J. Mol.
Biol.
(1991) 222, 881-884
Crystallization and Preliminary X-ray Analysis of Phosphoporin from the Outer Membrane of Escherichia coli Alec D. Tuckert,
Stephen JackmanS, Michael W. Parkers and Demetrius
Tsernoglou
European
Molecular Biology Laboratory Post&h 102209 D-6900 Heidelberg, Germany
(Received
29 May
1991; accepted 26 September
1991)
Phosphoporin is a pore-forming transmembrane protein that spans the outer membrane of Escherichia coli and facilitates the diffusion of phosphates and phosphorylated compounds. Phosphoporin has been crystallized in several different crystal forms, although only one appears to be suitabl? for X-ray analysis. These crystals, which are hexagonal plates, diffract X-rays to 3 A Tesolution and 0belong to the space-group P6,22, with unit cell dimensions a = b = 121 A and c = 111 A. Keywords: crystallization;
membrane protein; porin; ion channel; X-ray crystallography
Present knowledge of membrane protein structure at atomic resolution has until recently been limited to the reaction centre of photosynthetic bacteria (Deisenhofer et al., 1985) and bacteriorhodopsin (Henderson et al., 1990), both of which feature alpha-helical hydrophobic transmembrane domains. The three-dimensional structure of porin from Rhodobacter capsulatus has peen dletermined recently at a resolution of 1.8 A (1 A = 0.1 nm) (Weiss et aE., 1991). This is the first report of an integral membrane protein, solved at atomic resolution, with a mainly beta-sheet topology. In the past ten years, three-dimensional crystals from two other beta-sheet outer membrane proteins, matrix porin and maltoporin, from Escherichia coli have also been worked on extensively, mainly at the Biozentrum, Base1 (Garavito et aE., 1983; Pauptit et al., 1991; &auffer et al., 1990). For a recent review on porins and their properties see Jap & Walian (1990). In general, porins form trimers that are stable in detergents and are fairly resistant to proteases. Spectroscopic investigation indicates a high proportion of beta-sheet structure (Neuhaus, 1982a,b; Nabedryk et al., 1988). Primary structure analysis
indicates that there are no long stretches of hydrophobic residues that would be likely membrane spanning segments. Recently, an electron microscopereconstruction of phosphoporin at a resolution of 6 A has been published (Jap et al., 1991); it shows elliptical, ring-like structures that have been interpreted as the projected images of beta-sheet seen edge-on. Here we report the crystallization and preliminary X-ray analysis of phosphoporin, another member of this family of proteins from the outer membrane of E. coli. Phosphoporin spans the outer membrane of the bacterium and facilitates the diffusion of inorganic and organic phosphates (Lugtenberg & Van Alphen, 1983). The protein was extracted and purified from an overproducing E. coli strain (CE 1248, a kind gift from J. Tommassen) using the non-ionic detergent octylpolyoxyethylene (Garavito & Rosenbusch, 1986) in a purification scheme similar to that described for maltoporin and matrix porin (Neuhaus et aE., 1982a). Protein was judged to be pure by SDS-silver staining. Detergent was exchanged by repeated dilution and concentration using an Amicon dialysis cell. The drop test (Garavito et al., 1984) was used as a quick method to determine possible crystallization
t Present address: ICI Pharmaceuticals, Alderley Park, Macclesfield, Cheshire SHlO 4TG, England. $ Present address: School of Medicine, Yale University, CT 06511, U.S.A. 0 Present address: St Vincent’s Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065, Australia.
conditions.
A drop
(8 to 10 ~1) of protein
buffered
11Abbreviations with M, = 4000.
used: PEG 4000, polyethylene
glycol
881 0022-2836/91/240881-04
$03.00/O
in
a detergent solution was layered on top of another drop of buffered polyethylene glycol (PEGJJ) 4000
0
1991
Academic
Press
Limited
882
A. D. Tucker
et al.
