Interactions of Chaperone α-Crystallin with the Molten Globule State of Xylose Reductase
1998; Elsevier BV; Volume: 273; Issue: 16 Linguagem: Inglês
10.1074/jbc.273.16.9415
ISSN1083-351X
Autores Tópico(s)Heat shock proteins research
Resumoα-Crystallin is a multimeric protein that has been shown to function as a molecular chaperone. Present investigations were undertaken to understand its mechanism of chaperoning. For this functional in vitro analysis of α-crystallin we used xylose reductase (XR) from Neurospora crassa as the model system. Denaturation studies using the structure-perturbing agent guanidinium chloride indicated that XR folds through a partially folded state that resembles the molten globule. Fluorescence and delay experiments revealed that α-crystallin interacts with the molten globule state of XR (XR-m) and prevents its aggregation. Cold lability of α-crystallin·XR-m interaction was revealed by temperature shift experiments implicating the involvement of hydrophobic interactions in the formation of the complex. Reconstitution of active XR was observed on cooling the α-crystallin·XR-m complex to 4 °C or on addition of ATP at 37 °C. ATP hydrolysis is not a prerequisite for XR release since the nonhydrolyzable analogue 5′-adenylyl imidodiphosphate (AMP-PNP) was capable of reconstitution of active XR. Experimental evidence has been provided for temperature- and ATP-mediated structural changes in the α-crystallin·XR-m complex that shed some light on the mechanism of reconstitution of active XR by this chaperone. The relevance of our finding to the role of α-crystallin in vivo is discussed. α-Crystallin is a multimeric protein that has been shown to function as a molecular chaperone. Present investigations were undertaken to understand its mechanism of chaperoning. For this functional in vitro analysis of α-crystallin we used xylose reductase (XR) from Neurospora crassa as the model system. Denaturation studies using the structure-perturbing agent guanidinium chloride indicated that XR folds through a partially folded state that resembles the molten globule. Fluorescence and delay experiments revealed that α-crystallin interacts with the molten globule state of XR (XR-m) and prevents its aggregation. Cold lability of α-crystallin·XR-m interaction was revealed by temperature shift experiments implicating the involvement of hydrophobic interactions in the formation of the complex. Reconstitution of active XR was observed on cooling the α-crystallin·XR-m complex to 4 °C or on addition of ATP at 37 °C. ATP hydrolysis is not a prerequisite for XR release since the nonhydrolyzable analogue 5′-adenylyl imidodiphosphate (AMP-PNP) was capable of reconstitution of active XR. Experimental evidence has been provided for temperature- and ATP-mediated structural changes in the α-crystallin·XR-m complex that shed some light on the mechanism of reconstitution of active XR by this chaperone. The relevance of our finding to the role of α-crystallin in vivo is discussed. The folding and assembly of a protein into its biologically active conformation is a complex succession of reactions involving the formation of secondary and tertiary structures and domains, the pairing of domains, and the oligomerization of folded monomers (1Kim P.S. Baldwin R.L. Annu. Rev. Biochem. 1982; 51: 459-489Crossref PubMed Scopus (948) Google Scholar, 2Jaenicke R. Prog. Biophys. Mol. Biol. 1987; 49: 117-237Crossref PubMed Scopus (619) Google Scholar). Elucidation of the various processes that govern protein folding has been the focus of intense research for the past decades. Numerousin vitro protein folding experiments have demonstrated that many proteins successfully achieve their correct native structures in the complete absence of other cellular factors and without input of energy. This led to the expectation that protein folding is determined by the information encoded by the amino acid sequence and proceedsin vivo by the same spontaneous mechanism (1Kim P.S. Baldwin R.L. Annu. Rev. Biochem. 