J. Mol. Biol. (1991) 219, 593-594
Crystallization of the Calcium-activated Phosphoenolpyruvate Carboxykinase from Escherichia coli K12 Louis T. J. Delbaere’,
Margaret Vandonselaarl, Daniel Glaeske2, Corrine Jabs1 and Hughes GoWe Departments of ‘Biochemistry and 2Microbiology University of Saskatchewan, Saskatoon Saskatchewan S7N 0 WO, Canada
(Received 22 January
1991; accepted 7 March 1991)
Single crystals of phosphoenolpyruvate carboxykinase from Escherichia coli K12 have been grown in the orthorhombic crystal system. Single crystals developed to a maximum size of O-25 mm x 925 mm x 1.5 mm by the technique of washing and reseeding. The space group is there is one enzyme molecule p2,2,2,, with a= 77.24 A, b= 89.18 A, c=93*24 A and 2=4; per crystallographic asymmetric unit and the solvent content is estimated to be 59O/&. The crystals diffract to at least 2.8 A d spacings and decompose in t,he X-ray beam after approximately two days of exposure.
Keywords: gluconeogenesis;
enzyme;
calcium-activated;
$03.00/O
reseeding
sites (Teintze et al., 1988). Site-specific mutagenesis of residues in either site affected calcium binding of protein S and assembly of protein S into spores (Teintze et aE., 1988). The periplasmic galactosebinding protein of Escherichia coli has a single calcium binding loop, but the structure determined by X-ray crystallography does not have helical regions surrounding the loop (Vyas et al., 1987). The of a calcium-binding prot,ein from sequence Streptomyces erythreus contains four potential calcium-binding motifs similar to those of calmodulin: possible helix-loop-helix structures are predicted for three of the sites (Swan et al., 1987). Phosphoenolpyruvate carboxykinase (PCKaset), EC 4.1.1.49) catalyzes the phosphorylation and decarboxylation of the Krebs cycle intermediate. oxaloacetic acid, to form phosphoenolpyruvate, a key reaction in bacterial gluconeogenesis (Sanwal, 1970). The ATP-dependent PCKase of E. coli K12 is activated by calcium (Goldie & Sanwal, 1980). Although the physiological purpose of calcium activation is unknown, the mechanism is allosteric, since partial digestion with trypsin desensitizes the purified enzyme to calcium activation without changing other kinetic parameters, and binding of the fluorescent calcium analogue, terbium, is abolished by trypsin cleavage. To learn more about the structure and allosteric regulation of PCKase from E. co&, the pckA gene has been sequenced (Medina of al., 1990).
Calcium-binding proteins such as troponins C, calmodulins, parvalbumins and intestinal calciumbinding proteins, mediate regulation of cellular activity by calcium in eukaryotic organisms. C’alcium-binding sites in these proteins have a continuous polypeptide chain structure of about 30 residues, consisting of a helix-loop-helix and denoted the EF-hand (Kretsinger & ?Il’ockolds, 1973). Calcium-binding proteins typically have from two to four calcium-binding sites per polypeptide chain. For troponin C: one pair of low-affinity sites and one pair of high-affinity sites have been observed and positive co-operativity between each pair of sites for calcium-binding is suggested (Potter & Gergely. 1975). Conformational changes caused by binding of calcium to different sites and interactions hetween different sites have complicated the study of the molecular mechanism of calcium binding. In order to simplify the experimental system, fragments of the natural proteins containing single binding sites (Leavis et al., 1978; IVaIl et al., 1981; ?\‘ewton et al., 1984) and a synthetic analogue of a calcium-binding site have been constructed (Delbaere et al., 1989). However, such artificial constructs do not permit study of allosteric effects associated with calcium binding and may have somewhat altered binding mechanisms. Several bacterial proteins have been studied which appear to have calcium-binding sites somewhat similar to those of eukaryotic proteins. Development-specific protein S from Myzococcus santhus contains two calcium-binding sites resembling the loops of the eukaryotic calcium binding 0022%2836/Q1/12059342
crystallization;
t Abbreviations used: PCKase, phosphoenolp?iruvate carboxykinase. PEG. polyethylene glvrol. 593 0
1991
Academic
Press
Limited
594
L. T. J. DelbaeTe et, al
