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Structural and Functional Consequences of Substitutions at the Pro108–Arg14 Hydrogen Bond in Bovine Adrenodoxin
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
249, 933–937 (1998)
RC989225
Structural and Functional Consequences of Substitutions at the Pro108–Arg14 Hydrogen Bond in Bovine Adrenodoxin Asya Grinberg and Rita Bernhardt1 Fachbereich Pharmazie und Umwelttechnologie, Fachrichtung 12.4 Biochemie, Universita¨t des Saarlandes, P.O. Box 15 11 50, D-66041 Saarbru¨cken, Germany
Received July 13, 1998
Elimination of Pro108 in bovine adrenodoxin is known to result in the formation of a misfolded protein that is not able to incorporate a [2Fe-2S] cluster and rapidly degrades upon expression in E. coli. However, no experimental explanation for this phenomenon has been demonstrated so far. Using the recently obtained 3D structure of the truncated mutant Adx(4-108) we have studied the reasons of the protein stabilization by the proline residue by means of site-directed mutagenesis. Two main results have been obtained: (i) the conserved hydrogen bond Pro108-Arg14, that connects different structural domains of Adx, contributes 6 kJ/ mol into the protein stability and (ii) the presence of proline at position 108 provides a low conformational entropy of the unfolded state, supporting a gain in the Gibbs energy of 5.4 kJ/mol at 377C. q 1998 Academic Press
Bovine adrenal ferredoxin (adrenodoxin) is a small (14 kDa) iron-sulfur protein of the [2Fe-2S] type. It plays an essential role in the biosynthesis of steroid hormones by transferring the electrons from NADPHdependent Adx-reductase (AdR)** to mitochondrial cytochromes P450 (CYP11A1 and CYP11B1) [1]. Little is known about the folding of Adx or other ferredoxins and the mechanism of stabilization of these proteins. Recent studies of our laboratory have shown that Adx is a protein of rather low conformational stability (13 kJ/mol) at physiological temperatures [2]. The question
was raised, whether this marginal stability is favorable in forming electron transfer complexes with various redox partners. However, no simple correlation between conformational stability and functional properties of the wild-type Adx and mutants has been found. Proline 108 in adrenodoxin is an evolutionary conserved residue (Fig. 1) that was presumed to be important for the integrity of the iron-sulfur cluster and for the correct folding of Adx. Its deletion results in a formation of a misfolded protein that is not able to incorporate the cluster upon expression in E. coli. The protein, where proline is reserved, in opposite, folds correctly, successfully assembles an iron-sulfur cluster and reserves its function in the electron transport pathway [3]. However, this residue can be replaced by other amino acids without any effect on the cluster incorporation or functional activity, but with essential decrease in protein stability (Grinberg & Bernhardt, unpublished results). Recently, the crystal structure for the ˚ truncated form Adx(4-108) was obtained with 1.85 A resolution [4]. The steric requirements imposed by a proline residue direct the H-bonding between O of Pro108 and N1 of Arg14 (Fig. 2). To examine whether this hydrogen bond contributes significantly to the conformational stability of Adx and if its elimination is responsible for the formation of a misfolded protein upon Pro108 deletion, we replaced both residues by different amino acids and the biological and chemical properties of the mutated forms were investigated. MATERIALS AND METHODS
1
To whom correspondence should be addressed. Fax: /49 681-3024739; E-mail: [email protected]. Abbreviations used: Adx, bovine adrenodoxin; pseudo-WT, truncated mutant Adx(4-108); AdR, adrenodoxin reductase; CYP11A1, cytochrome P450scc ; CYP11B1, cytochrome P45011b ; Td , midpoint of the thermal unfolding transition; DHd(Td), the denaturation enthalpy of the transition determined at Td ; DGd(377C), Gibbs energy change at 377C; D(DGd(377C)), the difference in the Gibbs energy change between mutant and pseudo-WT at 377C.
Construction and purification of proteins. Mutant proteins were expressed using pKKHC plasmid bearing residues 4-108 of Adx with corresponded mutation as essentially described previously [5]. The optimal expression levels of the R14E, R14A, P108A mutants were achieved at 307C. The mutant proteins were isolated as given in [3] with slight modifications. The concentration of recombinant Adx was calculated using 1414Å9.8 (mMrcm)01 for bovine Adx [6]. AdR and CYP11A1 were isolated from bovine adrenals as previously described [7]. 0006-291X/98 $25.00
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FIG. 1. Structure-based amino acid sequence alignment of representative ferredoxins. (1) vertebrate-type: bovine adrenal, human mitochondrial, sheep, chicken, mouse; (2) plant type: Anabaena 7120 heterocyst, Anabaena variabilis vegetative; (3) bacterial-type: putidaredoxin, E.coli ferredoxin, Caulobacter crescentus ferredoxin. The sequences are taken from the Swiss-Prot Databank. Numbers refer to Adx structure. Highly conserved residues Arg14 and Pro108 (sites of mutations) are shown in bold.
