J. Mol. Biol. (1990) 211,
297-299
COMMUNICATIONS
Crystallization Kathrin
and Preliminary X-ray Characterization Maltoporin from Escherichia coli
A. StaufferJf, Malcolm G. P. Page, Ariane Hardmeyer, and Richard A. Pauptit’
of
Thomas A. Kelller
Departm,ent. of Microbiology and 1Department of Structural Biology Biocentre University of Base1 Klingelbergstr, 70 CH-4056 Basel, Switzerland (Received 13 July Crystals to 3 A which diffusion belong and c=
1989, and in revised form 9 September 1989)
of maltoporin (the bacteriophage 1 receptor of Escherichia co&) that diffract X-rays resolution can be grown reproducibly. Maltoporin is an integral membrane protein, forms a channel in the E. coli outer membrane that specifically facilitates the of maltose and maltodextrins. The crystals have a rhombic prismatic habit and to the orthorhombic space group C222, with unit cell dimensions a= 130 A, b= 213 A 216 A. X-ray structure determination is underway.
Current knowledge of high resolution structures of membrane proteins is limited.. The structures of the reaction centre of photosynthetic bacteria (Deisenhofer et al., 1985) and bacteriorhodopsin (Henderson & Unwin, 1975; Baldwin et al., 1988) feature cc-helical hydrophobic transmembrane domains, while the channel-forming porin from Escherichia coli contains predominantly p-structure (Kleffel et al., 1985). Over the past ten years it has become apparent that with effort the crystallization of membrane proteins can be successful, while the optimal choice of the necessary detergents remains empirical. Here we report the crystallization of maltoporin from E. coli. This protein, coded by the gene 1amB and first characterized through its role as the receptor of bacteriophage jl (Randall & Schwartz, 1973), has been shown to form a pore in the outer membrane that exhibits a molecular sieve property similar to that of other porins, but allows preferential diffusion of maltose and maltodextrins (Luckey & Nikaido, 1980; Dargent et al., 1987). of integral membrane As is characteristic proteins, maltoporin is insoluble in water and requires detergents for extraction from the outer membrane. It associates as a trimer (3 x 47,400 Da) which is stable over a pH range of 4 to 10 and at temperatures of up to 90°C (Neuhaus, 1982a). The
t Present address: Laboratory of Molecular Biology, Medical Research Council, Hills Road, Cambridge, Great Britain. Om2-2836/90/02029743
$03.00/O
primary structure, deduced from the 1amB gene sequence (Clement & Hofnung, 1981), does not contain long stretches of hydrophobic residues that would be obvious candidates for spanning the membrane. Electron microscopy of reconstituted arrays of maltoporin reveals a trimeric structure featuring a thplet of stain-filled indentations that represent channels (Lepault et al. 1988). At 25 A (1 A=O.l nm) resolution the structure resembles that obtained from arrays of the unspecific OmpF porin (Engel et al., 1985), despite lack of sequence homology. Circular dichroism measurements indicate a high proportion of p-structure (Neuhaus, 1982a,b), as is found for the other porins. The finding that pore-forming proteins unrelated in sequence might be structurally similar raises many questions concerning the principles of membrane protein folding. To approach such1 problems, and in order to understand the molecular basis of channel selectivity, it is important to albtain high-resolution structural data, which implies