Figure 1. Crystallization trials for phosphoporin. Conditions for outside reservoir: (a) 50 mM-phosphate (pH 7.6). 14’>0 (pH 62). 14% PEG 4000. PEG 4000, 100 mmNaCl, @S% beta-octylglucoside, @l T/o C,E,; (b) 50 m&I-phosphate @5 M-NaCl, @S% beta-octylglucoside, @2% C 8E 6 11; (c) 50 mlvr-acetate (pH 54. 1546 PEG 4000. @5 M-PiaCl, 0.7”& beta-octylglucoside, @2% C*E,-, 1; (d) 50 mw-phosphate (pH 62): 14:& PEG 4000, 100 mM-NaC1. o%(/, hrtaoctylglucoside, O-l% C&E,. The protein concentration in each case was 8 to 10 mg/ml. The bars represent, 50 pm.
and detergent. Crystallization trials were carried out in triplicate, drops were concentrated by vapour diffusion against an outside reservoir. See the legend to Figure 1 for details. As has been previously reported for matrix porin and maltoporin (Garavito et al., 1983), the effect of mixed detergents and changing NaCl concentration can have a dramatic effect on crystal morphology (Garavito et al., 1984). Crystals appeared in most cases after two to t,hree days at room temperature with incubation against a buffered PEG 4000/NaCl reservoir. Depending on the composition of the drops, crystals could appear with or without phase separation. No crystals were obtained by the hanging-drop method, although end conditions were similar. We then attempted to grow larger well-ordered crystals by microdialysis (Garavito et al., 1983; Pauptit et al., 1991; Stauffer et al., 1990), a method that has proved to be successful for matrix porin and maltoporin. Depending on the pH and deter-
gent mixture. one of three distinct crystal forms could be grown. In all cases, protein concentration was between 8 and 10 mg/ml. The protein solut.ion was prepared by overnight dialysis at room tcmperature against one of the following crystallization buffers. Crystal form 1 was produced when protein was initially dialysed as above against 50 rnM-phosphatr (pH 6.2), 100 mi%-NaCl, 7”/, (w/v) PEG 4000, 0.7”;, beta-octylglucoside, 0.2 y/b CsE,- , 1. For the microdialysis experiments, 50 ,uI of the protein solution equilibrated against the above was dialysed against 3.5 ml of t’he buffer containing 14 to 16 “/;, PEG 4000. Long, thick needles (not shown, but very similar to those in Fig. l(b) grew very rapidly over a period of three to five days at room temperature to a final size of 1.0 mm x 0.2 mm x0.1 mm. These crystals showed no diffraction. Crystal form 2 was produced by initial dialysis against 50 m&I-phosphate (pH 62), 100 mw-NaCl. 7% PEG 4000, @So/b beta-octylglucosidle. and (PI O;,
883
Communications
Figure02. Crystals of phosphoporin. (a) Well-formed 3 A. The bars represent 200 pm.
crystals with poor diffraction.
(b) Hexagonal
plates diffracting
to
about
CsE,. The microdialysis was carried out as above, in this buffer with the PEG 4000 concentration at 14%. The wedge-shaped crystals shown in Figure 2(a) grew at room temperature over a period of 10 to 14 days to a maximum size of 0.3 mm x 0.3 mm x 0.1 mm but showed no diffraction. Crystal form 3 was prepared by dialysis of protein in the same conditions as for crystal form 2 except that the pH was raised to 7.6. As can be seen in Figure 2(b), hexagonal plates are produced. These crystals grow to a maximum size of 05 mm x 0.5 mm x @OS mm over a period of two to three weeks at room temperature. Diffraction on “still” photographs extends to a resolution of about was determined to be P6,22 3.0 A. The space-g;oup with a = b = 121 A, c = 111 A. The crystals show little signs of radiation damage after 30 hours in t,he beam. A native data set to 3.0 A resolution was collected using a Siemens/Nicolet detector system with a Rsym of 8.9o/o. There appears to be one merging molecule in the asymmetric unit on the basis of volume per mass calculations (Matthews, 1968). The porins, when reasonably pure, appear to crystallize fairly readily, although the resulting crystals are not always suitable for detailed structural studies. We report here the successful crystallization of a third member of this family of integral membrane proteins from the outer membrane E=. coli, and, furthermore, that one of the crystal forms is suitable for X-ray crystallographic analysis.