Thermal denaturation. A Jasco 700 spectropolarimeter fitted with a Jasco-PTC348 temperature controller was used to monitor structural changes of pseudo-wild type Adx and mutant proteins during thermal denaturation. The thermal unfolding was monitored in a 1-cm hermetically closed cuvette by following the ellipticity at 440 nm over the temperature range 10-707 C with a temperature increment of 0.2 degrees at a heating rate of 507 C/hour. Adx solutions for CD experiments were prepared chromatographically prior to use on a 0.5110 cm Sephadex G-25 column equilibrated with buffer containing 40 mM glycine, 10 mM Na2S, 1 mM ascorbate, 10 mM b-mercaptoethanol [2]. The mathematical treatment of CD scans was made by a non-linear regression fit using the two-state model [8] with SigmaPlot software.
Pro108-Arg14 significantly contributes to the stability of adrenodoxin, we made neutral and charge-reversed mutants, P108A, R14A and R14E, and evaluated the structural role of this H-bond by means of thermal denaturation and limited proteolysis studies (Fig. 2). After expression of the mutants in E.coli protease deficient strain BL 21 various spectral and functional properties of the pseudo-WT (truncated mutant Adx(4-108) functionally active in electron transfer reactions [3]) and the mutants were analyzed to check, whether the
Proteolytic digestion. Unspecific proteolysis by thermolysin (EC 3.4.24.27) was performed in a range of temperatures between 20 and 607 C (ratio Adx:thermolysinÅ250:1) in a buffer, containing 40 mM glycine, 10 mM Na2S, 1 mM ascorbate, 10 mM b-mercaptoethanol (pH 9.2). Fluorescence emission spectra. Recorded between 285 and 360 nm on FluoroMax-2 (Jobin Ivon-SPEX Instruments S.A., Inc.) spectrofluorimeter by exciting at 270 nm. Measurements were carried out in 10 mM Tris-HCl or 10 mM potassium phosphate (pH 7.5) at room temperature. Enzyme activity and redox potential measurements. Performed as described in [9, 10].
RESULTS AND DISCUSSION The side-chain of Arg14 is hydrogen bonded to the main chain O of Pro108 as revealed by X-ray crystallography [4]. Both residues are highly conserved among vertebrate, many bacterial and some plant ferredoxins: proline is reserved at the correspondent positions, while the residue correspondent to position 14 carries a positive charge, being occupied by arginine, lysine or histidine (Fig.1). To prove, whether the hydrogen band
FIG. 2. Close-up view of the three-dimensional structure of adrenodoxin in the vicinity of structurally important residues Pro108 and Arg14. The distance between hydrogen bond donor and acceptor is ˚ . Hydrogen bond is indicated as dotted line. 2.9 A
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FIG. 3. CD spectra in far-UV range and fluorescence emission spectra (inset) of pseudo-WT (—), and mutant adrenodoxins P108A (---), R14A (rrr) and R14E (-rr-).