the preparation of crystals suitable for X-ray crystallographic analysis. For maltoporin, this is described below. The protein was purified from an overproducing E. coli strain (E. coli pop65lOpAC1, a kind gift from M. Hofnung) that contains the 1amB gene on a multicopy plasmid under control of the tat promoter. Cells were broken by passage through a French pressure cell. The resulting homogenate was centrifuged at 39,000g for 30 minutes. Membrane pellets were extracted twice with a’ buffer solution containing 20 m&r-sodium phosphate (pH 7.5),
297
0
1990
Academic
Press
Limited
K. A. Staufler
298
0.1 M-NaCl, 3 mw-NaNV,, 0.2 mM-dithiothreitol and 1 y. octyl-polyoxyethylene (octylPOE-i-), at 60 “C for 30 minutes. Then, in order to solubilize maltoporin, two extractions were performed under the same conditions except that, the buffer contained 3% octylPOE. The maltoporin was purified by two cycles of anion exchange chromatography using Whatman DE52 DEAE-cellulose columns. In the first’ cycle the extract was applied to the column, which was washed with extraction buffer containing 1 y. octylPOE. Maltoporin was eluted using the same buffer but with 1 M-NaGI. In the second cycle the pooled maltoporin fractions were applied in buffer containing O-05 M-NaCl and then eluted with a gradient of 0.05 M to 1.0 M-NaCl. The fractions containing maltoporin were pooled and concentrated by ultrafiltration. The concentrate was 25 mM-imidazole, 0.1 rnMequilibrated wit,h dithiothreitol and 1 y. octylPOE, adjusted to pH 7 with concentrated HCl and applied to a column of Pharmacia Polybuffer exchanger 94. Maltoporin was eluted with 12% Polybuffer 74 adjusted to pH 3 containing 1 oh octylPOE. Final purification was achieved on Sephadex G-150 equilibrated with 20 mM-sodium phosphate (pH 7), 61 M-hTaC1, 0.1 mM-dithiothreitol, 2 mM-NaEDTA, 3 mM-Nan’, and 1 y. octylPOE. By this procedure, which is based on that used for porin (Garavito & Rosenbusch, 1986), a batch of 50 g E. coli cells yielded 50 to 60 mg of pure maltoporin. Buffer exchange for crystallization was accomplished by precipitating the protein with ethanol pre-cooled to - 7O”C, washing the precipitate once with ethanol and once with water, followed by resolubilization in a buffer solution containing 20 mM-N-2-hydroxyethylpiperazine-N’-2-ethanesulphonic acid (Hepes), pH 7, 0.4% fi-decylmaltoside, 0.1 ye dodecyl-nonaoxyethylene (CL2E,), O-1M-M&X,, 3 mM-NaN3 and 7.5 y. polyethylene glycol 2000, by bath sonication. Crystals were grown at room temperature by microdialysis (Garavito & Rosenbusch; 1986). The protein concentration was 7.0 mg/ml. The protein compartment (50 ~1) was separated by a dialysis membrane from the reservoir chamber containing t,he crystallization buffer with 15 to 18% polyethylene glycol 2000. Crystals of a rhombic prismatic habit with typical dimensions of 0.25 mm x 0.25 mm x 0.4 mm appeared within a few days. Several crystals grew to lengths of 2 to 3 mm. The crystals sometimes exhibited aggregation or twinning to give misleading hexagonal habits. Under some conditions large wedge-shaped crystals were also noticed but these yielded no observable X-ray diffraction. The prismatic crystals were characterized by precession photography as belonging to the orthorhombic space group C222,, with unit cell dimensions a=130 A, b=213 A and c=216 A. Still photographs (1 h exposure to X-ra.ys from a t Abbreviation
used: octylPOE,
octyl-polyoxyethylene.
