of recombinant prolyl-4-hydroxylase (7Koivunen P. Pirneskoski A. Karvonen P. Ljung J. Helakoski T. Trontbohm H. Kivirikko K.J. EMBO, J. 1999; 18: 65-74Crossref PubMed Scopus (54) Google Scholar). Other investigators have reported that small molecular weight peptides bind to the b′ domain of PDI (8Klappa P. Ruddeck L.W. Darby N.J. Freedman R.B. EMBO J. 1998; 17: 927-935Crossref PubMed Scopus (295) Google Scholar). The possibility exists that more than one site in the structure of PDI is involved in recognition and refolding of protein substrates. The binding and hydrolysis of ATP by PDI has been reported by Guthapfelet al. (9Guthapfel R. Gueguen P. Quemeneur E. J. Biol. Chem. 1996; 271: 2663-2666Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar); strikingly, the ATPase reaction is stimulated in the presence of denatured polypeptides, whereas the disulfide oxidation of PDI is not influenced by ATP. However, the functional role played by ATP hydrolysis during the refolding of denatured proteins has not been investigated in detail. The aim of the present work is 2-fold: first, to study regions of the primary structure of PDI involved in recognition of unfolded protein substrates and, second, to investigate whether the free energy of hydrolysis of ATP is required for unfolding of misfolded protein substrates. PDI was purified by the method described in Ref. 10Hillson D.A. Lambert N. Freedman R.B. Methods Enzymol. 1994; 107: 281-294Crossref Scopus (188) Google Scholar with small modifications. Fresh porcine livers (600 g) were homogenized in 0.1 m phosphate (pH 7.5) containing 1% Triton X-100 and 5 mm EDTA. After centrifugation, the supernatant was treated with ammonium sulfate, and the fractions obtained between 55–85% saturation were suspended in 25 mm citrate buffer (pH 5.3), dialyzed against the same buffer (buffer A), applied to CM-Sephadex C-50 column and eluted with the same buffer. Fractions displaying PDI activity were pooled, dialyzed against 20 mm sodium phosphate (pH 6.3) (buffer B), and applied to a DEAE-Sepharose fast flow column, which was eluted using a linear gradient of 0–0.7 m NaCl in buffer B. Purified PDI was kept at 4 °C and used in subsequent studies. The concentration of PDI was determined using absorbance at 280 nm = 1 for 1 mg of protein/ml (11Gilbert H.F. Methods Enzymol. 1998; 290: 26-50Crossref PubMed Scopus (120) Google Scholar). The activity of PDI was determined using the insulin reduction assay as described in Ref. 11Gilbert H.F. Methods Enzymol. 1998; 290: 26-50Crossref PubMed Scopus (120) Google Scholar. The Escherichia coli GroEL-GroESL gene (12Goloubinoff P. Gatenby A.A. Lorimer G.H. Nature. 1994; 337: 44-47Crossref Scopus (523) Google Scholar), inserted in a plasmid provided by Dr. F. Larimer (Oak Ridge Laboratories), was expressed in E. coli strain BL 21 (DE 3) cells. The protein GroEL was purified by modifications of the procedure included in Ref.13Makino Y. Taguchi H. Yoshida M. FEBS Lett. 1993; 336: 363-367Crossref PubMed Scopus (34) Google Scholar. After ammonium sulfate fractionation, the protein was purified by means of three chromatographic steps: DEAE-Sephacel, gel filtration through Sepharose CL-4B, and affinity chromatography through red agarose (14Todd M.J. Lorimer G.H. Methods Enzymol. 1998; 290: 135-141Crossref PubMed Scopus (20) Google Scholar). The last step removes contaminating proteins trapped by GroEL. The protein concentration was calculated using absorbance at 280 nm = 0.15 for 2.5 mg of protein/ml. Porcine heart mMDH was purchased from Roche Molecular Biochemicals. The enzyme was dialyzed against 20 mm Tris/HCl buffer, pH 7.5, at 4 °C, applied to a DEAE-cellulose column, and eluted by means of a linear gradient made with the equilibrium buffer (20 mm) and the same volume of 100 mm Tris/HCl (pH 7.5). The active fractions were concentrated by ultrafiltration. The enzyme concentration was calculated using A 280 = 2.5 for a 1% solution. (15Li W. Churchich J.E. Eur. J. Biochem. 1997; 246: 127-132Crossref PubMed Scopus (9) Google Scholar). A solution of MDH (5 mg/ml) was prepared in 100 mm Tris/HCl, pH 7.5, containing 3m GdnHCl and allowed to denature at 25 °C for 1 h. The protein solution was 5-fold diluted with 100 mmTris/HCl, pH 7.5, and then passed through a Sephadex G-25 column to remove GdnHCl. Renaturation of MDH was initiated by diluting denatured protein (0.1 μm) in renaturation buffer and incubated for 3 h at 25 °C. The renaturation buffer consisted of 100 mm Tris/HCl, pH 7.5, dithiothreitol (1 mm), and KCl (0.1 m) with and without chaperone proteins. Aliquots of this renaturing reaction were withdrawn at specific times and assayed for MDH activity. The assay buffer consisted of 100 mm Tris/HCl, pH 7.5, 0.5 mm oxaloacetate, and 0.2 mm NADH. The initial rate of conversion of NADH to NAD was determined by measuring changes