Domain Identification of Hormone-sensitive Lipase by Circular Dichroism and Fluorescence Spectroscopy, Limited Proteolysis, and Mass Spectrometry
1999; Elsevier BV; Volume: 274; Issue: 22 Linguagem: Inglês
10.1074/jbc.274.22.15382
ISSN1083-351X
AutoresTorben Østerlund, Douglas J. Beussman, Karin Julenius, Pak H. Poon, Sara Linse, Jeffrey Shabanowitz, Donald F. Hunt, Michael C. Schotz, Zygmunt S. Derewenda, Cecilia Holm,
Tópico(s)Lipid metabolism and biosynthesis
ResumoStructure-function relationship analyses of hormone-sensitive lipase (HSL) have suggested that this metabolically important enzyme consists of several functional and at least two structural domains (Østerlund, T., Danielsson, B., Degerman, E., Contreras, J. A., Edgren, G., Davis, R. C., Schotz, M. C., and Holm, C. (1996) Biochem. J. 319, 411–420; Contreras, J. A., Karlsson, M., Østerlund, T., Laurell, H., Svensson, A., and Holm, C. (1996) J. Biol. Chem. 271, 31426–31430). To analyze the structural domain composition of HSL in more detail, we applied biophysical methods. Denaturation of HSL was followed by circular dichroism measurements and fluorescence spectroscopy, revealing that the unfolding of HSL is a two-step event. Using limited proteolysis in combination with mass spectrometry, several proteolytic fragments of HSL were identified, including one corresponding exactly to the proposed N-terminal domain. Major cleavage sites were found in the predicted hinge region between the two domains and in the regulatory module of the C-terminal, catalytic domain. Analyses of a hinge region cleavage mutant and calculations of the hydropathic pattern of HSL further suggest that the hinge region and regulatory module are exposed parts of HSL. Together, these data support our previous hypothesis that HSL consists of two major structural domains, encoded by exons 1–4 and 5–9, respectively, of which the latter contains an exposed regulatory module outside the catalytic α/β-hydrolase fold core. Structure-function relationship analyses of hormone-sensitive lipase (HSL) have suggested that this metabolically important enzyme consists of several functional and at least two structural domains (Østerlund, T., Danielsson, B., Degerman, E., Contreras, J. A., Edgren, G., Davis, R. C., Schotz, M. C., and Holm, C. (1996) Biochem. J. 319, 411–420; Contreras, J. A., Karlsson, M., Østerlund, T., Laurell, H., Svensson, A., and Holm, C. (1996) J. Biol. Chem. 271, 31426–31430). To analyze the structural domain composition of HSL in more detail, we applied biophysical methods. Denaturation of HSL was followed by circular dichroism measurements and fluorescence spectroscopy, revealing that the unfolding of HSL is a two-step event. Using limited proteolysis in combination with mass spectrometry, several proteolytic fragments of HSL were identified, including one corresponding exactly to the proposed N-terminal domain. Major cleavage sites were found in the predicted hinge region between the two domains and in the regulatory module of the C-terminal, catalytic domain. Analyses of a hinge region cleavage mutant and calculations of the hydropathic pattern of HSL further suggest that the hinge region and regulatory module are exposed parts of HSL. Together, these data support our previous hypothesis that HSL consists of two major structural domains, encoded by exons 1–4 and 5–9, respectively, of which the latter contains an exposed regulatory module outside the catalytic α/β-hydrolase fold core. The release of fatty acids from stored triglycerides in adipocytes is accomplished by hormone-sensitive lipase (HSL) 1The abbreviations used are: HSL, hormone-sensitive lipase; DTE, dithioerythritol; GdnCl, guanidine hydrochloride; SDS-PAGE, SDS-polyacrylamide gel electrophoresis1The abbreviations used are: HSL, hormone-sensitive lipase; DTE, dithioerythritol; GdnCl, guanidine hydrochloride; SDS-PAGE, SDS-polyacrylamide gel electrophoresis and monoglyceride lipase. Lipolysis is regulated by hormones and neurotransmitters, and the major target of this regulation is HSL (1Strålfors P. Olsson H. Belfrage P. Boyer P.D. Krebs E.G. The Enzymes. 18. Academic Press, New York1987: 147-177Google Scholar, 2Holm C. Langin D. Manganiello V. Belfrage P. Degerman E. Methods Enzymol. 