et a:
rotating anode generator operated at 38 kV and 38 mA) indicated that the intensity data were measurable at 3.0 A resolution. Data collection for crystals of the native protein and a search for heavy-atom derivatives are underway. It seems reasonable to assume that the trimeric functional unit’ is also present in the crystal. The occurrence of one trimer per asymmetric unit would correspond to a volume/mass ratio (V,) of 5.2 d’/Da (Matthews, 1968), or a protein content of 2476 in the erystal. This agrees well with values observed for other membrane prot’eins (Garavit,o et al.: 1983; Deisenhofer et al., 1984; Witt et al., 1988). The crystals may be related to one of the micmcrystal forms reported previously and tentatively space group C222, with a= 119 A, assigned b=218 A, c=203 A (Garavito et al.: 1984). The use of an overproducing strain of E. coli for large-scale production and thorough opt’imization of purificat’ion procedures has allowed the screening of numerous crystallization conditions that eventually resulted in the reproducible growth of large crystals. The prot,ein crystallized readily in a variety of buffers, precipitants and detergents, but only one of the crystal forms obtained was suitable for diffraction experiments. The appearance of the orthorhombic crystals was strongly dependent on detergent composition. For instance, use of dodecylmaltoside instead of decylmaltoside did not produce the same crystal habit. Moreover, decylmaltoside alone does not sustain erystal growth and small amounts of polyoxyethylene detergents were required. Of these, dodecyl-nonaoxyethylene ga,ve the best result,s. The choice of buffer and ionic strength did not appear to be critical; any buffer having a pH between 6.5 and 7.5 seemed satisfactory and, while concentrations in the range 0.05 to 025 M produced the best results, crystal formation occurred with salt, concentrations between 601 and 1 M. The use of MgCl, rather than ?iaCl greatly improved crystal morphology. With the procedures now established, crystals of maltoporin can be reliably produced in quantity, allowing structural analysis to proceed. We are grateful to Professors J. S. Jansonius and J. P. Rosenbusch for support and active interest. This work was made possible through Swiss National Science Foundation grants 3.656.80 and 3294.85 to J. P. Rosenbusch and 31.25712% to J. N. Jansonius.
References Baldwin, J. 31.: &nderson, R., Beckman, E. & Zemiin. F. (1988). J. iKoZ. Riol. 202. 585-591. ClBment, J. M. & IHofnung, M. (1981). Cell, 27, 507-514. Dargent, B., Rosenbusch, J. P. & Pattus, F. (1987). FEBX Letters, 220, 13G142. Deisenhofer, (1984). Deisenhofer, (1985).
J., Epp, J. Mol. J., Epp,
O., Miki,
K.,
Huber,
R. & Michel,
H.
K., Huber, R.. & Michel, 318, 618-624.
H.
Biol. 18(B, 385-398. O., Miki,
Na.ture (London),
Communications
Engel,
A., Massalski, A., Schindler, H., Dorset, D. L. & Rosenbusch, J. P. (1985). Nature (London), 317, 643-645. Garavito, R. M. & Rosenbusch, J. P. (1986). Methods Enzymol. 125, 309-328. Garavito, R. M.; Jenkins, J. A., Jansonius, J. N., Karlsson, R. 8: Rosenbusch, J. P. (1983) J. Mol. Biol.
164, 313-327. Garavito, R. M.; Hinz, U. 8.1 Neuhaus, J.-M. (1984). J. Biol. Chem. 259, 42544257. Henderson, R. 8: Unwin, P. N. T. (1975). Nature (London), 257, 28-32. Kleffel, B., Garavito, R. M., Baumeister, W. & Rosenbusch, J. P. (1985). EMBO J. 4, 1589-1592.
Edited
299
Lepault, J., Dargent, J. P., Leonard, K. 261-268. Luckey, M. & Nikaido, U.S.A. 77, 1677171. Matthews, B. (1968). J. Neuhaus, J.-M. (1982a). Neuhaus, J.-M. (1982b). 133A, 21-32. Randall, L. & Schwartz,
B., Tichelaar, & Pattus, F. H.
(1980).
Mol. Ph.D. Ann. M.
W.,
Proc.
Nat.
Biol. 33, 49149’7. thesis; University Microbial. (Inst. (1973).
Rosenbusch, J. 7,
(1988). EMBO
Acad.