We are grateful to Professor J. P. Rosenbusch for continued support of this work and to Dr Franc Pattus for encouragement. One of us, A.D.T., is especially grateful to Dr Mike Garavito for many helpful discussions. S.J. was a recipient of financial assistance from the DAAD.
References Deisenhofer, J., Epp, O., Miki, K., Huber, R. & Michel, H. (1985). Structure of the protein subunits in the viridis at ph$oreaction center of Rhodopseudomonus 3 A resolution. Nature (London), 318, 618-624. Garavito. R. M., & Rosenbusch, J. P. (1986). Isolation and crystallisation of bacterial porin. Methods Enzymol. 125, 309-328. Garavito, R. M., Jenkins, J., Jansonius, J. N., Karlsson, R. & Rosenbusch, J. P. (1983). X-ray diffraction analysis of matrix porin, an integral membrane protein from Escherichia coli outer membranes. J. Mol. Biol. 164, 313-327. Garavito, R. M., Hinz, U. & Neuhaus. J.-M. (1984). The crystallisation of outer membrane proteins from Escherichia
coli.
J. Biol.
Chem.
259,
4254-4257.
Henderson, R., Baldwin, J. M.. Ceska, T. A., Zemlin, F., Beckman, E. & Downing, K. H. (1990). Model for the structure of bacteriorhodopsin based on highresolution electron cryo-microscopy. J. Mol. Biol. 213, 899-929. Jap, B. K. & Walian, P. J. (1990). Biophysics of the structure and function of porins. &. Rev. Biophys. 23, 367-403. Jap, B. K., Walian, P. J. & Gehring, K. (1991). Structural architecture of an outer membrane channel as determined by electron crystallography. Nature (London), 350, 167-170. Lugtenberg, B. & Van Alphen, L. (1983). Molecular architecture and functioning of the outer membrane of Escherichia coli and other Gram-negative bacteria. Biochim. Biophys. Acta, 737, 51-115. Matthews, B. W. (1968). Solvent content of protein crystals. J. Mol. Biol. 33, 491-497. Nabedryk, E., Garavito, R. M. & Bret,on, J. (1988). The orientation of beta sheets in porin. 4 polarised Fourier transform infrared spectroscopic investigation. Biophys. J. 53, 671-676. Neuhaus, J.-M. (1982a). Ph.D. thesis, University of Basel. Neuhaus, J.-M. (19823). The receptor protein of phage lambda, purification, characterisation and preliminary electrical studies in planar lipid bilayers. Ann. Microbial. (Inst. Pasteur), 133A, 27-32. Pauptit, R. A., Zhang, H., Rummel. G.. Schirmer, T..
884
A. D. Tucker
Jansonius, J. H. & Rosenbuch, J. P. (1991). Trigonal crystals of porin from Escberichia coli. J. Mol. Biol. 218, 505-507. Stauffer, K. A., Page, M. G. P., Hardmeyer, A., Keller, T. A. $ Pauptit, R. A. (1990). Crystallization and preliminary X-ray characterization of maltoporin from Escherichia coli. J. Mol. Biol. 211, 297-299. Edited
et al. Weiss, M. S., Kreusch, A., Schiltz, E., Nestel, LT.. Welte. W., Weckesser, J. & Schulz, G. E. (1991). The structure of porin from Rh&oba&er capsulatus at 1% A resolution. FEBS Letters, 280, 379-382.
by R. Huber
Biol.