mutations affected the structure of the polypeptide chain and the conformation of [2Fe-2S] cluster as well as their redox properties. Absorption spectra of the oxidized mutants showed characterized peaks of Adx at 455, 414, and 320 nm, indicating that incorporation of the [2Fe-2S] cluster was not disturbed upon mutations (data not shown). Unchanged EPR spectra of the mutants, showing g⊥ of 1.94 and g\ of 2.03, provide additional evidence that the organization of reduced [2Fe2S] cluster was similar to pseudo-WT (data not shown). Moreover, no significant changes were observed in the visible region of CD spectra of the mutants indicating that no major changes in the environment of the cluster occurred. The CD spectra in far-UV were slightly affected, indicating some local conformation changes
(Fig. 3). This is especially the case of R14E, where the protein contains more random conformation. Obviously, the Adx fold does not tolerate the introduction of a negative charge at position 14. Similar conclusions can be drawn from the fluorescence measurements using Tyr82, which is situated in the immediate vicinity of Arg14 and Pro108, as an internal probe to follow local conformational changes. The fluorescence intensity of adrenodoxin R14E mutant (Fig. 3, inset) demonstrated a 50% increase compared with the pseudoWT. However, the CD spectrum of the mutant in the aromatic region (250-320 nm) was not significantly different from the pseudo-WT indicating that this mutation does not affect the fine structure around aromatic residues, but rather the distance between Tyr82 and the fluorescence energy acceptor, - the [2Fe-2S] cluster. The ability of adrenodoxin to accept and donate electrons is essentially dependent on the oxidation-reduction potential. Deletion of a positive charge (R14A), introduction of an additional negative charge (R14E) or exchange of the residue in position 108 (P108A) do not influence significantly the oxidation-reduction potential of Adx (Table I), thus proposing these residues to be of small importance for the regulation of the electron transfer properties of Adx. The catalytic activities of the mutant proteins were measured in the NADPHdependent cytochrome c reduction, testing the electron transfer from AdR to Adx, and in the CYP11A1 dependent conversion of cholesterol to pregnenolone. In line with the previous assumption, neither replacement of Pro108 nor Arg14, demonstrated any significant changes in interaction and electron transfer properties of mutant adrenodoxins with adrenodoxin reductase or CYP11A1 (Table I). These results are not surprising, since the residues participating in the direct recognition of the Adx ligands, involving the conserved acidic domain (Glu68-Asp86), are positioned on the other face of the Adx molecule [4]. The importance of the hydrogen bond between
TABLE I
Relative Kinetic Constantsa and Redox Potentials of Pseudo-WTb and Mutant Adrenodoxins Cholesterol side-chain cleavage
Cytochrome c reduction Protein
Km (nM)
Vmaxc
Km (mM)
Vmaxd
Redox potential E (mV)
pseudo-WT P108A R14A R14E
1.00 0.99 0.83 1.19
1.00 0.98 1.08 1.04
1.00 1.70 1.03 1.08
1.00 0.95 1.04 1.00
0343 0337 0331 0325
a Interaction with AdR was assayed by monitoring the reduction of cytochrome c at 550 nm. Catalytic activities of adrenodoxin mutants in CYP11A1 dependent substrate conversion were studied by analyzing the product of respective hydroxylation (pregnenolone). b Pseudo-WT is truncated mutant Adx(4-108), functionally active in electron transfer reactions [3]. c Expressed in nmol cytochrome c reduced/min. d Expressed in nmol pregnenolone produced/min/nmol CYP11A1.
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Thermodynamic Parameters of Adrenodoxin Mutantsa Mutant pseudo-WT P108A R14A R14E
Td (7C) 53.5 49.2 47.2 40.0
{ { { {
0.5 0.2 0.2 0.2
DHd(Td) kJ/mol
D(Td) kJ/mol
D(DHd(Td)) kJ/mol
DGd(377C)b kJ/mol
D(DGd(377C)) kJ/mol
Proteolysis temperaturec, (7C)
364 306 329 288
0 04.3 06.3 013.5
0 058 035 076
15.31 9.87 9.29 2.65
0 05.44 06.02 012.66
51–55 42–47 47–51 37–42
{ { { {
7 5 6 7
Note. Mathematic treatment of the protein unfolding curves, obtained by CD, was made using a non-linear regression fit, assuming a two-state model of protein denaturation. a 10 mM Na2S, 1 mM ascorbate, 10 mM 2-mercaptoethanol, 40 mM glycine, pH 9.2. b Calculated using individual Td and DHd values for mutant proteins and DCp Å 7.5 { 0.67 kJ/mol/K [2]. c From the thermolysin digestion data.