Sci.,
of Basel. Pasteur),
J. Bacterial.
116,
1436-1446. Witt,
I., Witt, H. T., Di Fiore, D., Riigner, M., Hinrichs, W., Saenger, W., Granzin, J., Betzel, C. & Dauter, Z. (1988). Ber. Bunsenges. Phys. Chem. 92, 150331506.
by R. Huber
297-299
COMMUNICATIONS
Crystallization Kathrin
and Preliminary X-ray Characterization Maltoporin from Escherichia coli
A. StaufferJf, Malcolm G. P. Page, Ariane Hardmeyer, and Richard A. Pauptit’
of
Thomas A. Kelller
Departm,ent. of Microbiology and 1Department of Structural Biology Biocentre University of Base1 Klingelbergstr, 70 CH-4056 Basel, Switzerland (Received 13 July Crystals to 3 A which diffusion belong and c=
1989, and in revised form 9 September 1989)
of maltoporin (the bacteriophage 1 receptor of Escherichia co&) that diffract X-rays resolution can be grown reproducibly. Maltoporin is an integral membrane protein, forms a channel in the E. coli outer membrane that specifically facilitates the of maltose and maltodextrins. The crystals have a rhombic prismatic habit and to the orthorhombic space group C222, with unit cell dimensions a= 130 A, b= 213 A 216 A. X-ray structure determination is underway.
Current knowledge of high resolution structures of membrane proteins is limited.. The structures of the reaction centre of photosynthetic bacteria (Deisenhofer et al., 1985) and bacteriorhodopsin (Henderson & Unwin, 1975; Baldwin et al., 1988) feature cc-helical hydrophobic transmembrane domains, while the channel-forming porin from Escherichia coli contains predominantly p-structure (Kleffel et al., 1985). Over the past ten years it has become apparent that with effort the crystallization of membrane proteins can be successful, while the optimal choice of the necessary detergents remains empirical. Here we report the crystallization of maltoporin from E. coli. This protein, coded by the gene 1amB and first characterized through its role as the receptor of bacteriophage jl (Randall & Schwartz, 1973), has been shown to form a pore in the outer membrane that exhibits a molecular sieve property similar to that of other porins, but allows preferential diffusion of maltose and maltodextrins (Luckey & Nikaido, 1980; Dargent et al., 1987). of integral membrane As is characteristic proteins, maltoporin is insoluble in water and requires detergents for extraction from the outer membrane. It associates as a trimer (3 x 47,400 Da) which is stable over a pH range of 4 to 10 and at temperatures of up to 90°C (Neuhaus, 1982a). The
t Present address: Laboratory of Molecular Biology, Medical Research Council, Hills Road, Cambridge, Great Britain. Om2-2836/90/02029743
$03.00/O
primary structure, deduced from the 1amB gene sequence (Clement & Hofnung, 1981), does not contain long stretches of hydrophobic residues that would be obvious candidates for spanning the membrane. Electron microscopy of reconstituted arrays of maltoporin reveals a trimeric structure featuring a thplet of stain-filled indentations that represent channels (Lepault et al. 1988). At 25 A (1 A=O.l nm) resolution the structure resembles that obtained from arrays of the unspecific OmpF porin (Engel et al., 1985), despite lack of sequence homology. Circular dichroism measurements indicate a high proportion of p-structure (Neuhaus, 1982a,b), as is found for the other porins. The finding that pore-forming proteins unrelated in sequence might be structurally similar raises many questions concerning the principles of membrane protein folding. To approach such1 problems, and in order to understand the molecular