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Structure of human carbonic anhydrase B
J. Mol.
Biol.
(1972) 63, 601-604
Structure of Human Carbonic Anhydrase B I. Crystallization
and Heavy Atom
Modifications
Human carbonic anhydrase B has been crystallized from 2.3 M-ammonium sulphate solution at pH 8.7. A method for reproducible crystallization is presented. The crystals are suitable for high-resolution X-ray diffraction studies. They belong to the orthorhombic space group P 2,2,2, with cell dimensions a = 81.5 A, b = 73.6 A, c = 37.1 A. The position of the essential zinc ion has been established from two projections. The zinc ion has been replaced by mercury to form one of the heavy atom derivatives.
Several forms of carbonic anhydrase have been isolated from human erythrocytes (for reviews see: Maren, 1967; Edsall, 1968; Forster, Edsall, Otis & Roughton, 1969). The two main types, usually denoted B and C, both have a molecular weight near 30,000 and consist of single polypeptide chains. There is one zinc ion per molecule, firmly bound and essential for catalytic activity. The enzymes catalyze the reversible hydrations of carbon dioxide and aldehydes and the hydrolysis of esters.Aromatic and heterocyclic sulphonamides,and monovalent anions, act as inhibitors. Form C, which is easierto crystallize, has been studied by X-ray diffraction, and the structure is now known to 2 A resolution (Liljas et al., 1972). F orm B, which predominates in human red cells, is distinctly different from form C in catalytic properties and inhibitor binding, as well as in amino-acid sequence.B detailed comparison of the structures, of the two forms would therefore be of considerableinterest. The present paper describes the preparation of crystals of human enzyme B aswell as preliminary results of X-ray diffraction studies on these crystals and someisomorphousheavy atom derivatives. Experiments to grow crystals were performed in dialysis bags or micro-diffusion cells (Zeppezauer, Eklund & Zeppezauer, 1968). The tendency for crystal formation was found to increase when lyophilization was avoided during the purification of the protein, and the treatment with organic solvents for removal of hemoglobin was replaced with more gentle methods such asgel filtration (Armstrong, Myers, Verpoorte & Edsall, 1966) or ion exchange chromatography (Armstrong et al., 1966; Nyman, 1961). These and previous experiences with carbonic anhydrase C (Strandberg, 1967) would suggest that the conformational homogeneity of the protein is of critical importance in the preparation of satisfactory crystals. The tendency for crystal formation increasedat alkaline pH, but at values above pH9 only amorphous precipitates were obtained and most experiments to grow crystals were carried out between pH 8 and 9. The presenceof the sulphonamide inhibitor acetazolamide (99% of total protein as enzyme-inhibitor complex) was found to 601
602
K.
K.
KANNAN
ET
AL.
a/2
b/;
0 L--l--T
a
t
Fro. 1. Difference Patterson projections at 2 A resolution between zinc-free enzyme treated with HgCl, (BAL + HgCls derivative) and zinc-free enzyme (BAL derivative). The arrows indicate the Harker vectors which are related to the general vector marked by a cross. The three vectors together correspond to heavy atom binding at a single site of the molecule. The dotted contours define the zero level. The full contours are drawn at an interval of 75 on an arbitrary scale where the origin peak is scaled to 999. The Harker peaks have a value of about 450 on this scale and the general peaks have a value of about 250. The background level is of the order of 125. Negative values are not contoured, the minimum value being - 150. The AF2 (000) term is not included in the calculation. (a) Projection along the c-axis. (b) Projection along the b-axis.