1997; 286: 45-67Crossref PubMed Scopus (50) Google Scholar). Catabolic hormones and neurotransmitters (e.g. norepinephrine) activate cAMP-dependent protein kinase, which, in turn, phosphorylates and activates HSL. The major antilipolytic hormone is insulin, which activates phosphodiesterase 3B that decreases cAMP levels and thereby deactivates cAMP-dependent protein kinase (3Degerman E. Belfrage P. Manganiello V.C. J. Biol. Chem. 1997; 272: 6823-6826Abstract Full Text Full Text PDF PubMed Scopus (383) Google Scholar). The regulation by reversible phosphorylation makes HSL unique among lipases. Phosphorylation of HSL by cAMP-dependent protein kinase occurs at three sites, the serines 563, 659, and 660, 2The residue numbering is for rat HSL.2The residue numbering is for rat HSL.both in vitro and in primary rat adipocytes (4Anthonsen M.W. Rönnstrand L. Wernstedt C. Degerman E. Holm C. J. Biol. Chem. 1998; 273: 215-221Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). A fourth site of phosphorylation, Ser-565, may be a substrate of the AMP-activated protein kinase, because this has been shown to be the case in vitro (5Garton A.J. Cambell D.G. Carling D. Hardie D.G. Colbran R.J. Yeaman S.J. Eur. J. Biochem. 1989; 129: 249-254Crossref Scopus (228) Google Scholar). Also, preincubations of primary rat adipocytes with the AMP-mimicking agent 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside inhibit isoprenaline-induced lipolysis (6Sullivan J.E. Brocklehurst K.J. Marley A.E. Carey F. Carling D. Beri R.K. FEBS Lett. 1994; 353: 33-36Crossref PubMed Scopus (410) Google Scholar), thus lending support for a role of AMP-activated protein kinase in the regulation of HSL.HSL is known to associate with phospholipid vesicles (7Holm C. Fredrikson G. Sundler R. Belfrage P. Lipids. 1990; 25: 254-259Crossref PubMed Scopus (10) Google Scholar) and to have an overall amphipathic character (8Holm C. Fredrikson G. Belfrage P. J. Biol. Chem. 1986; 261: 15659-15661Abstract Full Text PDF PubMed Google Scholar). This suggests that HSL has one or more sites at the surface, enabling interactions with detergent and membranes such as the surface of intracellular lipid droplets. Specific recognition of these lipid droplets, as opposed to other membrane surfaces, would constitute another unique feature of HSL among lipases.As for other lipases and esterases, it was expected that HSL, at least in part, harbors the α/β-hydrolase fold and has a catalytic triad (Ser, Asp/Glu, His) (9Derewenda Z.S. Schumaker V.N. Advances in Protein Chemistry. 45. Elsevier, Amsterdam1994: 1-52Google Scholar). To map these and other features in HSL, we have initiated analyses into its structural and functional domains by alignments to other lipases and esterases and by limited proteolysis, denaturation, site-directed mutagenesis, and molecular modeling. Analysis by limited proteolysis and denaturation suggested that HSL has at least two structural domains (10Østerlund T. Danielsson B. Degerman E. Contreras J.A. Edgren G. Davis R.C. Schotz M.C. Holm C. Biochem. J. 1996; 319: 411-420Crossref PubMed Scopus (137) Google Scholar). Those regions in the C-terminal part of the molecule that align to other lipases and esterases are thought to adopt the α/β-hydrolase fold and to harbor the catalytic triad (10Østerlund T. Danielsson B. Degerman E. Contreras J.A. Edgren G. Davis R.C. Schotz M.C. Holm C. Biochem. J. 1996; 319: 411-420Crossref PubMed Scopus (137) Google Scholar, 11Contreras J.A. Karlsson M. Østerlund T. Laurell H. Svensson A. Holm C. J. Biol. Chem. 1996; 271: 31426-31430Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). In fact, it was possible to build a model for the catalytic α/β-hydrolase fold core (11Contreras J.A. Karlsson M. Østerlund T. Laurell H. Svensson A. Holm C. J. Biol. Chem. 1996; 271: 31426-31430Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). The proposed residues of the catalytic triad have been probed by site-directed mutagenesis and found to be essential for activity (12Holm C. Davis R.C. Østerlund T. Schotz M.C. Fredrikson G. FEBS Lett. 1994; 344: 234-238Crossref PubMed Scopus (56) Google Scholar, 13Østerlund T. Contreras J.A. Holm C. FEBS Lett. 1997; 403: 259-262Crossref PubMed Scopus (47) Google Scholar), strongly supporting the structural model for the catalytic core. In the primary structure, this core is interrupted by approximately 200 residues, including the four phosphorylation sites, which are thought to form a regulatory module (10Østerlund T. Danielsson B. Degerman E. Contreras J.A. Edgren G. Davis R.C. Schotz M.C. Holm C. Biochem. J. 1996; 319: 411-420Crossref PubMed Scopus (137) Google Scholar, 11Contreras J.A. Karlsson M. Østerlund T. Laurell H. Svensson A. Holm C. J. Biol. Chem. 1996; 271: 31426-31430Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). This part is a substrate of kinases and phosphatases and has been predicted to contain only a few secondary structure elements (11Contreras J.A. Karlsson M. Østerlund T. Laurell H. Svensson A. Holm C. J. Biol. Chem. 1996; 271: 31426-31430Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Thus, it is suspected to be an exposed and rather flexible module. The N-terminal part of the protein (approximately 320 residues) is believed to constitute a separate structural