Pro108 and Arg14 was directly studied by investigating the conformational stability of the mutants. The asymmetric environment of the [2Fe-2S] cluster is primarily responsible for the positive ellipticity peak at 440 nm [6], thus providing a sensitive monitor of the denaturation of adrenodoxin. The thermal transitions were analyzed assuming a two-state model, and the values of the unfolding temperature, Td , and the unfolding enthalpy, DHd(Td), derived from this analysis are listed in Table II. The reversibility of the denaturation was demonstrated for all proteins by the recovery of the native ellipticity upon cooling of the heat denatured proteins. Upon proline replacement by alanine a significant decrease in the overall protein stability has been induced, as judged by lowering the transition temperature and enthalpy. Thus, the DTd between pseudoWT and P108A resembles 4.3 7C. DHd(Td) of this mutant is reduced by 58 kJ/mol relative to the pseudoWT. The Adx mutants at position 14 were also found to be less stable than pseudo-WT. The R14A mutant melted 6.3 degrees lower (Td Å 47.27C) than pseudoWT and R14E melted at Td Å 407C that was reduced by 13.5 degrees compared to pseudo-WT. The reduced stability of both these mutants is supposed to be a primary result of a loss of the hydrogen bond between Arg14 and Pro108. In case of ArgrGlu mutation the effect is more pronounced because of an altered local packing density and repulsion from the carboxyl group of Pro108. The physico-chemical data correlate well with the resistance against proteolysis at various temperatures (Table II). The stability differences of the mutant proteins expressed in terms of an increment of the Gibbs energy of unfolding, D(DGd) Å DGd, mut 0 DGd, pseudo-wt , at the physiological temperature are given in Table II. As shown, a significant drop in the Gibbs energy is observed for the position 108 and 14 mutants. The obtained results can be explained by taking a look at the very recently obtained structural data on pseudo-WT Adx [4]. Figure 2 shows that the N1 of Arg14 is positioned to form a hydrogen bond with the
˚ ), connecting two secondary structure Pro108 O (2.9 A elements, parallel b-sheets A and J of Adx polypeptide chain. As far as the conformations allowed for carboxyl groups of all amino acids are almost the same, the hydrogen bond between backbone O of residue 108 and Arg14 is supposed to be reserved in P108A mutant, but eliminated in case of R14A and R14E mutants. Correspondingly we attribute the destabilization, observed when Pro is replaced by Ala, to the increased entropy of the unfolded polypeptide chain. In fact, the theoretical estimation (05.4 kJ/mol) of the thermodynamical effect expected upon ProrAla substitution coincides fairy well with the experimentally observed value (05.4 kJ/mol). The mutations of Arg14 result in a destruction of the hydrogen bond between residues 108 and 14. Although the importance of H-bonds for the stability of the protein has been underestimated for a long time, however, now there are plenty of evidence that the hydrogen bonds contribute significantly to the stabilization of the native compact protein structure [11-12]. Pace and co-workers found that on average a hydrogen bonding residue contributes about 4.29.2 kJ/mol (expressed in D(DdG) relative to a non-hydrogen bonding residue in the corresponding position) [13]. Here we demonstrated that the intramolecular hydrogen bond even at the solvent accessible molecular surface is favorable and contributes 6 kJ/mol into the stability of bovine adrenodoxin holding together the Nand C-terminal regions of the protein. ACKNOWLEDGMENTS The authors thank Dr. V. Ru¨diger for his generous help with preparing Figure 2 and W. Reinle for excellent technical assistance in purification of mutant proteins. This work is supported by a grant from the Boehringer Ingelheim Fonds to A.G., by the Deutsche Forschungsgemeinschaft, Grant Be 1343/1-3, and by the Fonds der chemischen Industrie.
REFERENCES 1. Bernhardt, R. (1996) Rev. Physiol. Biochem. Pharmacol. 127, 137–221.
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2. Burova, T. V., Bernhardt, R., and Pfeil, W. (1995) Protein Sci. 4, 909–916. 3. Uhlmann, H., Kraft, R., and Bernhardt, R. (1994) J. Biol. Chem. 269, 22557–22564. 4. Mu¨ller, A., Mu¨ller, J. J., Muller, Y. A., Uhlmann, H., Bernhardt, R., and Heinemann, U. (1998) Structure 6, 269–280. 5. Uhlmann, H., Beckert, V., Schwarz, D., and Bernhardt, R. (1992) Biochem. Biophys. Res. Commun. 188, 1131–1138. 6. Kimura, T. (1968) in Structure and Bonding (Jorgensen, C. K., Neiland, J. B., Nyholm, R. S., Reinen, D., and Williams, R. J. P., Eds.), Vol. 5, pp. 1–40, Springer-Verlag, Berlin. 7. Akhrem, A. A., Lapko, V. N., Lapko, A. G., Shkumatov, V. M., and Chashin, V. L. (1979) Acta Biol. Med. Ger. 38, 257–274.
8. Privalov, P. L. (1979) Adv. Protein Chem. 33, 167–240. 9. Beckert, V., and Bernhardt, R. (1997) J. Biol. Chem. 272, 4883– 4888. 10. Beckert, V., Dettmer, R., and Bernhardt, R. (1994) J. Biol. Chem. 269, 2568–2573. 11. Makhatadze, G. I., and Privalov, P. L. (1995) Adv. Protein Chem. 47, 307–425. 12. Fersht, A. R., Shi, J. P., Jones, J., Lowe, D. M., Wilkinson, A. J., Blow, D. M., Brick, P., Carter, P., Waye, M. M., and Winter, G. (1985) Nature 314, 235–238. 13. Pace, C. N., Shirley, B. A., McNutt, M., and Gajiwala, K. (1996) FASEB J. 10, 75–83.