1982; 51: 459-489Crossref PubMed Scopus (948) Google Scholar, 2Jaenicke R. Prog. Biophys. Mol. Biol. 1987; 49: 117-237Crossref PubMed Scopus (619) Google Scholar). However,in vivo folding and assembly of proteins occur in a highly complex heterogeneous environment, in which high concentrations of proteins in various stages of folding and with potentially interactive surfaces coexist that may change the folding potentials inherent in the sequence. Recently, a number of accessory proteins have been identified that affect the folding and subsequent assembly of proteins. These include the protein isomerases catalyzingcis-trans-isomerization of peptide bonds or disulfide exchange (3Freedman R.B. Trends Biochem. Sci. 1984; 9: 438-441Abstract Full Text PDF Scopus (200) Google Scholar, 4Lang K. Schmid F.X. Fischer G. Nature. 1987; 329: 268-270Crossref PubMed Scopus (415) Google Scholar) and the polypeptide binding proteins termed as "molecular chaperones" (5Hendrick J.P. Hartl F.U. Annu. Rev. Biochem. 1993; 62: 349-384Crossref PubMed Scopus (1461) Google Scholar). The molecular mechanisms by which the protein isomerases accelerate the rate-determining step in the folding of proteins are understood but perhaps not in detail (3Freedman R.B. Trends Biochem. Sci. 1984; 9: 438-441Abstract Full Text PDF Scopus (200) Google Scholar, 4Lang K. Schmid F.X. Fischer G. Nature. 1987; 329: 268-270Crossref PubMed Scopus (415) Google Scholar). Elucidating the mechanistic details underlying the efficient refolding of proteins by chaperones now appears to be an important consideration for defining how proteins fold in vivo. The GroEL and GroES proteins from Escherichia coli are among the most detailed characterized chaperones (5Hendrick J.P. Hartl F.U. Annu. Rev. Biochem. 1993; 62: 349-384Crossref PubMed Scopus (1461) Google Scholar). Horwitz (6Horwitz J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10449-10453Crossref PubMed Scopus (1735) Google Scholar) and other workers (7Wang K. Spector A. J. Biol. Chem. 1994; 269: 13601-13608Abstract Full Text PDF PubMed Google Scholar, 8Raman B. Rao Ch. M. J. Biol. Chem. 1994; 269: 27264-27268Abstract Full Text PDF PubMed Google Scholar, 9Das K.P. Surewicz W.K. FEBS Lett. 1995; 369: 321-325Crossref PubMed Scopus (276) Google Scholar) have shown that α-crystallin acts as a molecular chaperone under various denaturing conditions including thermal inactivation, UV irradiation, or reduction of disulfide bonds. Until recently, α-crystallin was believed to be a lens-specific protein; however, now it has been reported to be present in many non-lenticular tissues (10Iwaki T. KumeIwaki A. Liem R. Goldman J.E. Cell. 1989; 57: 71-78Abstract Full Text PDF PubMed Scopus (484) Google Scholar, 11Klemenz R. Frohli E. Steiger R.H. Schafer R. Aoyama A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3652-3656Crossref PubMed Scopus (478) Google Scholar). The expression of α-crystallin has been shown to be induced by thermal (11Klemenz R. Frohli E. Steiger R.H. Schafer R. Aoyama A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3652-3656Crossref PubMed Scopus (478) Google Scholar) or hypertonic stress (12Dasgupta S. Hohman T.C. Carper D. Exp. Eye Res. 1992; 54: 461-470Crossref PubMed Scopus (179) Google Scholar). Numerous studies provide evidence that the ability of α-crystallin to suppress aggregation of damaged proteins plays a crucial role in maintaining the transparency of the ocular lens, and the failure of this function could contribute to the development of cataracts (13Groenen P.J. Merck K.B. de Jong W.W. Bloemendal H. Eur. J. Biochem. 1994; 225: 1-19Crossref PubMed Scopus (368) Google Scholar, 14Kelley M.J. David L. Iwasaki N. Wright J. Shearer T.R. J. Biol. Chem. 1993; 268: 18844-18849Abstract Full Text PDF PubMed Google Scholar). Important recent development is the finding that α-crystallin shows extensive structural similarity with small heat shock proteins that are known to act in vitro as molecular chaperones (15Merck K.B. Groenen P.J. Voorter C.E.M. de Haard-Hoekman W.A. Horwitz J. Bloemendal H. de Jong W. J. Biol. Chem. 1993; 268: 1046-1052Abstract Full Text PDF PubMed Google Scholar, 16Jakob U. Gaestel M. Engel K. Buchner J. J. Biol. Chem. 1993; 268: 1517-1520Abstract Full Text PDF PubMed Google Scholar). α-Crystallin has been shown to be functionally equivalent to the small heat shock proteins namely murine Hsp25 and human Hsp27 in refolding of α-glucosidase and citrate synthase in vitro (16Jakob U. Gaestel M. Engel K. Buchner J. J. Biol. Chem. 1993; 268: 1517-1520Abstract Full Text PDF PubMed Google Scholar). It was, however, unable to refold rhodanase denatured in 6 mGdmCl 1The abbreviations used are: GdmCl, guanidinium chloride; XR, xylose reductase; XR-u, unfolded state of XR; XR-m, molten globule state of XR; ANS, 8-anilinonaphthalene-1-sulfonic acid; AMP-PNP, 5′-adenylyl imidodiphosphate. 