in absorbance for 1 min at 25 °C. Porcine livers were obtained from a local slaughter house. DEAE-Sephacel, DEAE-Sepharose, CM-cellulose, DEAE-cellulose, and Sephadex-G-25 were purchased from Amersham Pharmacia Biotech. Insulin, GSH, NADPH, DTT, gluthione reductase, and scrambled RNase A were purchased from Sigma. The polypeptide mastoparan was obtained from Sigma. The reagent iodoacetamide fluorescein was purchased from Molecular Probes, Inc. (Eugene, OR). PDI (1 mg/ml) was reacted with iodoacetamide fluorescein (IAF) at a final concentration of 0.1 mm in 0.1 m potassium phosphate buffer (pH 7.4) containing 0.5 m GdnHCl at 4 °C for 12 h. Under this set of experimental conditions, SH groups located in the central domain of the protein are exposed to the alkylating reagent. The labeled protein was dialyzed against 0.1 m potassium phosphate (pH 7.4) to remove unreacted dye, followed by gel filtration chromatography on Sephadex-G-25. The labeled protein displayed catalytic activity when assayed using insulin as a substrate, suggesting that alkylation on the thioredoxin domain has not taken place. The degree of labeling (1.3 mol of IAF/mol of monomer) was determined by using an extinction coefficient of 4.9 × 104m−1 cm−1 at 490 nm. The modified protein was denatured in 0.8 ml of 6m guanidinium chloride containing dithiothreitol (1 mm) for 1 h at 37 °C. A freshly prepared solution of 20 mm iodoacetic acid was then added, and the mixture was incubated in the dark at room temperature for 30 min. The mixture was then dialyzed against 2 liters of 0.1 m ammonium bicarbonate. The labeled protein (100 nmol) was suspended in 0.8 ml of 0.1m ammonium bicarbonate, pH 8, and digested with trypsin for 24 h at 37 °C at a substrate/trypsin ratio of 40:1 (by mass). To 0.8 ml of tryptic digest, 50 μl of acetic acid was added, and the precipitate was removed by centrifugation. The solution was then lyophilized, and the peptides were separated by reverse-phase HPLC (Vydac C18 column). The separation was performed with a linear gradient of 10–80% B over 70 min at a flow rate of 0.5 ml/min. Eluant A was 0.1% trifluoroacetic acid, and eluant B was 0.1% trifluoroacetic acid in 80:20 acetonitrile/H2O. Absorbance was monitored at 220 nm, and fluorescence was monitored at 535 nm for the detection of labeled peptides. The fluorescent peptides were further purified with a linear gradient of 5–60% B over 30 min at a flow rate of 0.5 ml/min. The sequence of the isolated peptide, labeled with fluorescein, was determined by Edman degradation using an Applied Biosystems model sequencer (model 492 cLC). Emission spectra were recorded in a Perkin-Elmer LS-50B spectrofluorimeter. For fluorescein-labeled protein, the excitation was 480 nm, whereas for unlabeled proteins the excitation was set at 295 nm. Excitation and emission slits were set at 2.5 nm. The results of the fluorescence titration experiments were fitted to Equation 1 as follows, α/1−α=[mMDH]F/KDEquation 1 Where α = (F −F o )/(F M −F o ). F is the observed fluorescence,F o and F M the fluorescence intensities of free and bound IAF-PDI, respectively. In the analysis of the results, it was assumed that the stoichiometry of binding is 1 mol of IAF-PDI/1 mol of denatured mMDH. Since PDI is a multifunctional enzyme endowed with ATPase and chaperone activities, it was thought to be of interest to investigate whether both catalytic activities are linked during the process of refolding of a protein substrate. Is the ATPase activity enhanced during the refolding of the protein substrate? As a protein substrate of PDI, we have chosen an intermediate of denatured mMDH generated by GdnHCl denaturation of the wild type protein. Partially folded mMDH, containing 12% α-helix, exhibits exposed hydrophobic amino acid residues and is devoid of catalytic activity (15Li W. Churchich J.E. Eur. J. Biochem. 1997; 246: 127-132Crossref PubMed Scopus (9) Google Scholar). Moreover, the unfolded conformations of mMDH recognizes GroEL and regains a good deal of its catalytic activity (90%) in the presence of Mg ATP (16Hutchinson J.P. Elthaner T.S.H. Miller A.D. Biochem. J. 1994; 302: 405-410Crossref PubMed Scopus (30) Google Scholar, 17Churchich J.E. J. Biol. Chem. 1997; 272: 19645-19648Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, 18Ranson N.A. Burston S.E. Clarke A.R. J. Mol. Biol. 1997; 266: 656-664Crossref PubMed Scopus (80) Google Scholar). The results included in Fig. 1 show the time course of recovery of dehydrogenase activity in the presence of 1 mm DTT. Under this set of experimental conditions, the recovery of catalytic activity is due to spontaneous refolding of the protein after reduction of disulfide bonds generated during GdnHCl treatment (15Li W. Churchich J.E. Eur. J. Biochem. 