domain and shows no significant sequence similarity to other proteins (10Østerlund T. Danielsson B. Degerman E. Contreras J.A. Edgren G. Davis R.C. Schotz M.C. Holm C. Biochem. J. 1996; 319: 411-420Crossref PubMed Scopus (137) Google Scholar).To establish the structural domain composition of HSL, we have performed spectroscopic analyses during denaturation. Data from these analyses support the idea that HSL has two major structural domains. Limited proteolysis analyses (10Østerlund T. Danielsson B. Degerman E. Contreras J.A. Edgren G. Davis R.C. Schotz M.C. Holm C. Biochem. J. 1996; 319: 411-420Crossref PubMed Scopus (137) Google Scholar) have been extended through identification of generated peptides by mass spectrometry to identify exposed regions of the intact protein and to identify more stable fragments of the HSL molecule. Both the suggested hinge region and the regulatory module are exposed to proteolysis, whereas the N-terminal domain appears to be highly resistant to cleavage. Analysis by proteolysis of a HSL mutant with an engineered Factor X site in the predicted hinge confirms these results, and further support is provided by analysis of the hydropathic pattern of HSL. Based on the generated results, we present a more complete overall structural model for HSL.DISCUSSIONIn this study, further evidence for our previously proposed model for the domain structure of HSL is provided (10Østerlund T. Danielsson B. Degerman E. Contreras J.A. Edgren G. Davis R.C. Schotz M.C. Holm C. Biochem. J. 1996; 319: 411-420Crossref PubMed Scopus (137) Google Scholar, 11Contreras J.A. Karlsson M. Østerlund T. Laurell H. Svensson A. Holm C. J. Biol. Chem. 1996; 271: 31426-31430Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). The denaturation analyses support that HSL has at least two (and probably only two) structural domains. The fluorescence data clearly suggest the unfolding of two individually folded domains. Although HSL appears to be a homodimer in solution, it is unlikely that the dissociation of dimers contributes significantly to the loss in fluorescence at low GdnCl concentrations. CD measurements during denaturation by GdnCl and heat indicate that the unfolding of secondary structure elements occurs in two major steps. It cannot be ruled out that some protein aggregation occurs in the thermal denaturation. However, it does not seem to have any significant influence on the CD signal.From both spectroscopic data and proteolytic analyses, it appears that one domain is more fragile than the other. The fragile domain is unfolded by low GdnCl concentrations and probably represents the C-terminal domain, because all HSL activities are lost concomitant with its unfolding (10Østerlund T. Danielsson B. Degerman E. Contreras J.A. Edgren G. Davis R.C. Schotz M.C. Holm C. Biochem. J. 1996; 319: 411-420Crossref PubMed Scopus (137) Google Scholar). This domain is also highly sensitive to proteolytic digestion, especially in the regulatory module, as shown by proteolytic cleavage by both EndoL and Factor X. The second unfolding, which occurs at high GdnCl concentrations, probably represents the N-terminal domain. Because detergent and perhaps glycerol had a more protective effect on this domain than on the other domain, it is speculated that some sites of detergent and membrane interactions are located here. Other sites might be found in the α/β-hydrolase fold core, although most hydrophobic residues of this part are not exposed (11Contreras J.A. Karlsson M. Østerlund T. Laurell H. Svensson A. Holm C. J. Biol. Chem. 1996; 271: 31426-31430Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). To directly compare the pattern of hydrophobicity in the primary structure of HSL with the proposed domain structure, the mean hydropathic index was calculated for every 60 residues, according to Kyte and Doolittle (19Kyte J. Doolittle R.F. J. Mol. Biol. 1982; 157: 105-132Crossref PubMed Scopus (17007) Google Scholar). Fig. 8 shows the calculated hydropathy indices with indications of the localization of suggested domains in the primary structure. Hydrophilic regions correlate well with the anticipated hinge region and regulatory module.Arrows indicate the major cleavage sites as identified in Fig. 6. The most hydrophobic regions are those of the α/β-hydrolase fold, whereas the N-terminal domain has an amphipathic character. Because the regulatory module is probably located at the surface of the C-terminal domain, and most of the hydrophobic residues of the α/β-hydrolase fold are located in the core of the fold (11Contreras J.A. Karlsson M. Østerlund T. Laurell H. Svensson A. Holm C. J. Biol. Chem. 1996; 271: 31426-31430Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), there are probably only a few exposed hydrophobic