basis of channel selectivity, it is important to albtain high-resolution structural data, which implies the preparation of crystals suitable for X-ray crystallographic analysis. For maltoporin, this is described below. The protein was purified from an overproducing E. coli strain (E. coli pop65lOpAC1, a kind gift from M. Hofnung) that contains the 1amB gene on a multicopy plasmid under control of the tat promoter. Cells were broken by passage through a French pressure cell. The resulting homogenate was centrifuged at 39,000g for 30 minutes. Membrane pellets were extracted twice with a’ buffer solution containing 20 m&r-sodium phosphate (pH 7.5),
297
0
1990
Academic
Press
Limited
K. A. Staufler
298
0.1 M-NaCl, 3 mw-NaNV,, 0.2 mM-dithiothreitol and 1 y. octyl-polyoxyethylene (octylPOE-i-), at 60 “C for 30 minutes. Then, in order to solubilize maltoporin, two extractions were performed under the same conditions except that, the buffer contained 3% octylPOE. The maltoporin was purified by two cycles of anion exchange chromatography using Whatman DE52 DEAE-cellulose columns. In the first’ cycle the extract was applied to the column, which was washed with extraction buffer containing 1 y. octylPOE. Maltoporin was eluted using the same buffer but with 1 M-NaGI. In the second cycle the pooled maltoporin fractions were applied in buffer containing O-05 M-NaCl and then eluted with a gradient of 0.05 M to 1.0 M-NaCl. The fractions containing maltoporin were pooled and concentrated by ultrafiltration. The concentrate was 25 mM-imidazole, 0.1 rnMequilibrated wit,h dithiothreitol and 1 y. octylPOE, adjusted to pH 7 with concentrated HCl and applied to a column of Pharmacia Polybuffer exchanger 94. Maltoporin was eluted with 12% Polybuffer 74 adjusted to pH 3 containing 1 oh octylPOE. Final purification was achieved on Sephadex G-150 equilibrated with 20 mM-sodium phosphate (pH 7), 61 M-hTaC1, 0.1 mM-dithiothreitol, 2 mM-NaEDTA, 3 mM-Nan’, and 1 y. octylPOE. By this procedure, which is based on that used for porin (Garavito & Rosenbusch, 1986), a batch of 50 g E. coli cells yielded 50 to 60 mg of pure maltoporin. Buffer exchange for crystallization was accomplished by precipitating the protein with ethanol pre-cooled to - 7O”C, washing the precipitate once with ethanol and once with water, followed by resolubilization in a buffer solution containing 20 mM-N-2-hydroxyethylpiperazine-N’-2-ethanesulphonic acid (Hepes), pH 7, 0.4% fi-decylmaltoside, 0.1 ye dodecyl-nonaoxyethylene (CL2E,), O-1M-M&X,, 3 mM-NaN3 and 7.5 y. polyethylene glycol 2000, by bath sonication. Crystals were grown at room temperature by microdialysis (Garavito & Rosenbusch; 1986). The protein concentration was 7.0 mg/ml. The protein compartment (50 ~1) was separated by a dialysis membrane from the reservoir chamber containing t,he crystallization buffer with 15 to 18% polyethylene glycol 2000. Crystals of a rhombic prismatic habit with typical dimensions of 0.25 mm x 0.25 mm x 0.4 mm appeared within a few days. Several crystals grew to lengths of 2 to 3 mm. The crystals sometimes exhibited aggregation or twinning to give misleading hexagonal habits. Under some conditions large wedge-shaped crystals were also noticed but these yielded no observable X-ray diffraction. The prismatic crystals were characterized by precession photography as belonging to the orthorhombic space group C222,, with unit cell dimensions a=130 A, b=213 A and c=216 A. Still photographs (1 h exposure to X-ra.ys from a t Abbreviation
used: octylPOE,
octyl-polyoxyethylene.