(1991) 222, 881-884
Crystallization and Preliminary X-ray Analysis of Phosphoporin from the Outer Membrane of Escherichia coli Alec D. Tuckert,
Stephen JackmanS, Michael W. Parkers and Demetrius
Tsernoglou
European
Molecular Biology Laboratory Post&h 102209 D-6900 Heidelberg, Germany
(Received
29 May
1991; accepted 26 September
1991)
Phosphoporin is a pore-forming transmembrane protein that spans the outer membrane of Escherichia coli and facilitates the diffusion of phosphates and phosphorylated compounds. Phosphoporin has been crystallized in several different crystal forms, although only one appears to be suitabl? for X-ray analysis. These crystals, which are hexagonal plates, diffract X-rays to 3 A Tesolution and 0belong to the space-group P6,22, with unit cell dimensions a = b = 121 A and c = 111 A. Keywords: crystallization;
membrane protein; porin; ion channel; X-ray crystallography
Present knowledge of membrane protein structure at atomic resolution has until recently been limited to the reaction centre of photosynthetic bacteria (Deisenhofer et al., 1985) and bacteriorhodopsin (Henderson et al., 1990), both of which feature alpha-helical hydrophobic transmembrane domains. The three-dimensional structure of porin from Rhodobacter capsulatus has peen dletermined recently at a resolution of 1.8 A (1 A = 0.1 nm) (Weiss et aE., 1991). This is the first report of an integral membrane protein, solved at atomic resolution, with a mainly beta-sheet topology. In the past ten years, three-dimensional crystals from two other beta-sheet outer membrane proteins, matrix porin and maltoporin, from Escherichia coli have also been worked on extensively, mainly at the Biozentrum, Base1 (Garavito et aE., 1983; Pauptit et al., 1991; &auffer et al., 1990). For a recent review on porins and their properties see Jap & Walian (1990). In general, porins form trimers that are stable in detergents and are fairly resistant to proteases. Spectroscopic investigation indicates a high proportion of beta-sheet structure (Neuhaus, 1982a,b; Nabedryk et al., 1988). Primary structure analysis
indicates that there are no long stretches of hydrophobic residues that would be likely membrane spanning segments. Recently, an electron microscopereconstruction of phosphoporin at a resolution of 6 A has been published (Jap et al., 1991); it shows elliptical, ring-like structures that have been interpreted as the projected images of beta-sheet seen edge-on. Here we report the crystallization and preliminary X-ray analysis of phosphoporin, another member of this family of proteins from the outer membrane of E. coli. Phosphoporin spans the outer membrane of the bacterium and facilitates the diffusion of inorganic and organic phosphates (Lugtenberg & Van Alphen, 1983). The protein was extracted and purified from an overproducing E. coli strain (CE 1248, a kind gift from J. Tommassen) using the non-ionic detergent octylpolyoxyethylene (Garavito & Rosenbusch, 1986) in a purification scheme similar to that described for maltoporin and matrix porin (Neuhaus et aE., 1982a). Protein was judged to be pure by SDS-silver staining. Detergent was exchanged by repeated dilution and concentration using an Amicon dialysis cell. The drop test (Garavito et al., 1984) was used as a quick method to determine possible crystallization
t Present address: ICI Pharmaceuticals, Alderley Park, Macclesfield, Cheshire SHlO 4TG, England. $ Present address: School of Medicine, Yale University, CT 06511, U.S.A. 0 Present address: St Vincent’s Institute of Medical Research, 41 Victoria Parade, Fitzroy, Victoria 3065, Australia.
conditions.
A drop
(8 to 10 ~1) of protein
buffered
11Abbreviations with M, = 4000.
used: PEG 4000, polyethylene
glycol
881 0022-2836/91/240881-04
$03.00/O
in
a detergent solution was layered on top of another drop of buffered polyethylene glycol (PEGJJ) 4000
0
1991
Academic
Press
Limited
882
A. D. Tucker
et al.