patches in this part. The membrane/detergent binding sites are presumably located at these patches as well as at hydrophobic patches in the N-terminal domain. Overall, the hydropathy calculations support the suggested domain structure, particularly by demonstrating that the exposed regions are markedly hydrophilic.The exact location of the hinge region in the primary structure has been the subject of some speculation (10Østerlund T. Danielsson B. Degerman E. Contreras J.A. Edgren G. Davis R.C. Schotz M.C. Holm C. Biochem. J. 1996; 319: 411-420Crossref PubMed Scopus (137) Google Scholar, 11Contreras J.A. Karlsson M. Østerlund T. Laurell H. Svensson A. Holm C. J. Biol. Chem. 1996; 271: 31426-31430Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). The cleavage at Lys-323 and the hydrophilic character of this region support the concept that the N- and C-terminal domains are separated at the hinge region (residues 315–335) as suggested from alignments (10Østerlund T. Danielsson B. Degerman E. Contreras J.A. Edgren G. Davis R.C. Schotz M.C. Holm C. Biochem. J. 1996; 319: 411-420Crossref PubMed Scopus (137) Google Scholar). Cleavage of HSLFacX by Factor X shows that this region is immediately accessible to the protease. Our current view and working hypothesis of the domain structure is illustrated in Fig.9 with indications of cleavage sites and action of GdnCl at different concentrations. The secondary structure elements of the α/β-hydrolase fold are outlined (11Contreras J.A. Karlsson M. Østerlund T. Laurell H. Svensson A. Holm C. J. Biol. Chem. 1996; 271: 31426-31430Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar).Figure 9Hypothetical structural model of HSL . It is suggested that HSL has two major structural domains: one N-terminal domain (dark gray) formed approximately by residues 1–320, and a C-terminal catalytic domain (frame). One part of the C-terminal domain harbors the α/β-hydrolase fold that is shown outlined by its secondary structure elements (β-strands aregray arrows, α-helices are rectangles), as they were identified previously (11Contreras J.A. Karlsson M. Østerlund T. Laurell H. Svensson A. Holm C. J. Biol. Chem. 1996; 271: 31426-31430Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). The other part of the C-terminal domain is believed to form a regulatory module (light gray). Residues of particular importance are shown. These include members of the catalytic triad (Ser-423, Asp-703, and His-733) (10Østerlund T. Danielsson B. Degerman E. Contreras J.A. Edgren G. Davis R.C. Schotz M.C. Holm C. Biochem. J. 1996; 319: 411-420Crossref PubMed Scopus (137) Google Scholar, 11Contreras J.A. Karlsson M. Østerlund T. Laurell H. Svensson A. Holm C. J. Biol. Chem. 1996; 271: 31426-31430Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 12Holm C. Davis R.C. Østerlund T. Schotz M.C. Fredrikson G. FEBS Lett. 1994; 344: 234-238Crossref PubMed Scopus (56) Google Scholar, 13Østerlund T. Contreras J.A. Holm C. FEBS Lett. 1997; 403: 259-262Crossref PubMed Scopus (47) Google Scholar) and phosphorylation sites (Ser-563, Ser-565, Ser-659, and Ser-660) (4Anthonsen M.W. Rönnstrand L. Wernstedt C. Degerman E. Holm C. J. Biol. Chem. 1998; 273: 215-221Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar).Black arrows indicate the identified major cleavage sites, and C represents the C terminus.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In conclusion, the present study provides additional support for the proposal that HSL is composed of two major structural domains encoded by exons 1–4 and 5–9, respectively. The latter is the catalytic domain formed by an α/β-hydrolase fold core (11Contreras J.A. Karlsson M. Østerlund T. Laurell H. Svensson A. Holm C. J. Biol. Chem. 1996; 271: 31426-31430Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), which is interrupted in the primary structure by the insertion of a regulatory module. Both the regulatory module and the suggested hinge are exposed hydrophilic parts. Additional investigations of HSLFacXbefore and after cleavage and the expression of individual domains are underway. Purified domains and fragments will be valuable tools for structural and functional analyses as well as the determination of the three-dimensional structure of HSL. The release of fatty acids from stored triglycerides in adipocytes is accomplished by hormone-sensitive lipase (HSL) 1The abbreviations used are: HSL, hormone-sensitive lipase; DTE, dithioerythritol; GdnCl, guanidine hydrochloride; SDS-PAGE, SDS-polyacrylamide gel electrophoresis1The abbreviations used are: HSL, hormone-sensitive lipase; DTE, dithioerythritol; GdnCl, guanidine hydrochloride; SDS-PAGE, SDS-polyacrylamide gel electrophoresis and monoglyceride lipase. Lipolysis is regulated by hormones and neurotransmitters, and the major target of this regulation is HSL (1Strålfors P. Olsson H. Belfrage P. Boyer P.D. Krebs E.G. The Enzymes. 