et a:
rotating anode generator operated at 38 kV and 38 mA) indicated that the intensity data were measurable at 3.0 A resolution. Data collection for crystals of the native protein and a search for heavy-atom derivatives are underway. It seems reasonable to assume that the trimeric functional unit’ is also present in the crystal. The occurrence of one trimer per asymmetric unit would correspond to a volume/mass ratio (V,) of 5.2 d’/Da (Matthews, 1968), or a protein content of 2476 in the erystal. This agrees well with values observed for other membrane prot’eins (Garavit,o et al.: 1983; Deisenhofer et al., 1984; Witt et al., 1988). The crystals may be related to one of the micmcrystal forms reported previously and tentatively space group C222, with a= 119 A, assigned b=218 A, c=203 A (Garavito et al.: 1984). The use of an overproducing strain of E. coli for large-scale production and thorough opt’imization of purificat’ion procedures has allowed the screening of numerous crystallization conditions that eventually resulted in the reproducible growth of large crystals. The prot,ein crystallized readily in a variety of buffers, precipitants and detergents, but only one of the crystal forms obtained was suitable for diffraction experiments. The appearance of the orthorhombic crystals was strongly dependent on detergent composition. For instance, use of dodecylmaltoside instead of decylmaltoside did not produce the same crystal habit. Moreover, decylmaltoside alone does not sustain erystal growth and small amounts of polyoxyethylene detergents were required. Of these, dodecyl-nonaoxyethylene ga,ve the best result,s. The choice of buffer and ionic strength did not appear to be critical; any buffer having a pH between 6.5 and 7.5 seemed satisfactory and, while concentrations in the range 0.05 to 025 M produced the best results, crystal formation occurred with salt, concentrations between 601 and 1 M. The use of MgCl, rather than ?iaCl greatly improved crystal morphology. With the procedures now established, crystals of maltoporin can be reliably produced in quantity, allowing structural analysis to proceed. We are grateful to Professors J. S. Jansonius and J. P. Rosenbusch for support and active interest. This work was made possible through Swiss National Science Foundation grants 3.656.80 and 3294.85 to J. P. Rosenbusch and 31.25712% to J. N. Jansonius.
References Baldwin, J. 31.: &nderson, R., Beckman, E. & Zemiin. F. (1988). J. iKoZ. Riol. 202. 585-591. ClBment, J. M. & IHofnung, M. (1981). Cell, 27, 507-514. Dargent, B., Rosenbusch, J. P. & Pattus, F. (1987). FEBX Letters, 220, 13G142. Deisenhofer, (1984). Deisenhofer, (1985).
J., Epp, J. Mol. J., Epp,
O., Miki,
K.,
Huber,
R. & Michel,
H.
K., Huber, R.. & Michel, 318, 618-624.
H.
Biol. 18(B, 385-398. O., Miki,
Na.ture (London),
Communications
Engel,
A., Massalski, A., Schindler, H., Dorset, D. L. & Rosenbusch, J. P. (1985). Nature (London), 317, 643-645. Garavito, R. M. & Rosenbusch, J. P. (1986). Methods Enzymol. 125, 309-328. Garavito, R. M.; Jenkins, J. A., Jansonius, J. N., Karlsson, R. 8: Rosenbusch, J. P. (1983) J. Mol. Biol.
164, 313-327. Garavito, R. M.; Hinz, U. 8.1 Neuhaus, J.-M. (1984). J. Biol. Chem. 259, 42544257. Henderson, R. 8: Unwin, P. N. T. (1975). Nature (London), 257, 28-32. Kleffel, B., Garavito, R. M., Baumeister, W. & Rosenbusch, J. P. (1985). EMBO J. 4, 1589-1592.
Edited
299
Lepault, J., Dargent, J. P., Leonard, K. 261-268. Luckey, M. & Nikaido, U.S.A. 77, 1677171. Matthews, B. (1968). J. Neuhaus, J.-M. (1982a). Neuhaus, J.-M. (1982b). 133A, 21-32. Randall, L. & Schwartz,
B., Tichelaar, & Pattus, F. H.
(1980).
Mol. Ph.D. Ann. M.
W.,
Proc.
Nat.
Biol. 33, 49149’7. thesis; University Microbial. (Inst. (1973).
Rosenbusch, J. 7,
(1988). EMBO
Acad.
Sci.,
of Basel. Pasteur),
J. Bacterial.
116,
1436-1446. Witt,
I., Witt, H. T., Di Fiore, D., Riigner, M., Hinrichs, W., Saenger, W., Granzin, J., Betzel, C. & Dauter, Z. (1988). Ber. Bunsenges. Phys. Chem. 92, 150331506.
by R. Huber