1997; 246: 127-132Crossref PubMed Scopus (9) Google Scholar). A significant recovery of catalytic activity was observed in the presence of increasing concentrations of PDI. Maximum recovery of dehydrogenase activity, which amounts to approximately 50% of the wild-type protein, takes place when the concentration of PDI (2.2 μm) was 20-fold higher than the concentration of the protein substrate. A further increase in PDI concentration has no effect either on the rate or extent of recovery of catalytic activity. No antichaperone activity was detected at PDI concentrations above 2.0 μm. When similar reactivation experiments were performed in the presence of Mg-ATP (1 mm) at the optimal mixing molar ration of PDI/protein substrate of 20:1, the final recovery of catalytic activity was practically identical to that observed in the absence of Mg-ATP (Fig. 1). To ascertain whether the binding of the protein substrate influences ATPase activity displayed by PDI, the hydrolysis of ATP (1 mm) in the absence and presence of partially folded mMDH was measured using a coupled enzymatic assay consisting of pyruvate kinase and lactate dehydrogenase. As shown by the results included in Fig.2, the ATPase activity (k CAT = 0.26 min−1) characteristic of PDI remains practically invariant upon increasing the concentration of partially folded mMDH from 2 to 8 μm. In marked contrast to the lack of inhibition of ATPase activity by the binding of mMDH, the reduction of insulin is by small concentrations of partially folded mMDH as shown in Fig. that insulin and denatured mMDH binding on PDI. partially folded mMDH has been used in of the reported in this it be that the rate of reactivation of dehydrogenase is not by the of the initial unfolded used in the refolding the of reactivation of partially folded mMDH, prepared as described is similar to that observed when 3 m guanidinium enzyme is diluted the renaturation buffer containing PDI PDI which a emission at nm when at 295 nm. The of the emission is enhanced by the addition of amounts of denatured mMDH (Fig. mMDH not and the experimental chosen for the the enzyme not emission at nm. A increase in fluorescence is observed when the concentration of denatured protein from to μm. As shown in the of Fig. the fluorescence a at a mixing molar ratio of the proteins of approximately the molecular of mMDH and PDI as and respectively. In to the affinity of mMDH for PDI at a concentration with used in the reactivation it was to PDI with a fluorescent probe characterized by fluorescence at protein concentrations from to 0.1 μm. exhibits a of fluorescence at 535 nm due to the presence of the fluorescence The fluorescence of the probe = in the located from the amino acid residues and be used to the presence of in solution. Moreover, is bound to which be on the polypeptide by the protein. The emission of the at 480 nm is upon the addition of increasing concentrations of denatured proteins. shows the emission spectra of μm) recorded after the addition of denatured mMDH and scrambled RNase A. To the affinity of for partially folded mMDH, containing μm) and increasing concentrations of denatured protein were allowed to for 2 min at 25 °C, and their spectra were As shown in Fig. the fluorescence by is it remains practically constant when the concentration of partially folded mMDH is than μm. a stoichiometry of binding of the dissociation constant of the was to be 0.2 μm. To ascertain whether labeling of PDI by the probe has taken place in the central domains of the protein, derivatized PDI was to trypsin and the peptides were separated by HPLC as separation of tryptic peptides by HPLC using a reverse-phase column, one labeled peptide was (Fig. of the labeled peptide with the same column using a in further The characterized by a fluorescence at 535 nm, due to the presence of the fluorescein, were pooled, lyophilized, and Edman degradation the amino acid of the determined sequence of the modified peptide with the reported primary structure of PDI Nature. PubMed Scopus Google Scholar) that the labeled peptide to amino acid residues of the The as in the peptide sequence to in the The absence of labeled peptide the sequence to the active site of that the reaction with is to free groups in the central domain b′ of PDI. It has been reported that of PDI are in equilibrium with and at pH in m NaCl (9Guthapfel R. Gueguen P. Quemeneur E. J. Biol. Chem. 1996; 271: 2663-2666Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). The possibility that molecular of PDI bind small molecular weight peptides was investigated by gel filtration chromatography using 0.1 m Tris/HCl, pH as the When PDI, at 4 °C, was to gel one was The time of PDI is from characterized by a molecular of (Fig. The addition of the peptide mastoparan not in the of PDI, at concentrations required for saturation of the enzyme P. T. R. L.W. Freedman R.B. Eur. J. Biochem. 