18. Academic Press, New York1987: 147-177Google Scholar, 2Holm C. Langin D. Manganiello V. Belfrage P. Degerman E. Methods Enzymol. 1997; 286: 45-67Crossref PubMed Scopus (50) Google Scholar). Catabolic hormones and neurotransmitters (e.g. norepinephrine) activate cAMP-dependent protein kinase, which, in turn, phosphorylates and activates HSL. The major antilipolytic hormone is insulin, which activates phosphodiesterase 3B that decreases cAMP levels and thereby deactivates cAMP-dependent protein kinase (3Degerman E. Belfrage P. Manganiello V.C. J. Biol. Chem. 1997; 272: 6823-6826Abstract Full Text Full Text PDF PubMed Scopus (383) Google Scholar). The regulation by reversible phosphorylation makes HSL unique among lipases. Phosphorylation of HSL by cAMP-dependent protein kinase occurs at three sites, the serines 563, 659, and 660, 2The residue numbering is for rat HSL.2The residue numbering is for rat HSL.both in vitro and in primary rat adipocytes (4Anthonsen M.W. Rönnstrand L. Wernstedt C. Degerman E. Holm C. J. Biol. Chem. 1998; 273: 215-221Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). A fourth site of phosphorylation, Ser-565, may be a substrate of the AMP-activated protein kinase, because this has been shown to be the case in vitro (5Garton A.J. Cambell D.G. Carling D. Hardie D.G. Colbran R.J. Yeaman S.J. Eur. J. Biochem. 1989; 129: 249-254Crossref Scopus (228) Google Scholar). Also, preincubations of primary rat adipocytes with the AMP-mimicking agent 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside inhibit isoprenaline-induced lipolysis (6Sullivan J.E. Brocklehurst K.J. Marley A.E. Carey F. Carling D. Beri R.K. FEBS Lett. 1994; 353: 33-36Crossref PubMed Scopus (410) Google Scholar), thus lending support for a role of AMP-activated protein kinase in the regulation of HSL. HSL is known to associate with phospholipid vesicles (7Holm C. Fredrikson G. Sundler R. Belfrage P. Lipids. 1990; 25: 254-259Crossref PubMed Scopus (10) Google Scholar) and to have an overall amphipathic character (8Holm C. Fredrikson G. Belfrage P. J. Biol. Chem. 1986; 261: 15659-15661Abstract Full Text PDF PubMed Google Scholar). This suggests that HSL has one or more sites at the surface, enabling interactions with detergent and membranes such as the surface of intracellular lipid droplets. Specific recognition of these lipid droplets, as opposed to other membrane surfaces, would constitute another unique feature of HSL among lipases. As for other lipases and esterases, it was expected that HSL, at least in part, harbors the α/β-hydrolase fold and has a catalytic triad (Ser, Asp/Glu, His) (9Derewenda Z.S. Schumaker V.N. Advances in Protein Chemistry. 45. Elsevier, Amsterdam1994: 1-52Google Scholar). To map these and other features in HSL, we have initiated analyses into its structural and functional domains by alignments to other lipases and esterases and by limited proteolysis, denaturation, site-directed mutagenesis, and molecular modeling. Analysis by limited proteolysis and denaturation suggested that HSL has at least two structural domains (10Østerlund T. Danielsson B. Degerman E. Contreras J.A. Edgren G. Davis R.C. Schotz M.C. Holm C. Biochem. J. 1996; 319: 411-420Crossref PubMed Scopus (137) Google Scholar). Those regions in the C-terminal part of the molecule that align to other lipases and esterases are thought to adopt the α/β-hydrolase fold and to harbor the catalytic triad (10Østerlund T. Danielsson B. Degerman E. Contreras J.A. Edgren G. Davis R.C. Schotz M.C. Holm C. Biochem. J. 1996; 319: 411-420Crossref PubMed Scopus (137) Google Scholar, 11Contreras J.A. Karlsson M. Østerlund T. Laurell H. Svensson A. Holm C. J. Biol. Chem. 1996; 271: 31426-31430Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). In fact, it was possible to build a model for the catalytic α/β-hydrolase fold core (11Contreras J.A. Karlsson M. Østerlund T. Laurell H. Svensson A. Holm C. J. Biol. Chem. 1996; 271: 31426-31430Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). The proposed residues of the catalytic triad have been probed by site-directed mutagenesis and found to be essential for activity (12Holm C. Davis R.C. Østerlund T. Schotz M.C. Fredrikson G. FEBS Lett. 1994; 344: 234-238Crossref PubMed Scopus (56) Google Scholar, 13Østerlund T. Contreras J.A. Holm C. FEBS Lett. 1997; 403: 259-262Crossref PubMed Scopus (47) Google Scholar), strongly supporting the structural model for the catalytic core. In the primary structure, this core is interrupted by approximately 200 residues, including the four phosphorylation sites, which are thought to form a regulatory module (10Østerlund T. Danielsson