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Structural and Functional Consequences of Substitutions at the Pro108–Arg14 Hydrogen Bond in Bovine Adrenodoxin Asya Grinberg and Rita Bernhardt1 Fachbereich Pharmazie und Umwelttechnologie, Fachrichtung 12.4 Biochemie, Universita¨t des Saarlandes, P.O. Box 15 11 50, D-66041 Saarbru¨cken, Germany
Received July 13, 1998
Elimination of Pro108 in bovine adrenodoxin is known to result in the formation of a misfolded protein that is not able to incorporate a [2Fe-2S] cluster and rapidly degrades upon expression in E. coli. However, no experimental explanation for this phenomenon has been demonstrated so far. Using the recently obtained 3D structure of the truncated mutant Adx(4-108) we have studied the reasons of the protein stabilization by the proline residue by means of site-directed mutagenesis. Two main results have been obtained: (i) the conserved hydrogen bond Pro108-Arg14, that connects different structural domains of Adx, contributes 6 kJ/ mol into the protein stability and (ii) the presence of proline at position 108 provides a low conformational entropy of the unfolded state, supporting a gain in the Gibbs energy of 5.4 kJ/mol at 377C. q 1998 Academic Press
Bovine adrenal ferredoxin (adrenodoxin) is a small (14 kDa) iron-sulfur protein of the [2Fe-2S] type. It plays an essential role in the biosynthesis of steroid hormones by transferring the electrons from NADPHdependent Adx-reductase (AdR)** to mitochondrial cytochromes P450 (CYP11A1 and CYP11B1) [1]. Little is known about the folding of Adx or other ferredoxins and the mechanism of stabilization of these proteins. Recent studies of our laboratory have shown that Adx is a protein of rather low conformational stability (13 kJ/mol) at physiological temperatures [2]. The question
was raised, whether this marginal stability is favorable in forming electron transfer complexes with various redox partners. However, no simple correlation between conformational stability and functional properties of the wild-type Adx and mutants has been found. Proline 108 in adrenodoxin is an evolutionary conserved residue (Fig. 1) that was presumed to be important for the integrity of the iron-sulfur cluster and for the correct folding of Adx. Its deletion results in a formation of a misfolded protein that is not able to incorporate the cluster upon expression in E. coli. The protein, where proline is reserved, in opposite, folds correctly, successfully assembles an iron-sulfur cluster and reserves its function in the electron transport pathway [3]. However, this residue can be replaced by other amino acids without any effect on the cluster incorporation or functional activity, but with essential decrease in protein stability (Grinberg & Bernhardt, unpublished results). Recently, the crystal structure for the ˚ truncated form Adx(4-108) was obtained with 1.85 A resolution [4]. The steric requirements imposed by a proline residue direct the H-bonding between O of Pro108 and N1 of Arg14 (Fig. 2). To examine whether this hydrogen bond contributes significantly to the conformational stability of Adx and if its elimination is responsible for the formation of a misfolded protein upon Pro108 deletion, we replaced both residues by different amino acids and the biological and chemical properties of the mutated forms were investigated. MATERIALS AND METHODS
1
To whom correspondence should be addressed. Fax: /49 681-3024739; E-mail: [email protected]. Abbreviations used: Adx, bovine adrenodoxin; pseudo-WT, truncated mutant Adx(4-108); AdR, adrenodoxin reductase; CYP11A1, cytochrome P450scc ; CYP11B1, cytochrome P45011b ; Td , midpoint of the thermal unfolding transition; DHd(Td), the denaturation enthalpy of the transition determined at Td ; DGd(377C), Gibbs energy change at 377C; D(DGd(377C)), the difference in the Gibbs energy change between mutant and pseudo-WT at 377C.
Construction and purification of proteins. Mutant proteins were expressed using pKKHC plasmid bearing residues 4-108 of Adx with corresponded mutation as essentially described previously [5]. The optimal expression levels of the R14E, R14A, P108A mutants were achieved at 307C. The mutant proteins were isolated as given in [3] with slight modifications. The concentration of recombinant Adx was calculated using 1414Å9.8 (mMrcm)01 for bovine Adx [6]. AdR and CYP11A1 were isolated from bovine adrenals as previously described [7]. 0006-291X/98 $25.00
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FIG. 1. Structure-based amino acid sequence alignment of representative ferredoxins. (1) vertebrate-type: bovine adrenal, human mitochondrial, sheep, chicken, mouse; (2) plant type: Anabaena 7120 heterocyst, Anabaena variabilis vegetative; (3) bacterial-type: putidaredoxin, E.coli ferredoxin, Caulobacter crescentus ferredoxin. The sequences are taken from the Swiss-Prot Databank. Numbers refer to Adx structure. Highly conserved residues Arg14 and Pro108 (sites of mutations) are shown in bold.