IV-Terminal sequences of the purified enzyme and of proteolytic fragments confirm the identity of t,he protein product, and the translational start, site. The Mr value calculated from the amino acid sequence is consistent with a monomeric structure for this allosteric enzyme. The amino acid sequence of PCKase from E. COZY:. is 54 yO and 42:{, identical to the sequences predicted from the genes for other ATP-dependen t PCKase from Trypanosow~a hruwi and Saccharomyces cereaisiae, respectively. PCKase from T. bruwi and S. cere~isiar are not activated I)>, calcium and each have only partial homology t’o a calcium-binding site. Also, a sequence from the NIH gene bank, which is likely part of the Snlmonella typhi,muriunz PCKase gene? has been ident,ified and parGal sequence of the predicted prot,ein is X7 ‘:O homologous 60 t(he CI-terminal half of E. coli P(,‘Kase wit’h conserva.tion of the hypothet’ica,l calcium binding site. Calcium activation of 8. typhimwiunr PCKase appears to be identical to that observed for the /i:. coli enzyme. The cryst,al structure of’ a monomeric. clalciumbinding enzyme should help us learn more ahout thv mechanisms of calcium-binding and allost,eric* regula,tion as well as providing the tertiary stru&ure of this key gluconeogenic enzyme. ~Z’Vreport hertb the crystallization and preliminary studies on T’(‘Kase from E. roli KIT. PCKastt was isolated and purified as desc*ribed (Goldir & Sanwal, 1SW). (‘rystals of T’(tKase ~\‘f*r(’ grown hy the hanging drop vapor diffusion twhnique. ?\;eedle clusters first appeared when a drop caontaining 5 mg prot,ein/ml. 12*;‘,(!i, (w/v) plyethylene ,&-col 8000, and 25 mM-c~it’rat~-r)hoslha.t.~ huffw (pH 5.0) was equilibrated wit.h a reservoir of Z,rio{, PEG 8000. 50 mM-clitrat,e-phosphate buffer (pH .50) and sodium azide for six days ;~t 11 O(‘. Large c*rystals suitable for X-ray diffract.ion wer( grown by trashing and reseeditlg single c*rysta.ls selected from the needle clusters. according to the by Thaller it n,l. (1981). procedure developed Washed crystals were placed into 20 p_LIdrops containing 6 mg protein/ml, lOO;, PIGi 8000. 0.1 bT-ac>etate buffer (pH 5.4) and O*Ol’~;, (W/v) sodium axide. The seeded drops werp equilibra8ted over reservoirs containing 17 “;o 1’lN: X000 and 0.1 m-acetate buffer (pH 53) at room ternperat,urc. The washed and reseeded crystals grew to a maximum size of 0%~ mm x 0.25 mm x l-5 mm in two weeks time. The crystals exhibit the forms (101) and (011). P recession photographs of h0Z X-ray diffraction from crystals of PCKase were obtained using approximately 24 hours of exposure at 15°C with N-filtered copper radiation from a standard focus high intensity sealed tube operated at 50 kV, 40 mA. Reflections are absent for hO0, h#2n; 04~0, k # 2n; OOI, Z#2n. The Laue group is 222 and the space group is PZ12,2,, with unit cell dimensions n~77.24 8, b-89.18 A, c=93*24 A (1 A=0-1 nm) and 2~4; there is one PCKase molecule per asymmetric unit. The volume/unit mass (Matthews. 1968) is 3.029 A3/dalton and the solvent content, is
Crystallization of the Calcium-activated Phosphoenolpyruvate Carboxykinase from Escherichia coli K12 Louis T. J. Delbaere’,
Margaret Vandonselaarl, Daniel Glaeske2, Corrine Jabs1 and Hughes GoWe Departments of ‘Biochemistry and 2Microbiology University of Saskatchewan, Saskatoon Saskatchewan S7N 0 WO, Canada
(Received 22 January
1991; accepted 7 March 1991)
Single crystals of phosphoenolpyruvate carboxykinase from Escherichia coli K12 have been grown in the orthorhombic crystal system. Single crystals developed to a maximum size of O-25 mm x 925 mm x 1.5 mm by the technique of washing and reseeding. The space group is there is one enzyme molecule p2,2,2,, with a= 77.24 A, b= 89.18 A, c=93*24 A and 2=4; per crystallographic asymmetric unit and the solvent content is estimated to be 59O/&. The crystals diffract to at least 2.8 A d spacings and decompose in t,he X-ray beam after approximately two days of exposure.
Keywords: gluconeogenesis;
enzyme;
calcium-activated;
$03.00/O
reseeding
sites (Teintze et al., 1988). Site-specific mutagenesis of residues in either site affected calcium binding of protein S and assembly of protein S into spores (Teintze et aE., 1988). The periplasmic galactosebinding protein of Escherichia coli has a single calcium binding loop, but the structure determined by X-ray crystallography does not have helical regions surrounding the loop (Vyas et al., 1987). The of a calcium-binding prot,ein from sequence Streptomyces erythreus contains four potential calcium-binding motifs similar to those of calmodulin: possible helix-loop-helix structures are predicted for three of the sites (Swan et al., 1987). Phosphoenolpyruvate carboxykinase (PCKaset), EC 4.1.1.49) catalyzes the phosphorylation and decarboxylation of the Krebs cycle intermediate. oxaloacetic acid, to form phosphoenolpyruvate, a key reaction in bacterial gluconeogenesis (Sanwal, 1970). The ATP-dependent PCKase of E. coli K12 is activated by calcium (Goldie & Sanwal, 1980). Although the physiological purpose of calcium activation is unknown, the mechanism is allosteric, since partial digestion with trypsin desensitizes the purified enzyme to calcium activation without changing other kinetic parameters, and binding of the fluorescent calcium analogue, terbium, is abolished by trypsin cleavage. To learn more about the structure and allosteric regulation of PCKase from E. co&, the pckA gene has been sequenced (Medina of al., 1990).