B. Degerman E. Contreras J.A. Edgren G. Davis R.C. Schotz M.C. Holm C. Biochem. J. 1996; 319: 411-420Crossref PubMed Scopus (137) Google Scholar, 11Contreras J.A. Karlsson M. Østerlund T. Laurell H. Svensson A. Holm C. J. Biol. Chem. 1996; 271: 31426-31430Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). This part is a substrate of kinases and phosphatases and has been predicted to contain only a few secondary structure elements (11Contreras J.A. Karlsson M. Østerlund T. Laurell H. Svensson A. Holm C. J. Biol. Chem. 1996; 271: 31426-31430Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Thus, it is suspected to be an exposed and rather flexible module. The N-terminal part of the protein (approximately 320 residues) is believed to constitute a separate structural domain and shows no significant sequence similarity to other proteins (10Østerlund T. Danielsson B. Degerman E. Contreras J.A. Edgren G. Davis R.C. Schotz M.C. Holm C. Biochem. J. 1996; 319: 411-420Crossref PubMed Scopus (137) Google Scholar). To establish the structural domain composition of HSL, we have performed spectroscopic analyses during denaturation. Data from these analyses support the idea that HSL has two major structural domains. Limited proteolysis analyses (10Østerlund T. Danielsson B. Degerman E. Contreras J.A. Edgren G. Davis R.C. Schotz M.C. Holm C. Biochem. J. 1996; 319: 411-420Crossref PubMed Scopus (137) Google Scholar) have been extended through identification of generated peptides by mass spectrometry to identify exposed regions of the intact protein and to identify more stable fragments of the HSL molecule. Both the suggested hinge region and the regulatory module are exposed to proteolysis, whereas the N-terminal domain appears to be highly resistant to cleavage. Analysis by proteolysis of a HSL mutant with an engineered Factor X site in the predicted hinge confirms these results, and further support is provided by analysis of the hydropathic pattern of HSL. Based on the generated results, we present a more complete overall structural model for HSL. DISCUSSIONIn this study, further evidence for our previously proposed model for the domain structure of HSL is provided (10Østerlund T. Danielsson B. Degerman E. Contreras J.A. Edgren G. Davis R.C. Schotz M.C. Holm C. Biochem. J. 1996; 319: 411-420Crossref PubMed Scopus (137) Google Scholar, 11Contreras J.A. Karlsson M. Østerlund T. Laurell H. Svensson A. Holm C. J. Biol. Chem. 1996; 271: 31426-31430Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). The denaturation analyses support that HSL has at least two (and probably only two) structural domains. The fluorescence data clearly suggest the unfolding of two individually folded domains. Although HSL appears to be a homodimer in solution, it is unlikely that the dissociation of dimers contributes significantly to the loss in fluorescence at low GdnCl concentrations. CD measurements during denaturation by GdnCl and heat indicate that the unfolding of secondary structure elements occurs in two major steps. It cannot be ruled out that some protein aggregation occurs in the thermal denaturation. However, it does not seem to have any significant influence on the CD signal.From both spectroscopic data and proteolytic analyses, it appears that one domain is more fragile than the other. The fragile domain is unfolded by low GdnCl concentrations and probably represents the C-terminal domain, because all HSL activities are lost concomitant with its unfolding (10Østerlund T. Danielsson B. Degerman E. Contreras J.A. Edgren G. Davis R.C. Schotz M.C. Holm C. Biochem. J. 1996; 319: 411-420Crossref PubMed Scopus (137) Google Scholar). This domain is also highly sensitive to proteolytic digestion, especially in the regulatory module, as shown by proteolytic cleavage by both EndoL and Factor X. The second unfolding, which occurs at high GdnCl concentrations, probably represents the N-terminal domain. Because detergent and perhaps glycerol had a more protective effect on this domain than on the other domain, it is speculated that some sites of detergent and membrane interactions are located here. Other sites might be found in the α/β-hydrolase fold core, although most hydrophobic residues of this part are not exposed (11Contreras J.A. Karlsson M. Østerlund T. Laurell H. Svensson A. Holm C. J. Biol. Chem. 1996; 271: 31426-31430Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). To directly compare the pattern of hydrophobicity in the primary structure of HSL with the proposed domain structure, the mean hydropathic index was calculated for every 60 residues, according to Kyte and Doolittle (19Kyte J. Doolittle R.F. J. Mol. Biol. 1982; 157: 105-132Crossref PubMed Scopus (17007) Google Scholar). Fig. 8 shows the calculated