1998; PubMed Scopus Google Scholar). PDI not a process of in the absence or presence of molecular weight peptides. When the same experiments were performed with with mMDH and the were monitored by fluorescence measurements at 535 nm, we were to to of PDI not results not show or aggregation of PDI at The with other be to the of of purified PDI, it has been reported that a is by the in phosphate buffer N.A. H.F. A. 1983; Scopus Google Scholar). a protein, in containing GdnHCl. In the presence of 3m the protein shows and of the denatured protein with buffer a of the (15Li W. Churchich J.E. Eur. J. Biochem. 1997; 246: 127-132Crossref PubMed Scopus (9) Google Scholar). The partially folded of the catalytic activity after the addition of 1 mm DTT. The rate of recovery of catalytic activity of the dehydrogenase is influenced by the presence of PDI in the renaturation buffer. Under optimal of temperature and PDI concentration (2.2 the recovery of activity amounts to 50% of the wild-type protein. other characterized chaperone PDI by unfolding misfolded protein it that binding of the protein substrate to the the free energy required for of experimental are with this first, the rate of reactivation of partially folded mMDH is not influenced by the presence of Mg-ATP with PDI in the refolding buffer, and second, the ATPase activity of PDI is not by its with the protein substrate. In with the results by other laboratories (16Hutchinson J.P. Elthaner T.S.H. Miller A.D. Biochem. J. 1994; 302: 405-410Crossref PubMed Scopus (30) Google Scholar, 17Churchich J.E. J. Biol. Chem. 1997; 272: 19645-19648Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar), it was that unfolded mMDH a good deal of its catalytic activity (90%) when the denatured protein substrate with GroEL and Mg-ATP at concentrations of the proteins. In of these results, it is to PDI is than GroEL in assisting the refolding of partially folded mMDH. Based on the reported by other laboratories on the binding of small molecular weight peptides to PDI, it has been suggested that affinity of PDI for denatured proteins recovery of catalytic activity. on mMDH that derivatized binds denatured mMDH with a dissociation constant of 0.2 which is 5-fold higher than the dissociation constant determined for denatured mMDH bound to GroEL in the absence of Mg-ATP J.E. J. Biol. Chem. 1997; 272: 19645-19648Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). a 20-fold molar excess of PDI over the denatured protein substrate (0.1 μm) be to binding and reactivation of mMDH. However, the reactivation experiments in the presence of a 20-fold excess of PDI have shown recovery of catalytic activity. are of the of chaperone proteins with unfolded substrates that be in the analysis of the of PDI. GroEL with denatured mMDH, and the binding of Mg-ATP the chaperone through a functional in which refolding of the protein substrate the of GroEL N.A. Burston S.E. Clarke A.R. J. Mol. Biol. 1997; 266: 656-664Crossref PubMed Scopus (80) Google Scholar). of the protein substrate a that conformational and no with other proteins during the chaperone PDI not a for of a protein substrate of the of mMDH. On the other hand, the binding of small molecular weight polypeptides not of PDI of molecular weight that of the protein substrate to the Several laboratories have reported that the of misfolded proteins scrambled RNase with PDI with the binding of peptides (11Gilbert H.F. Methods Enzymol. 1998; 290: 26-50Crossref PubMed Scopus (120) Google Scholar, P. T. R. L.W. Freedman R.B. Eur. J. Biochem. 1998; PubMed Scopus Google Scholar). Based on these it has been suggested that the peptide binding site to a site at which PDI with unfolded regions of proteins during its in the the is the domain with peptide binding has not been it was reported that deletion of amino acid residues of the domain peptide binding and chaperone activity (6Dai Y. Wang C.C. J. Biol. Chem. 1997; 272 (27576): 27572Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). have shown that the deletion amino acid residues to the domain and a of the domain (7Koivunen P. Pirneskoski A. Karvonen P. Ljung J. Helakoski T. Trontbohm H. Kivirikko K.J. EMBO, J. 1999; 18: 65-74Crossref PubMed Scopus (54) Google Scholar). it has been reported that the b′ domain the peptide binding site of PDI (8Klappa P. Ruddeck L.W. Darby N.J. Freedman R.B. EMBO J. 1998; 17: 927-935Crossref PubMed Scopus (295) Google Scholar). In of these it that amino acid residues the domain to the of PDI with the protein substrate. fluorescence results have shown that binding of the protein substrate conformational changes in PDI propagated to domains of its the fluorescent probe in the b′ residues the a and a the conformational changes by the protein substrate. It is that the of the protein an role in as of disulfide bonds and refolding of protein substrates. the of in the of the tryptic peptides.