Thermal denaturation. A Jasco 700 spectropolarimeter fitted with a Jasco-PTC348 temperature controller was used to monitor structural changes of pseudo-wild type Adx and mutant proteins during thermal denaturation. The thermal unfolding was monitored in a 1-cm hermetically closed cuvette by following the ellipticity at 440 nm over the temperature range 10-707 C with a temperature increment of 0.2 degrees at a heating rate of 507 C/hour. Adx solutions for CD experiments were prepared chromatographically prior to use on a 0.5110 cm Sephadex G-25 column equilibrated with buffer containing 40 mM glycine, 10 mM Na2S, 1 mM ascorbate, 10 mM b-mercaptoethanol [2]. The mathematical treatment of CD scans was made by a non-linear regression fit using the two-state model [8] with SigmaPlot software.
Pro108-Arg14 significantly contributes to the stability of adrenodoxin, we made neutral and charge-reversed mutants, P108A, R14A and R14E, and evaluated the structural role of this H-bond by means of thermal denaturation and limited proteolysis studies (Fig. 2). After expression of the mutants in E.coli protease deficient strain BL 21 various spectral and functional properties of the pseudo-WT (truncated mutant Adx(4-108) functionally active in electron transfer reactions [3]) and the mutants were analyzed to check, whether the
Proteolytic digestion. Unspecific proteolysis by thermolysin (EC 3.4.24.27) was performed in a range of temperatures between 20 and 607 C (ratio Adx:thermolysinÅ250:1) in a buffer, containing 40 mM glycine, 10 mM Na2S, 1 mM ascorbate, 10 mM b-mercaptoethanol (pH 9.2). Fluorescence emission spectra. Recorded between 285 and 360 nm on FluoroMax-2 (Jobin Ivon-SPEX Instruments S.A., Inc.) spectrofluorimeter by exciting at 270 nm. Measurements were carried out in 10 mM Tris-HCl or 10 mM potassium phosphate (pH 7.5) at room temperature. Enzyme activity and redox potential measurements. Performed as described in [9, 10].
RESULTS AND DISCUSSION The side-chain of Arg14 is hydrogen bonded to the main chain O of Pro108 as revealed by X-ray crystallography [4]. Both residues are highly conserved among vertebrate, many bacterial and some plant ferredoxins: proline is reserved at the correspondent positions, while the residue correspondent to position 14 carries a positive charge, being occupied by arginine, lysine or histidine (Fig.1). To prove, whether the hydrogen band
FIG. 2. Close-up view of the three-dimensional structure of adrenodoxin in the vicinity of structurally important residues Pro108 and Arg14. The distance between hydrogen bond donor and acceptor is ˚ . Hydrogen bond is indicated as dotted line. 2.9 A
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FIG. 3. CD spectra in far-UV range and fluorescence emission spectra (inset) of pseudo-WT (—), and mutant adrenodoxins P108A (---), R14A (rrr) and R14E (-rr-).