1The abbreviations used are: GdmCl, guanidinium chloride; XR, xylose reductase; XR-u, unfolded state of XR; XR-m, molten globule state of XR; ANS, 8-anilinonaphthalene-1-sulfonic acid; AMP-PNP, 5′-adenylyl imidodiphosphate. (17Das K.P. Surewicz W.K. Biochem. J. 1995; 311: 367-370Crossref PubMed Scopus (78) Google Scholar). Recently α-crystallin has been reported to bind the temperature-induced molten globule state of proteins (18Rajaraman K. Raman B. Rao Ch. M. J. Biol. Chem. 1996; 271: 27595-27600Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 19Das K.P. Petrash J.M. Surewicz W.K. J. Biol. Chem. 1996; 271: 10449-10452Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar) and prevent photoaggregation of γ-crystallin by providing hydrophobic surfaces (8Raman B. Rao Ch. M. J. Biol. Chem. 1994; 269: 27264-27268Abstract Full Text PDF PubMed Google Scholar). Despite the growing interest in the chaperone action of α-crystallin, little is known about its mechanism of chaperoning. For this functional in vitro analysis of α-crystallin we used xylose reductase (XR), from Neurospora crassa as the model system. This oxidoreductase plays a crucial role in the fermentation of xylose to ethanol (20Rawat U.B. Bodhe A. Deshpande V. Rao M. Biotechnol. Lett. 1993; 15: 1173-1178Crossref Scopus (18) Google Scholar) and has recently gained more importance due to its application in the synthesis of xylitol, an acariogenic non-caloric sweetener used in food products (21Bernd N. Wilfried N. Kulbe K.D. Klaus D. Biotechnol. Bioeng. 1996; 52: 387-396PubMed Google Scholar). Earlier we have undertaken structure-function studies to understand the contribution of essential amino acids in the catalytic mechanism of XR, an enzyme that belongs to the important class of oxidoreductases; the conformation and microenvironment of the active site has also been assessed using fluorescent chemoaffinity labeling (22Rawat U.B. Rao M.B. Biochim. Biophys. Acta. 1996; 1293: 222-230Crossref PubMed Scopus (24) Google Scholar, 23Rawat U.B. Rao M. Eur. J. Biochem. 1997; 246: 344-349Crossref PubMed Scopus (11) Google Scholar, 24Rawat U.B. Rao M. Biochem. Biophys. Res. Commun. 1997; 239: 789-793Crossref PubMed Scopus (3) Google Scholar). Various studies on XR will assist in its biotechnological exploitation. Experimental evidence presented in this paper serves to implicate that the chaperone α-crystallin stabilizes the molten globule state of XR and thus restrains the non-native conformer from exploring unproductive pathways. Lowering the temperature to 4 °C or presence of ATP at 37 °C induces a conformational change in α-crystallin·XR-m complex that is accompanied by a concomitant internalization of hydrophobic surfaces previously exposed. This acts to reduce the hydrophobic interactions that facilitates the dissociation of the complex further allowing reconstitution of the active XR. This paper reports for the first time the mechanism of α-crystallin-mediated reconstitution of an active enzyme and the role of ATP in the function of α-crystallin as a molecular chaperone. The folding and assembly of a protein into its biologically active conformation is a complex succession of reactions involving the formation of secondary and tertiary structures and domains, the pairing of domains, and the oligomerization of folded monomers (1Kim P.S. Baldwin R.L. Annu. Rev. Biochem. 1982; 51: 459-489Crossref PubMed Scopus (948) Google Scholar, 2Jaenicke R. Prog. Biophys. Mol. Biol. 1987; 49: 117-237Crossref PubMed Scopus (619) Google Scholar). Elucidation of the various processes that govern protein folding has been the focus of intense research for the past decades. Numerousin vitro protein folding experiments have demonstrated that many proteins successfully achieve their correct native structures in the complete absence of other cellular factors and without input of energy. This led to the expectation that protein folding is determined by the information encoded by the amino acid sequence and proceedsin vivo by the same spontaneous mechanism (1Kim P.S. Baldwin R.L. Annu. Rev. Biochem. 