Calcium-binding proteins such as troponins C, calmodulins, parvalbumins and intestinal calciumbinding proteins, mediate regulation of cellular activity by calcium in eukaryotic organisms. C’alcium-binding sites in these proteins have a continuous polypeptide chain structure of about 30 residues, consisting of a helix-loop-helix and denoted the EF-hand (Kretsinger & ?Il’ockolds, 1973). Calcium-binding proteins typically have from two to four calcium-binding sites per polypeptide chain. For troponin C: one pair of low-affinity sites and one pair of high-affinity sites have been observed and positive co-operativity between each pair of sites for calcium-binding is suggested (Potter & Gergely. 1975). Conformational changes caused by binding of calcium to different sites and interactions hetween different sites have complicated the study of the molecular mechanism of calcium binding. In order to simplify the experimental system, fragments of the natural proteins containing single binding sites (Leavis et al., 1978; IVaIl et al., 1981; ?\‘ewton et al., 1984) and a synthetic analogue of a calcium-binding site have been constructed (Delbaere et al., 1989). However, such artificial constructs do not permit study of allosteric effects associated with calcium binding and may have somewhat altered binding mechanisms. Several bacterial proteins have been studied which appear to have calcium-binding sites somewhat similar to those of eukaryotic proteins. Development-specific protein S from Myzococcus santhus contains two calcium-binding sites resembling the loops of the eukaryotic calcium binding 0022%2836/Q1/12059342
crystallization;
t Abbreviations used: PCKase, phosphoenolp?iruvate carboxykinase. PEG. polyethylene glvrol. 593 0
1991
Academic
Press
Limited
594
L. T. J. DelbaeTe et, al
IV-Terminal sequences of the purified enzyme and of proteolytic fragments confirm the identity of t,he protein product, and the translational start, site. The Mr value calculated from the amino acid sequence is consistent with a monomeric structure for this allosteric enzyme. The amino acid sequence of PCKase from E. COZY:. is 54 yO and 42:{, identical to the sequences predicted from the genes for other ATP-dependen t PCKase from Trypanosow~a hruwi and Saccharomyces cereaisiae, respectively. PCKase from T. bruwi and S. cere~isiar are not activated I)>, calcium and each have only partial homology t’o a calcium-binding site. Also, a sequence from the NIH gene bank, which is likely part of the Snlmonella typhi,muriunz PCKase gene? has been ident,ified and parGal sequence of the predicted prot,ein is X7 ‘:O homologous 60 t(he CI-terminal half of E. coli P(,‘Kase wit’h conserva.tion of the hypothet’ica,l calcium binding site. Calcium activation of 8. typhimwiunr PCKase appears to be identical to that observed for the /i:. coli enzyme. The cryst,al structure of’ a monomeric. clalciumbinding enzyme should help us learn more ahout thv mechanisms of calcium-binding and allost,eric* regula,tion as well as providing the tertiary stru&ure of this key gluconeogenic enzyme. ~Z’Vreport hertb the crystallization and preliminary studies on T’(‘Kase from E. roli KIT. PCKastt was isolated and purified as desc*ribed (Goldir & Sanwal, 1SW). (‘rystals of T’(tKase ~\‘f*r(’ grown hy the hanging drop vapor diffusion twhnique. ?\;eedle clusters first appeared when a drop caontaining 5 mg prot,ein/ml. 12*;‘,(!i, (w/v) plyethylene ,&-col 8000, and 25 mM-c~it’rat~-r)hoslha.t.~ huffw (pH 5.0) was equilibrated wit.h a reservoir of Z,rio{, PEG 8000. 50 mM-clitrat,e-phosphate buffer (pH .50) and sodium azide for six days ;~t 11 O(‘. Large c*rystals suitable for X-ray diffract.ion wer( grown by trashing and reseeditlg single c*rysta.ls selected from the needle clusters. according to the by Thaller it n,l. (1981). procedure developed Washed crystals were placed into 20 p_LIdrops containing 6 mg protein/ml, lOO;, PIGi 8000. 0.1 bT-ac>etate buffer (pH 5.4) and O*Ol’~;, (W/v) sodium axide. The seeded drops werp equilibra8ted over reservoirs containing 17 “;o 1’lN: X000 and 0.1 m-acetate buffer (pH 53) at room ternperat,urc. The washed and reseeded crystals grew to a maximum size of 0%~ mm x 0.25 mm x l-5 mm in two weeks time. The crystals exhibit the forms (101) and (011). P recession photographs of h0Z X-ray diffraction from crystals of PCKase were obtained using approximately 24 hours of exposure at 15°C with N-filtered copper radiation from a standard focus high intensity sealed tube operated at 50 kV, 40 mA. Reflections are absent for hO0, h#2n; 04~0, k # 2n; OOI, Z#2n. The Laue group is 222 and the space group is PZ12,2,, with unit cell dimensions n~77.24 8, b-89.18 A, c=93*24 A (1 A=0-1 nm) and 2~4; there is one PCKase molecule per asymmetric unit. The volume/unit mass (Matthews. 1968) is 3.029 A3/dalton and the solvent content, is