mutations affected the structure of the polypeptide chain and the conformation of [2Fe-2S] cluster as well as their redox properties. Absorption spectra of the oxidized mutants showed characterized peaks of Adx at 455, 414, and 320 nm, indicating that incorporation of the [2Fe-2S] cluster was not disturbed upon mutations (data not shown). Unchanged EPR spectra of the mutants, showing g⊥ of 1.94 and g\ of 2.03, provide additional evidence that the organization of reduced [2Fe2S] cluster was similar to pseudo-WT (data not shown). Moreover, no significant changes were observed in the visible region of CD spectra of the mutants indicating that no major changes in the environment of the cluster occurred. The CD spectra in far-UV were slightly affected, indicating some local conformation changes
(Fig. 3). This is especially the case of R14E, where the protein contains more random conformation. Obviously, the Adx fold does not tolerate the introduction of a negative charge at position 14. Similar conclusions can be drawn from the fluorescence measurements using Tyr82, which is situated in the immediate vicinity of Arg14 and Pro108, as an internal probe to follow local conformational changes. The fluorescence intensity of adrenodoxin R14E mutant (Fig. 3, inset) demonstrated a 50% increase compared with the pseudoWT. However, the CD spectrum of the mutant in the aromatic region (250-320 nm) was not significantly different from the pseudo-WT indicating that this mutation does not affect the fine structure around aromatic residues, but rather the distance between Tyr82 and the fluorescence energy acceptor, - the [2Fe-2S] cluster. The ability of adrenodoxin to accept and donate electrons is essentially dependent on the oxidation-reduction potential. Deletion of a positive charge (R14A), introduction of an additional negative charge (R14E) or exchange of the residue in position 108 (P108A) do not influence significantly the oxidation-reduction potential of Adx (Table I), thus proposing these residues to be of small importance for the regulation of the electron transfer properties of Adx. The catalytic activities of the mutant proteins were measured in the NADPHdependent cytochrome c reduction, testing the electron transfer from AdR to Adx, and in the CYP11A1 dependent conversion of cholesterol to pregnenolone. In line with the previous assumption, neither replacement of Pro108 nor Arg14, demonstrated any significant changes in interaction and electron transfer properties of mutant adrenodoxins with adrenodoxin reductase or CYP11A1 (Table I). These results are not surprising, since the residues participating in the direct recognition of the Adx ligands, involving the conserved acidic domain (Glu68-Asp86), are positioned on the other face of the Adx molecule [4]. The importance of the hydrogen bond between
TABLE I
Relative Kinetic Constantsa and Redox Potentials of Pseudo-WTb and Mutant Adrenodoxins Cholesterol side-chain cleavage
Cytochrome c reduction Protein
Km (nM)
Vmaxc
Km (mM)
Vmaxd
Redox potential E (mV)
pseudo-WT P108A R14A R14E
1.00 0.99 0.83 1.19
1.00 0.98 1.08 1.04
1.00 1.70 1.03 1.08
1.00 0.95 1.04 1.00
0343 0337 0331 0325
a Interaction with AdR was assayed by monitoring the reduction of cytochrome c at 550 nm. Catalytic activities of adrenodoxin mutants in CYP11A1 dependent substrate conversion were studied by analyzing the product of respective hydroxylation (pregnenolone). b Pseudo-WT is truncated mutant Adx(4-108), functionally active in electron transfer reactions [3]. c Expressed in nmol cytochrome c reduced/min. d Expressed in nmol pregnenolone produced/min/nmol CYP11A1.
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Thermodynamic Parameters of Adrenodoxin Mutantsa Mutant pseudo-WT P108A R14A R14E
Td (7C) 53.5 49.2 47.2 40.0
{ { { {
0.5 0.2 0.2 0.2
DHd(Td) kJ/mol
D(Td) kJ/mol
D(DHd(Td)) kJ/mol
DGd(377C)b kJ/mol
D(DGd(377C)) kJ/mol
Proteolysis temperaturec, (7C)
364 306 329 288
0 04.3 06.3 013.5
0 058 035 076
15.31 9.87 9.29 2.65
0 05.44 06.02 012.66
51–55 42–47 47–51 37–42
{ { { {
7 5 6 7
Note. Mathematic treatment of the protein unfolding curves, obtained by CD, was made using a non-linear regression fit, assuming a two-state model of protein denaturation. a 10 mM Na2S, 1 mM ascorbate, 10 mM 2-mercaptoethanol, 40 mM glycine, pH 9.2. b Calculated using individual Td and DHd values for mutant proteins and DCp Å 7.5 { 0.67 kJ/mol/K [2]. c From the thermolysin digestion data.