LETTERS
TO
THE
EDITOR
603
markedly increasecrystallizability, but it hasonly twice beenpossibleto obtain crystals of the complex with dimensionssuitable for X-ray studiesby dialysis from ammonium sulphate solutions. These crystals were obtained in 2.5 M-(NH&SO,, 0.05 M-Tris. HCl (pH 8*7), after a few months at 4°C. Similar results have been reported by Coleman (1968). However, by using small amounts of the crystals as seeds,a more rapid and convenient procedure for production of crystals has been developed which will be described below. An 8% solution of the enzyme, native or complexed with inhibitors, was dialysed in 2.3 M-(NH&SO,, 0.05 M-Tris*HCl (pH 8.7). After three or four days the solution was centrifuged to remove amorphous precipitate, and the supernatant solution, containing about 5% protein, was diluted to about 3% in 2.3 M-(NH&SO,, and sucked up into a thick-walled glasscapillary (50 ~1.). One end of the capillary was then sealed.Selectedcrystals of the native enzyme, or in complex with inhibitors, were then crushed into small bits and washed in 2.3 M-(NH&SO,. Two or three of these bits were inserted through the open end of the capillary into the protein solution and the capillary sealed. In about six days these seedscan be seento heal and exhibit welldeveloped faces and increase in dimensions (O-05-mmseedshave grown to O-20-mm crystals). Crystals with dimensionsof about 2 mm have been obtained from such seeds in about a month. Zinc-free enzyme in the crystalline state (BAL derivative) was obtained by removing the zinc ion with 2,3-dimercaptopropanol-1 (BAL) according to the method described for the human C enzyme (Tilander, Strandberg & Pridborg, 1965). Crystals, of zinc-free enzyme were soaked in 2.3 M-(NH&SO, containing 0.001 M-HgCl, for one week to prepare the BAL + HgCl, derivative. The crystals of human carbonic anhydrase B are orthorhombic with a = 81.5, b = 73.6, and c = 37.1 A as measuredfrom precessionphotographs, and have a unit, cell volume of 223,000 A3. The space group is P 2,2,21 as determined from syste. matic absences.The dimensionsof the human B and C isozymes are very nearly the samefrom hydrodynamic data (Derrien & Laurent, 1969). By a comparison with the molecular dimensions of the human C enzyme as determined by X-ray diffraction. (Pridborg et al., 1967; Liljas et al., 1972) it can be concluded that there are four molecules per unit cell. The crystals of native enzyme and its derivatives are all isomorphous as judged from a comparison of cell dimensions. Difference Patterson calculations were performedintwoprojections. Figure 1 (a) and. (b) gives these at 2 d resolution along the c- and b-axes, respectively, between the BAL + HgCI, derivative and the BAL derivative. The low background level compared to the peak heights for the three vectors expected for a single heavy atom, site per asymmetric unit shows the good quality of the difference Patterson. The difference Patterson projections for the BAL derivative against the native enzyme give co-ordinates for the zinc ion which coincide with the mercury site in the BAL + HgCl, derivative. We are grateful for various types of assistance from U. Bengtsson, U. Carlbom, A. C. Carlsson and S. Nilsson. Generous gifts of Sephadex from Pharmacia AB are gratefully acknowledged. This work has been supported by grants from the National Institutes of Health, U.S. Public Health Service (AI GM 07382 and GM 12280-03), the Swedish Computing Authorities (Statskontoret, Stockholm), the Swedish Medical and Natural Science Research Councils and the Knut and Alice Wallenberg Foundation, Sweden.
604
K. K. KANNAN
ET
AL.
Wallenberg Laboratory University of Uppsala Uppsala, Sweden
K.K. K~NNAN K. FRIDBORQ P.-C. BEROSTI?N A. LILJAS S.LGVGREN M. PETEF B. STRANDBERG I. WAARA
Department of Biochemistry University of Giiteborg and Chalmers Institute of Technology Goteborg, Sweden
L. ADLER S.O.FALKBRINC P.O. G~THE P.O. NYMAN
Received
16 August
1971, and in revised form 5 November
1971
REFERENCES Armstrong, J. McD., Myers, D. V., Verpoorte, J. A. & Edsall, J. T. (1966). J. Biol. Chem. 241, 5137. Coleman, J. E. (1968). J. Biol. Chem. 243, 4574. Derrien, Y. & Laurent, G. (1969). Exposds Annuels de Biochimie Mddicale, 29, 167. Ed&l, J. T. (1968). Harvey Lectures, series 62, p. 191. Forster, R. E., Edsall, J. T., Otis, A. B. & Roughton, F. J. W. (editors) (1969). CO,: Chemical, Biochemical and Physiological Aspects. Washington DC. National Aeronautics and Space Administration. Fridborg, K., Kanuan, K. K., Liljas, A., Lundin, J., Strandberg, B., Strandberg, R., Tilander, B. & Wiren, G. (1967). J. Mol. Biol. 25, 505. Liljas, A., Kannan, K. K., Bergsten, P.-C., Waara, I., Fridborg, K., Strandberg, B., Carlbom, U., J&up, L., Liivgren, S. & Petef, M. (1972). Nature, in the press. Maren, T. H. (1967). Phyaiol. Rev. 47, 595. Nyman, P. 0. (1961). Biochim. biophys. Acta, 52, 1. Strandberg, B. (1967). Arkiw Kemi, 28, 1. Tilander, B., Strandberg, B. & Fridborg, K. (1965). J. Mol. Biol. 12, 740. Zeppezauer, M., Eklund, H. & Zeppezauer, E. (1968). Arch. B&hem. Biophys. 126, 564.