hydropathy indices with indications of the localization of suggested domains in the primary structure. Hydrophilic regions correlate well with the anticipated hinge region and regulatory module.Arrows indicate the major cleavage sites as identified in Fig. 6. The most hydrophobic regions are those of the α/β-hydrolase fold, whereas the N-terminal domain has an amphipathic character. Because the regulatory module is probably located at the surface of the C-terminal domain, and most of the hydrophobic residues of the α/β-hydrolase fold are located in the core of the fold (11Contreras J.A. Karlsson M. Østerlund T. Laurell H. Svensson A. Holm C. J. Biol. Chem. 1996; 271: 31426-31430Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), there are probably only a few exposed hydrophobic patches in this part. The membrane/detergent binding sites are presumably located at these patches as well as at hydrophobic patches in the N-terminal domain. Overall, the hydropathy calculations support the suggested domain structure, particularly by demonstrating that the exposed regions are markedly hydrophilic.The exact location of the hinge region in the primary structure has been the subject of some speculation (10Østerlund T. Danielsson B. Degerman E. Contreras J.A. Edgren G. Davis R.C. Schotz M.C. Holm C. Biochem. J. 1996; 319: 411-420Crossref PubMed Scopus (137) Google Scholar, 11Contreras J.A. Karlsson M. Østerlund T. Laurell H. Svensson A. Holm C. J. Biol. Chem. 1996; 271: 31426-31430Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). The cleavage at Lys-323 and the hydrophilic character of this region support the concept that the N- and C-terminal domains are separated at the hinge region (residues 315–335) as suggested from alignments (10Østerlund T. Danielsson B. Degerman E. Contreras J.A. Edgren G. Davis R.C. Schotz M.C. Holm C. Biochem. J. 1996; 319: 411-420Crossref PubMed Scopus (137) Google Scholar). Cleavage of HSLFacX by Factor X shows that this region is immediately accessible to the protease. Our current view and working hypothesis of the domain structure is illustrated in Fig.9 with indications of cleavage sites and action of GdnCl at different concentrations. The secondary structure elements of the α/β-hydrolase fold are outlined (11Contreras J.A. Karlsson M. Østerlund T. Laurell H. Svensson A. Holm C. J. Biol. Chem. 1996; 271: 31426-31430Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar).In conclusion, the present study provides additional support for the proposal that HSL is composed of two major structural domains encoded by exons 1–4 and 5–9, respectively. The latter is the catalytic domain formed by an α/β-hydrolase fold core (11Contreras J.A. Karlsson M. Østerlund T. Laurell H. Svensson A. Holm C. J. Biol. Chem. 1996; 271: 31426-31430Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), which is interrupted in the primary structure by the insertion of a regulatory module. Both the regulatory module and the suggested hinge are exposed hydrophilic parts. Additional investigations of HSLFacXbefore and after cleavage and the expression of individual domains are underway. Purified domains and fragments will be valuable tools for structural and functional analyses as well as the determination of the three-dimensional structure of HSL. In this study, further evidence for our previously proposed model for the domain structure of HSL is provided (10Østerlund T. Danielsson B. Degerman E. Contreras J.A. Edgren G. Davis R.C. Schotz M.C. Holm C. Biochem. J. 1996; 319: 411-420Crossref PubMed Scopus (137) Google Scholar, 11Contreras J.A. Karlsson M. Østerlund T. Laurell H. Svensson A. Holm C. J. Biol. Chem. 1996; 271: 31426-31430Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). The denaturation analyses support that HSL has at least two (and probably only two) structural domains. The fluorescence data clearly suggest the unfolding of two individually folded domains. Although HSL appears to be a homodimer in solution, it is unlikely that the dissociation of dimers contributes significantly to the loss in fluorescence at low GdnCl concentrations. CD measurements during denaturation by GdnCl and heat indicate that the unfolding of secondary structure elements occurs in two major steps. It cannot be ruled out that some protein aggregation occurs in the thermal denaturation. However, it does not seem to have any significant influence on the CD signal. From both spectroscopic data and proteolytic analyses, it appears that one domain is more fragile than the other. The fragile domain is unfolded by low GdnCl concentrations and probably represents the C-terminal domain, because all HSL activities are lost concomitant with its unfolding (10Østerlund T. Danielsson B. Degerman E. Contreras J.A. Edgren G. Davis