Pro108 and Arg14 was directly studied by investigating the conformational stability of the mutants. The asymmetric environment of the [2Fe-2S] cluster is primarily responsible for the positive ellipticity peak at 440 nm [6], thus providing a sensitive monitor of the denaturation of adrenodoxin. The thermal transitions were analyzed assuming a two-state model, and the values of the unfolding temperature, Td , and the unfolding enthalpy, DHd(Td), derived from this analysis are listed in Table II. The reversibility of the denaturation was demonstrated for all proteins by the recovery of the native ellipticity upon cooling of the heat denatured proteins. Upon proline replacement by alanine a significant decrease in the overall protein stability has been induced, as judged by lowering the transition temperature and enthalpy. Thus, the DTd between pseudoWT and P108A resembles 4.3 7C. DHd(Td) of this mutant is reduced by 58 kJ/mol relative to the pseudoWT. The Adx mutants at position 14 were also found to be less stable than pseudo-WT. The R14A mutant melted 6.3 degrees lower (Td Å 47.27C) than pseudoWT and R14E melted at Td Å 407C that was reduced by 13.5 degrees compared to pseudo-WT. The reduced stability of both these mutants is supposed to be a primary result of a loss of the hydrogen bond between Arg14 and Pro108. In case of ArgrGlu mutation the effect is more pronounced because of an altered local packing density and repulsion from the carboxyl group of Pro108. The physico-chemical data correlate well with the resistance against proteolysis at various temperatures (Table II). The stability differences of the mutant proteins expressed in terms of an increment of the Gibbs energy of unfolding, D(DGd) Å DGd, mut 0 DGd, pseudo-wt , at the physiological temperature are given in Table II. As shown, a significant drop in the Gibbs energy is observed for the position 108 and 14 mutants. The obtained results can be explained by taking a look at the very recently obtained structural data on pseudo-WT Adx [4]. Figure 2 shows that the N1 of Arg14 is positioned to form a hydrogen bond with the
˚ ), connecting two secondary structure Pro108 O (2.9 A elements, parallel b-sheets A and J of Adx polypeptide chain. As far as the conformations allowed for carboxyl groups of all amino acids are almost the same, the hydrogen bond between backbone O of residue 108 and Arg14 is supposed to be reserved in P108A mutant, but eliminated in case of R14A and R14E mutants. Correspondingly we attribute the destabilization, observed when Pro is replaced by Ala, to the increased entropy of the unfolded polypeptide chain. In fact, the theoretical estimation (05.4 kJ/mol) of the thermodynamical effect expected upon ProrAla substitution coincides fairy well with the experimentally observed value (05.4 kJ/mol). The mutations of Arg14 result in a destruction of the hydrogen bond between residues 108 and 14. Although the importance of H-bonds for the stability of the protein has been underestimated for a long time, however, now there are plenty of evidence that the hydrogen bonds contribute significantly to the stabilization of the native compact protein structure [11-12]. Pace and co-workers found that on average a hydrogen bonding residue contributes about 4.29.2 kJ/mol (expressed in D(DdG) relative to a non-hydrogen bonding residue in the corresponding position) [13]. Here we demonstrated that the intramolecular hydrogen bond even at the solvent accessible molecular surface is favorable and contributes 6 kJ/mol into the stability of bovine adrenodoxin holding together the Nand C-terminal regions of the protein. ACKNOWLEDGMENTS The authors thank Dr. V. Ru¨diger for his generous help with preparing Figure 2 and W. Reinle for excellent technical assistance in purification of mutant proteins. This work is supported by a grant from the Boehringer Ingelheim Fonds to A.G., by the Deutsche Forschungsgemeinschaft, Grant Be 1343/1-3, and by the Fonds der chemischen Industrie.
REFERENCES 1. Bernhardt, R. (1996) Rev. Physiol. Biochem. Pharmacol. 127, 137–221.
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2. Burova, T. V., Bernhardt, R., and Pfeil, W. (1995) Protein Sci. 4, 909–916. 3. Uhlmann, H., Kraft, R., and Bernhardt, R. (1994) J. Biol. Chem. 269, 22557–22564. 4. Mu¨ller, A., Mu¨ller, J. J., Muller, Y. A., Uhlmann, H., Bernhardt, R., and Heinemann, U. (1998) Structure 6, 269–280. 5. Uhlmann, H., Beckert, V., Schwarz, D., and Bernhardt, R. (1992) Biochem. Biophys. Res. Commun. 188, 1131–1138. 6. Kimura, T. (1968) in Structure and Bonding (Jorgensen, C. K., Neiland, J. B., Nyholm, R. S., Reinen, D., and Williams, R. J. P., Eds.), Vol. 5, pp. 1–40, Springer-Verlag, Berlin. 7. Akhrem, A. A., Lapko, V. N., Lapko, A. G., Shkumatov, V. M., and Chashin, V. L. (1979) Acta Biol. Med. Ger. 38, 257–274.
8. Privalov, P. L. (1979) Adv. Protein Chem. 33, 167–240. 9. Beckert, V., and Bernhardt, R. (1997) J. Biol. Chem. 272, 4883– 4888. 10. Beckert, V., Dettmer, R., and Bernhardt, R. (1994) J. Biol. Chem. 269, 2568–2573. 11. Makhatadze, G. I., and Privalov, P. L. (1995) Adv. Protein Chem. 47, 307–425. 12. Fersht, A. R., Shi, J. P., Jones, J., Lowe, D. M., Wilkinson, A. J., Blow, D. M., Brick, P., Carter, P., Waye, M. M., and Winter, G. (1985) Nature 314, 235–238. 13. Pace, C. N., Shirley, B. A., McNutt, M., and Gajiwala, K. (1996) FASEB J. 10, 75–83.
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