Biol.
(1972) 63, 601-604
Structure of Human Carbonic Anhydrase B I. Crystallization
and Heavy Atom
Modifications
Human carbonic anhydrase B has been crystallized from 2.3 M-ammonium sulphate solution at pH 8.7. A method for reproducible crystallization is presented. The crystals are suitable for high-resolution X-ray diffraction studies. They belong to the orthorhombic space group P 2,2,2, with cell dimensions a = 81.5 A, b = 73.6 A, c = 37.1 A. The position of the essential zinc ion has been established from two projections. The zinc ion has been replaced by mercury to form one of the heavy atom derivatives.
Several forms of carbonic anhydrase have been isolated from human erythrocytes (for reviews see: Maren, 1967; Edsall, 1968; Forster, Edsall, Otis & Roughton, 1969). The two main types, usually denoted B and C, both have a molecular weight near 30,000 and consist of single polypeptide chains. There is one zinc ion per molecule, firmly bound and essential for catalytic activity. The enzymes catalyze the reversible hydrations of carbon dioxide and aldehydes and the hydrolysis of esters.Aromatic and heterocyclic sulphonamides,and monovalent anions, act as inhibitors. Form C, which is easierto crystallize, has been studied by X-ray diffraction, and the structure is now known to 2 A resolution (Liljas et al., 1972). F orm B, which predominates in human red cells, is distinctly different from form C in catalytic properties and inhibitor binding, as well as in amino-acid sequence.B detailed comparison of the structures, of the two forms would therefore be of considerableinterest. The present paper describes the preparation of crystals of human enzyme B aswell as preliminary results of X-ray diffraction studies on these crystals and someisomorphousheavy atom derivatives. Experiments to grow crystals were performed in dialysis bags or micro-diffusion cells (Zeppezauer, Eklund & Zeppezauer, 1968). The tendency for crystal formation was found to increase when lyophilization was avoided during the purification of the protein, and the treatment with organic solvents for removal of hemoglobin was replaced with more gentle methods such asgel filtration (Armstrong, Myers, Verpoorte & Edsall, 1966) or ion exchange chromatography (Armstrong et al., 1966; Nyman, 1961). These and previous experiences with carbonic anhydrase C (Strandberg, 1967) would suggest that the conformational homogeneity of the protein is of critical importance in the preparation of satisfactory crystals. The tendency for crystal formation increasedat alkaline pH, but at values above pH9 only amorphous precipitates were obtained and most experiments to grow crystals were carried out between pH 8 and 9. The presenceof the sulphonamide inhibitor acetazolamide (99% of total protein as enzyme-inhibitor complex) was found to 601
602
K.
K.
KANNAN
ET
AL.
a/2
b/;
0 L--l--T
a
t
Fro. 1. Difference Patterson projections at 2 A resolution between zinc-free enzyme treated with HgCl, (BAL + HgCls derivative) and zinc-free enzyme (BAL derivative). The arrows indicate the Harker vectors which are related to the general vector marked by a cross. The three vectors together correspond to heavy atom binding at a single site of the molecule. The dotted contours define the zero level. The full contours are drawn at an interval of 75 on an arbitrary scale where the origin peak is scaled to 999. The Harker peaks have a value of about 450 on this scale and the general peaks have a value of about 250. The background level is of the order of 125. Negative values are not contoured, the minimum value being - 150. The AF2 (000) term is not included in the calculation. (a) Projection along the c-axis. (b) Projection along the b-axis.