R.C. Schotz M.C. Holm C. Biochem. J. 1996; 319: 411-420Crossref PubMed Scopus (137) Google Scholar). This domain is also highly sensitive to proteolytic digestion, especially in the regulatory module, as shown by proteolytic cleavage by both EndoL and Factor X. The second unfolding, which occurs at high GdnCl concentrations, probably represents the N-terminal domain. Because detergent and perhaps glycerol had a more protective effect on this domain than on the other domain, it is speculated that some sites of detergent and membrane interactions are located here. Other sites might be found in the α/β-hydrolase fold core, although most hydrophobic residues of this part are not exposed (11Contreras J.A. Karlsson M. Østerlund T. Laurell H. Svensson A. Holm C. J. Biol. Chem. 1996; 271: 31426-31430Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). To directly compare the pattern of hydrophobicity in the primary structure of HSL with the proposed domain structure, the mean hydropathic index was calculated for every 60 residues, according to Kyte and Doolittle (19Kyte J. Doolittle R.F. J. Mol. Biol. 1982; 157: 105-132Crossref PubMed Scopus (17007) Google Scholar). Fig. 8 shows the calculated hydropathy indices with indications of the localization of suggested domains in the primary structure. Hydrophilic regions correlate well with the anticipated hinge region and regulatory module.Arrows indicate the major cleavage sites as identified in Fig. 6. The most hydrophobic regions are those of the α/β-hydrolase fold, whereas the N-terminal domain has an amphipathic character. Because the regulatory module is probably located at the surface of the C-terminal domain, and most of the hydrophobic residues of the α/β-hydrolase fold are located in the core of the fold (11Contreras J.A. Karlsson M. Østerlund T. Laurell H. Svensson A. Holm C. J. Biol. Chem. 1996; 271: 31426-31430Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), there are probably only a few exposed hydrophobic patches in this part. The membrane/detergent binding sites are presumably located at these patches as well as at hydrophobic patches in the N-terminal domain. Overall, the hydropathy calculations support the suggested domain structure, particularly by demonstrating that the exposed regions are markedly hydrophilic. The exact location of the hinge region in the primary structure has been the subject of some speculation (10Østerlund T. Danielsson B. Degerman E. Contreras J.A. Edgren G. Davis R.C. Schotz M.C. Holm C. Biochem. J. 1996; 319: 411-420Crossref PubMed Scopus (137) Google Scholar, 11Contreras J.A. Karlsson M. Østerlund T. Laurell H. Svensson A. Holm C. J. Biol. Chem. 1996; 271: 31426-31430Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). The cleavage at Lys-323 and the hydrophilic character of this region support the concept that the N- and C-terminal domains are separated at the hinge region (residues 315–335) as suggested from alignments (10Østerlund T. Danielsson B. Degerman E. Contreras J.A. Edgren G. Davis R.C. Schotz M.C. Holm C. Biochem. J. 1996; 319: 411-420Crossref PubMed Scopus (137) Google Scholar). Cleavage of HSLFacX by Factor X shows that this region is immediately accessible to the protease. Our current view and working hypothesis of the domain structure is illustrated in Fig.9 with indications of cleavage sites and action of GdnCl at different concentrations. The secondary structure elements of the α/β-hydrolase fold are outlined (11Contreras J.A. Karlsson M. Østerlund T. Laurell H. Svensson A. Holm C. J. Biol. Chem. 1996; 271: 31426-31430Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). In conclusion, the present study provides additional support for the proposal that HSL is composed of two major structural domains encoded by exons 1–4 and 5–9, respectively. The latter is the catalytic domain formed by an α/β-hydrolase fold core (11Contreras J.A. Karlsson M. Østerlund T. Laurell H. Svensson A. Holm C. J. Biol. Chem. 1996; 271: 31426-31430Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), which is interrupted in the primary structure by the insertion of a regulatory module. Both the regulatory module and the suggested hinge are exposed hydrophilic parts. Additional investigations of HSLFacXbefore and after cleavage and the expression of individual domains are underway. Purified domains and fragments will be valuable tools for structural and functional analyses as well as the determination of the three-dimensional structure of HSL. We thank Birgitta Danielsson for excellent technical assistance regarding the expression and purification of wild-type HSL and Drs. Howard Wong and Henry Choy for contributing unpublished data on the subunit structure of HSL.
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