1982; 51: 459-489Crossref PubMed Scopus (948) Google Scholar, 2Jaenicke R. Prog. Biophys. Mol. Biol. 1987; 49: 117-237Crossref PubMed Scopus (619) Google Scholar). However,in vivo folding and assembly of proteins occur in a highly complex heterogeneous environment, in which high concentrations of proteins in various stages of folding and with potentially interactive surfaces coexist that may change the folding potentials inherent in the sequence. Recently, a number of accessory proteins have been identified that affect the folding and subsequent assembly of proteins. These include the protein isomerases catalyzingcis-trans-isomerization of peptide bonds or disulfide exchange (3Freedman R.B. Trends Biochem. Sci. 1984; 9: 438-441Abstract Full Text PDF Scopus (200) Google Scholar, 4Lang K. Schmid F.X. Fischer G. Nature. 1987; 329: 268-270Crossref PubMed Scopus (415) Google Scholar) and the polypeptide binding proteins termed as "molecular chaperones" (5Hendrick J.P. Hartl F.U. Annu. Rev. Biochem. 1993; 62: 349-384Crossref PubMed Scopus (1461) Google Scholar). The molecular mechanisms by which the protein isomerases accelerate the rate-determining step in the folding of proteins are understood but perhaps not in detail (3Freedman R.B. Trends Biochem. Sci. 1984; 9: 438-441Abstract Full Text PDF Scopus (200) Google Scholar, 4Lang K. Schmid F.X. Fischer G. Nature. 1987; 329: 268-270Crossref PubMed Scopus (415) Google Scholar). Elucidating the mechanistic details underlying the efficient refolding of proteins by chaperones now appears to be an important consideration for defining how proteins fold in vivo. The GroEL and GroES proteins from Escherichia coli are among the most detailed characterized chaperones (5Hendrick J.P. Hartl F.U. Annu. Rev. Biochem. 1993; 62: 349-384Crossref PubMed Scopus (1461) Google Scholar). Horwitz (6Horwitz J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10449-10453Crossref PubMed Scopus (1735) Google Scholar) and other workers (7Wang K. Spector A. J. Biol. Chem. 1994; 269: 13601-13608Abstract Full Text PDF PubMed Google Scholar, 8Raman B. Rao Ch. M. J. Biol. Chem. 1994; 269: 27264-27268Abstract Full Text PDF PubMed Google Scholar, 9Das K.P. Surewicz W.K. FEBS Lett. 1995; 369: 321-325Crossref PubMed Scopus (276) Google Scholar) have shown that α-crystallin acts as a molecular chaperone under various denaturing conditions including thermal inactivation, UV irradiation, or reduction of disulfide bonds. Until recently, α-crystallin was believed to be a lens-specific protein; however, now it has been reported to be present in many non-lenticular tissues (10Iwaki T. KumeIwaki A. Liem R. Goldman J.E. Cell. 1989; 57: 71-78Abstract Full Text PDF PubMed Scopus (484) Google Scholar, 11Klemenz R. Frohli E. Steiger R.H. Schafer R. Aoyama A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3652-3656Crossref PubMed Scopus (478) Google Scholar). The expression of α-crystallin has been shown to be induced by thermal (11Klemenz R. Frohli E. Steiger R.H. Schafer R. Aoyama A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3652-3656Crossref PubMed Scopus (478) Google Scholar) or hypertonic stress (12Dasgupta S. Hohman T.C. Carper D. Exp. Eye Res. 1992; 54: 461-470Crossref PubMed Scopus (179) Google Scholar). Numerous studies provide evidence that the ability of α-crystallin to suppress aggregation of damaged proteins plays a crucial role in maintaining the transparency of the ocular lens, and the failure of this function could contribute to the development of cataracts (13Groenen P.J. Merck K.B. de Jong W.W. Bloemendal H. Eur. J. Biochem. 