Figure 1. Crystallization trials for phosphoporin. Conditions for outside reservoir: (a) 50 mM-phosphate (pH 7.6). 14’>0 (pH 62). 14% PEG 4000. PEG 4000, 100 mmNaCl, @S% beta-octylglucoside, @l T/o C,E,; (b) 50 m&I-phosphate @5 M-NaCl, @S% beta-octylglucoside, @2% C 8E 6 11; (c) 50 mlvr-acetate (pH 54. 1546 PEG 4000. @5 M-PiaCl, 0.7”& beta-octylglucoside, @2% C*E,-, 1; (d) 50 mw-phosphate (pH 62): 14:& PEG 4000, 100 mM-NaC1. o%(/, hrtaoctylglucoside, O-l% C&E,. The protein concentration in each case was 8 to 10 mg/ml. The bars represent, 50 pm.
and detergent. Crystallization trials were carried out in triplicate, drops were concentrated by vapour diffusion against an outside reservoir. See the legend to Figure 1 for details. As has been previously reported for matrix porin and maltoporin (Garavito et al., 1983), the effect of mixed detergents and changing NaCl concentration can have a dramatic effect on crystal morphology (Garavito et al., 1984). Crystals appeared in most cases after two to t,hree days at room temperature with incubation against a buffered PEG 4000/NaCl reservoir. Depending on the composition of the drops, crystals could appear with or without phase separation. No crystals were obtained by the hanging-drop method, although end conditions were similar. We then attempted to grow larger well-ordered crystals by microdialysis (Garavito et al., 1983; Pauptit et al., 1991; Stauffer et al., 1990), a method that has proved to be successful for matrix porin and maltoporin. Depending on the pH and deter-
gent mixture. one of three distinct crystal forms could be grown. In all cases, protein concentration was between 8 and 10 mg/ml. The protein solut.ion was prepared by overnight dialysis at room tcmperature against one of the following crystallization buffers. Crystal form 1 was produced when protein was initially dialysed as above against 50 rnM-phosphatr (pH 6.2), 100 mi%-NaCl, 7”/, (w/v) PEG 4000, 0.7”;, beta-octylglucoside, 0.2 y/b CsE,- , 1. For the microdialysis experiments, 50 ,uI of the protein solution equilibrated against the above was dialysed against 3.5 ml of t’he buffer containing 14 to 16 “/;, PEG 4000. Long, thick needles (not shown, but very similar to those in Fig. l(b) grew very rapidly over a period of three to five days at room temperature to a final size of 1.0 mm x 0.2 mm x0.1 mm. These crystals showed no diffraction. Crystal form 2 was produced by initial dialysis against 50 m&I-phosphate (pH 62), 100 mw-NaCl. 7% PEG 4000, @So/b beta-octylglucosidle. and (PI O;,
883
Communications
Figure02. Crystals of phosphoporin. (a) Well-formed 3 A. The bars represent 200 pm.
crystals with poor diffraction.
(b) Hexagonal
plates diffracting
to
about
CsE,. The microdialysis was carried out as above, in this buffer with the PEG 4000 concentration at 14%. The wedge-shaped crystals shown in Figure 2(a) grew at room temperature over a period of 10 to 14 days to a maximum size of 0.3 mm x 0.3 mm x 0.1 mm but showed no diffraction. Crystal form 3 was prepared by dialysis of protein in the same conditions as for crystal form 2 except that the pH was raised to 7.6. As can be seen in Figure 2(b), hexagonal plates are produced. These crystals grow to a maximum size of 05 mm x 0.5 mm x @OS mm over a period of two to three weeks at room temperature. Diffraction on “still” photographs extends to a resolution of about was determined to be P6,22 3.0 A. The space-g;oup with a = b = 121 A, c = 111 A. The crystals show little signs of radiation damage after 30 hours in t,he beam. A native data set to 3.0 A resolution was collected using a Siemens/Nicolet detector system with a Rsym of 8.9o/o. There appears to be one merging molecule in the asymmetric unit on the basis of volume per mass calculations (Matthews, 1968). The porins, when reasonably pure, appear to crystallize fairly readily, although the resulting crystals are not always suitable for detailed structural studies. We report here the successful crystallization of a third member of this family of integral membrane proteins from the outer membrane E=. coli, and, furthermore, that one of the crystal forms is suitable for X-ray crystallographic analysis.