LETTERS
TO
THE
EDITOR
603
markedly increasecrystallizability, but it hasonly twice beenpossibleto obtain crystals of the complex with dimensionssuitable for X-ray studiesby dialysis from ammonium sulphate solutions. These crystals were obtained in 2.5 M-(NH&SO,, 0.05 M-Tris. HCl (pH 8*7), after a few months at 4°C. Similar results have been reported by Coleman (1968). However, by using small amounts of the crystals as seeds,a more rapid and convenient procedure for production of crystals has been developed which will be described below. An 8% solution of the enzyme, native or complexed with inhibitors, was dialysed in 2.3 M-(NH&SO,, 0.05 M-Tris*HCl (pH 8.7). After three or four days the solution was centrifuged to remove amorphous precipitate, and the supernatant solution, containing about 5% protein, was diluted to about 3% in 2.3 M-(NH&SO,, and sucked up into a thick-walled glasscapillary (50 ~1.). One end of the capillary was then sealed.Selectedcrystals of the native enzyme, or in complex with inhibitors, were then crushed into small bits and washed in 2.3 M-(NH&SO,. Two or three of these bits were inserted through the open end of the capillary into the protein solution and the capillary sealed. In about six days these seedscan be seento heal and exhibit welldeveloped faces and increase in dimensions (O-05-mmseedshave grown to O-20-mm crystals). Crystals with dimensionsof about 2 mm have been obtained from such seeds in about a month. Zinc-free enzyme in the crystalline state (BAL derivative) was obtained by removing the zinc ion with 2,3-dimercaptopropanol-1 (BAL) according to the method described for the human C enzyme (Tilander, Strandberg & Pridborg, 1965). Crystals, of zinc-free enzyme were soaked in 2.3 M-(NH&SO, containing 0.001 M-HgCl, for one week to prepare the BAL + HgCl, derivative. The crystals of human carbonic anhydrase B are orthorhombic with a = 81.5, b = 73.6, and c = 37.1 A as measuredfrom precessionphotographs, and have a unit, cell volume of 223,000 A3. The space group is P 2,2,21 as determined from syste. matic absences.The dimensionsof the human B and C isozymes are very nearly the samefrom hydrodynamic data (Derrien & Laurent, 1969). By a comparison with the molecular dimensions of the human C enzyme as determined by X-ray diffraction. (Pridborg et al., 1967; Liljas et al., 1972) it can be concluded that there are four molecules per unit cell. The crystals of native enzyme and its derivatives are all isomorphous as judged from a comparison of cell dimensions. Difference Patterson calculations were performedintwoprojections. Figure 1 (a) and. (b) gives these at 2 d resolution along the c- and b-axes, respectively, between the BAL + HgCI, derivative and the BAL derivative. The low background level compared to the peak heights for the three vectors expected for a single heavy atom, site per asymmetric unit shows the good quality of the difference Patterson. The difference Patterson projections for the BAL derivative against the native enzyme give co-ordinates for the zinc ion which coincide with the mercury site in the BAL + HgCl, derivative. We are grateful for various types of assistance from U. Bengtsson, U. Carlbom, A. C. Carlsson and S. Nilsson. Generous gifts of Sephadex from Pharmacia AB are gratefully acknowledged. This work has been supported by grants from the National Institutes of Health, U.S. Public Health Service (AI GM 07382 and GM 12280-03), the Swedish Computing Authorities (Statskontoret, Stockholm), the Swedish Medical and Natural Science Research Councils and the Knut and Alice Wallenberg Foundation, Sweden.
604
K. K. KANNAN
ET
AL.
Wallenberg Laboratory University of Uppsala Uppsala, Sweden
K.K. K~NNAN K. FRIDBORQ P.-C. BEROSTI?N A. LILJAS S.LGVGREN M. PETEF B. STRANDBERG I. WAARA
Department of Biochemistry University of Giiteborg and Chalmers Institute of Technology Goteborg, Sweden
L. ADLER S.O.FALKBRINC P.O. G~THE P.O. NYMAN
Received
16 August
1971, and in revised form 5 November
1971
REFERENCES Armstrong, J. McD., Myers, D. V., Verpoorte, J. A. & Edsall, J. T. (1966). J. Biol. Chem. 241, 5137. Coleman, J. E. (1968). J. Biol. Chem. 243, 4574. Derrien, Y. & Laurent, G. (1969). Exposds Annuels de Biochimie Mddicale, 29, 167. Ed&l, J. T. (1968). Harvey Lectures, series 62, p. 191. Forster, R. E., Edsall, J. T., Otis, A. B. & Roughton, F. J. W. (editors) (1969). CO,: Chemical, Biochemical and Physiological Aspects. Washington DC. National Aeronautics and Space Administration. Fridborg, K., Kanuan, K. K., Liljas, A., Lundin, J., Strandberg, B., Strandberg, R., Tilander, B. & Wiren, G. (1967). J. Mol. Biol. 25, 505. Liljas, A., Kannan, K. K., Bergsten, P.-C., Waara, I., Fridborg, K., Strandberg, B., Carlbom, U., J&up, L., Liivgren, S. & Petef, M. (1972). Nature, in the press. Maren, T. H. (1967). Phyaiol. Rev. 47, 595. Nyman, P. 0. (1961). Biochim. biophys. Acta, 52, 1. Strandberg, B. (1967). Arkiw Kemi, 28, 1. Tilander, B., Strandberg, B. & Fridborg, K. (1965). J. Mol. Biol. 12, 740. Zeppezauer, M., Eklund, H. & Zeppezauer, E. (1968). Arch. B&hem. Biophys. 126, 564.