1994; 225: 1-19Crossref PubMed Scopus (368) Google Scholar, 14Kelley M.J. David L. Iwasaki N. Wright J. Shearer T.R. J. Biol. Chem. 1993; 268: 18844-18849Abstract Full Text PDF PubMed Google Scholar). Important recent development is the finding that α-crystallin shows extensive structural similarity with small heat shock proteins that are known to act in vitro as molecular chaperones (15Merck K.B. Groenen P.J. Voorter C.E.M. de Haard-Hoekman W.A. Horwitz J. Bloemendal H. de Jong W. J. Biol. Chem. 1993; 268: 1046-1052Abstract Full Text PDF PubMed Google Scholar, 16Jakob U. Gaestel M. Engel K. Buchner J. J. Biol. Chem. 1993; 268: 1517-1520Abstract Full Text PDF PubMed Google Scholar). α-Crystallin has been shown to be functionally equivalent to the small heat shock proteins namely murine Hsp25 and human Hsp27 in refolding of α-glucosidase and citrate synthase in vitro (16Jakob U. Gaestel M. Engel K. Buchner J. J. Biol. Chem. 1993; 268: 1517-1520Abstract Full Text PDF PubMed Google Scholar). It was, however, unable to refold rhodanase denatured in 6 mGdmCl 1The abbreviations used are: GdmCl, guanidinium chloride; XR, xylose reductase; XR-u, unfolded state of XR; XR-m, molten globule state of XR; ANS, 8-anilinonaphthalene-1-sulfonic acid; AMP-PNP, 5′-adenylyl imidodiphosphate. 1The abbreviations used are: GdmCl, guanidinium chloride; XR, xylose reductase; XR-u, unfolded state of XR; XR-m, molten globule state of XR; ANS, 8-anilinonaphthalene-1-sulfonic acid; AMP-PNP, 5′-adenylyl imidodiphosphate. (17Das K.P. Surewicz W.K. Biochem. J. 1995; 311: 367-370Crossref PubMed Scopus (78) Google Scholar). Recently α-crystallin has been reported to bind the temperature-induced molten globule state of proteins (18Rajaraman K. Raman B. Rao Ch. M. J. Biol. Chem. 1996; 271: 27595-27600Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 19Das K.P. Petrash J.M. Surewicz W.K. J. Biol. Chem. 1996; 271: 10449-10452Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar) and prevent photoaggregation of γ-crystallin by providing hydrophobic surfaces (8Raman B. Rao Ch. M. J. Biol. Chem. 1994; 269: 27264-27268Abstract Full Text PDF PubMed Google Scholar). Despite the growing interest in the chaperone action of α-crystallin, little is known about its mechanism of chaperoning. For this functional in vitro analysis of α-crystallin we used xylose reductase (XR), from Neurospora crassa as the model system. This oxidoreductase plays a crucial role in the fermentation of xylose to ethanol (20Rawat U.B. Bodhe A. Deshpande V. Rao M. Biotechnol. Lett. 1993; 15: 1173-1178Crossref Scopus (18) Google Scholar) and has recently gained more importance due to its application in the synthesis of xylitol, an acariogenic non-caloric sweetener used in food products (21Bernd N. Wilfried N. Kulbe K.D. Klaus D. Biotechnol. Bioeng. 1996; 52: 387-396PubMed Google Scholar). Earlier we have undertaken structure-function studies to understand the contribution of essential amino acids in the catalytic mechanism of XR, an enzyme that belongs to the important class of oxidoreductases; the conformation and microenvironment of the active site has also been assessed using fluorescent chemoaffinity labeling (22Rawat U.B. Rao M.B. Biochim. Biophys. Acta. 1996; 1293: 222-230Crossref PubMed Scopus (24) Google Scholar, 23Rawat U.B. Rao M. Eur. J. Biochem. 1997; 246: 344-349Crossref PubMed Scopus (11) Google Scholar, 24Rawat U.B. Rao M. Biochem. Biophys. Res. Commun. 1997; 239: 789-793Crossref PubMed Scopus (3) Google Scholar). Various studies on XR will assist in its biotechnological exploitation. Experimental evidence presented in this paper serves to implicate that the chaperone α-crystallin stabilizes the molten globule state of XR and thus restrains the non-native conformer from exploring unproductive pathways. Lowering the temperature to 4 °C or presence of ATP at 37 °C induces a conformational change in α-crystallin·XR-m complex that is accompanied by a concomitant internalization of hydrophobic surfaces previously exposed. This acts to reduce the hydrophobic interactions that facilitates the dissociation of the complex further allowing reconstitution of the active XR. This paper reports for the first time the mechanism of α-crystallin-mediated reconstitution of an active enzyme and the role of ATP in the function of α-crystallin as a molecular chaperone. We thank Prof. S. Mitra, Tata Institute of Fundamental Research, Bombay, for permitting the use of CD facility and Dr. K. N. Ganesh, National Chemical Laboratory, for the use of spectrofluorometer.
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