We are grateful to Professor J. P. Rosenbusch for continued support of this work and to Dr Franc Pattus for encouragement. One of us, A.D.T., is especially grateful to Dr Mike Garavito for many helpful discussions. S.J. was a recipient of financial assistance from the DAAD.
References Deisenhofer, J., Epp, O., Miki, K., Huber, R. & Michel, H. (1985). Structure of the protein subunits in the viridis at ph$oreaction center of Rhodopseudomonus 3 A resolution. Nature (London), 318, 618-624. Garavito. R. M., & Rosenbusch, J. P. (1986). Isolation and crystallisation of bacterial porin. Methods Enzymol. 125, 309-328. Garavito, R. M., Jenkins, J., Jansonius, J. N., Karlsson, R. & Rosenbusch, J. P. (1983). X-ray diffraction analysis of matrix porin, an integral membrane protein from Escherichia coli outer membranes. J. Mol. Biol. 164, 313-327. Garavito, R. M., Hinz, U. & Neuhaus. J.-M. (1984). The crystallisation of outer membrane proteins from Escherichia
coli.
J. Biol.
Chem.
259,
4254-4257.
Henderson, R., Baldwin, J. M.. Ceska, T. A., Zemlin, F., Beckman, E. & Downing, K. H. (1990). Model for the structure of bacteriorhodopsin based on highresolution electron cryo-microscopy. J. Mol. Biol. 213, 899-929. Jap, B. K. & Walian, P. J. (1990). Biophysics of the structure and function of porins. &. Rev. Biophys. 23, 367-403. Jap, B. K., Walian, P. J. & Gehring, K. (1991). Structural architecture of an outer membrane channel as determined by electron crystallography. Nature (London), 350, 167-170. Lugtenberg, B. & Van Alphen, L. (1983). Molecular architecture and functioning of the outer membrane of Escherichia coli and other Gram-negative bacteria. Biochim. Biophys. Acta, 737, 51-115. Matthews, B. W. (1968). Solvent content of protein crystals. J. Mol. Biol. 33, 491-497. Nabedryk, E., Garavito, R. M. & Bret,on, J. (1988). The orientation of beta sheets in porin. 4 polarised Fourier transform infrared spectroscopic investigation. Biophys. J. 53, 671-676. Neuhaus, J.-M. (1982a). Ph.D. thesis, University of Basel. Neuhaus, J.-M. (19823). The receptor protein of phage lambda, purification, characterisation and preliminary electrical studies in planar lipid bilayers. Ann. Microbial. (Inst. Pasteur), 133A, 27-32. Pauptit, R. A., Zhang, H., Rummel. G.. Schirmer, T..
884
A. D. Tucker
Jansonius, J. H. & Rosenbuch, J. P. (1991). Trigonal crystals of porin from Escberichia coli. J. Mol. Biol. 218, 505-507. Stauffer, K. A., Page, M. G. P., Hardmeyer, A., Keller, T. A. $ Pauptit, R. A. (1990). Crystallization and preliminary X-ray characterization of maltoporin from Escherichia coli. J. Mol. Biol. 211, 297-299. Edited
et al. Weiss, M. S., Kreusch, A., Schiltz, E., Nestel, LT.. Welte. W., Weckesser, J. & Schulz, G. E. (1991). The structure of porin from Rh&oba&er capsulatus at 1% A resolution. FEBS Letters, 280, 379-382.
by R. Huber
