.I. Mol. Biol.
(1979) 133, 181-184
LETTERS TO THE EDITOR
Preliminary Crystallographic Data of Receptors for Transport and Chemotaxis in Escherichia coli: D-Galactose and Maltose-binding Proteins We have obtained single crystals of maltose-binding protein (M, = 40,500) and n-galactose-binding protein (M, = 32,000), chemoreceptors for active transport and chemotaxis in Escherichia coli. This brings to a total of five the binding proteins that we have thus far crystallized; they include the L-arabinose-binding prot)ein, the leucine, isoleucine, va.line-binding protein from Escherichia coli, and a sulfate-binding protein from SaZrnoneZZatyphimurium. The crystal structure of t.he L-arabinose-binding prot,ein has been d&ermined at 2.8 A resolution (Quioch~~ et al., 1977a).
laboratory is engaged in the structural analysis of a family of proteins, called binding proteins, which are essential components of high-affinity active transport systems and chemotaxis in gram negative bacteria. We have recently solved the 2.8 A resolution structure of the L-arabinose-binding protein from Escherichia coli B/r (Quiocho et al., 1977a), and published the preliminary crystallographic data for a leucine, isoleucine, valine-binding protein from E. coli K12 (Meador & Quiocho. 1978). Here we present preliminary crystal data for the maltose-binding and ngalactose-binding proteins from E. wli. Maltose-binding protein was isolated from osmotic shock fluid of E. coli K12 (strain LA 3400 glp - l, ma1 + , phoA ; kindly provided by Dr Winfried Boos) using a DE-23 batch column and two subsequent column chromatographic steps with DE-52 caellulose and QAE-Sephadex A-25 (unpublished procedure). This procedure is an extensive modification of the method of Kellermann & Szmelcman (1974). Crystals were obtained from a 6 mg/ml solution of protein in IS:/, (w/v) polyethylene glycol 6000, 10 mnr-sodium citrate (pH 4.2) by the microdiffusion vapor technique. Purified D-galactose-binding protein was a byproduct of the isolation of bhe Larabinose-binding protein from E. coli K12 (Parsons & Hogg, 1974a,6). Crystals were obtained by dialysis of an 8 mg/ml solution of protein in 13% (w/v) polyethylene glycol, 40 mM-N&l, 6 mM-sodium citrate (pH 4.9) against 1474 (w/v) polyethylene glycol 6000, 40 mM-NaC1, 6 mm-sodium citrate (pH 4.9). The crystals are tabular prismatic, with an average size of 1 mm x 0.5 mm x 0.5 mm. For X-ray diffraction experiments, a crystal was mounted in a thin-walled glass capillary (1 mm diam.) in the usual way. Diffraction patterns of the central projections were recorded with a Buerger precession camera (Ni-filtered CuKa radiation from an Elliot rotating anode generator). The crystal-to-film distance is 10 cm. Crystals of both the maltose-binding protein and the u-galactose-binding protein belong to t’he monoclinic crystal system. The unit cell parameters are shown in Table 1. Since only even-order reflections appear on Olco, the space group for both crystals is P2,, with 2n molecules per unit cell, where n is the number of molecules 181 0 1979 Acatlemic Press Inc. (Lonikm) Ltd. Our
weight
B=
t Measured with a carbon $ Crystals obtained using results).
VM (A3/daltons) Density (g/cm3)t Number of molecules/unit cell References
properties
paper
paper
density gradient column calibrated technique with polyethylene glycol
This
2
wit,h droplets aa precipitant
Quiocho
4
2.01 1.24
Ile, Val-binding protein
4 Meador 8: Quiocho (1978)
2.30 1.29
39.68 70.77 116.01
P2,2,2,
E. coli K12 36,000 Leu = Ile > Val Thr > Ala 1 x 10-e
Leu,
proteins
>
:
4
2.17 1.25
40.9 49.6 141.5
P2,2,2,
“0 x 10-s
unln~blished
8s. typhimurium 33,000 Sulfate :. chromate
Sulfate-binding protein
of NaBr solution of known density. (M. Willcutts, H. Isihara & F. A. Quiocho,
ef cd. (1974)
65.8 36.7 61.4 106”17’ 2.22 1.28
57.7 65.1 44.7 100”40’ 2.04 1.23
2 This
P2,2,21
p21
P2, 55.44 71.72 77.64
E. coli S/r 33,100 L-arabinose = ugalactose > fucose 0.1 x 10-G
L-Arabinose-bincling protein
of crystals of binding
E. coli B/r 32,000 D-galactose = glucose >> L-arabinose 0.35 x 10-e
D-Galactose-binding protein
and chemical
E. coli K12 40,500 Maltose = several maltodextrins 1.5 x 10-e
Maltose-binding protein
tet,rachloride/xylene microdiffusion vapor
K, of primary substrate (M) Crystallographic data Space group Unit cell dimensions (A) : (I, E b= c z
Molecular Specificity
Source
Protein
Physical
TABLET
LETTERS
TO
THE
EDITOR
IA3
per asymmetric unit. The value of n was evaluated by comparing values of J’,, the crystal volume per unit cell of protein molecular weight, for values of n within the range (1.68 to 3.53 A3/daltons) normally observed for protein crystals (Matthews. 1968). As indicated in Table 1, only at n = 1 do the values of Vv, for both binding protein crystals fall within this range. We concluded that for crystals of D-galactosc and maltose-binding proteins the asymmetric unit contains one protein molecule. This conclusion and the crystal densities shown in Table 1 yield solvent contents that compare well with values obtained for other proteins (Matthews, 1968). The same number of molecules per asymmetric unit has been determined for the other three! binding protein crystals listed in Table 1. All five binding proteins have similar molecular weights (Table 1). proteins Of the five binding proteins (Table l), maltose and D-gnlactose-binding have been shown to be components of the shock-sensitive high-affinity active transport and of chemotaxis; the others are apparently important only for transport (Boos. 1974). However, similarities between binding proteins have been indicated. For instance, despite the fact that the L-arabinose-binding protein and D-galactosehinding protein biosyntheses in E. coli are controlled by distinctly different genes. nrnC (Hogg & Engelsberg, 1969) and mgZR (Lengeler et aE., 1971), respectively, the cross-reactivity of antibodies for each shows that both proteins share some regions of similar tertiary structures (Parsons & Hogg, 19743). Furthermore. we ha,ve shown t,hat the dye 2’,4’.5’.7’-tetraiodofluorescein, a chromophoric probe for the nucleot’ideIbinding sit,e in a variety of proteins (Wasserman & Lentz, 1971 :
(1979) 133, 181-184
LETTERS TO THE EDITOR
Preliminary Crystallographic Data of Receptors for Transport and Chemotaxis in Escherichia coli: D-Galactose and Maltose-binding Proteins We have obtained single crystals of maltose-binding protein (M, = 40,500) and n-galactose-binding protein (M, = 32,000), chemoreceptors for active transport and chemotaxis in Escherichia coli. This brings to a total of five the binding proteins that we have thus far crystallized; they include the L-arabinose-binding prot)ein, the leucine, isoleucine, va.line-binding protein from Escherichia coli, and a sulfate-binding protein from SaZrnoneZZatyphimurium. The crystal structure of t.he L-arabinose-binding prot,ein has been d&ermined at 2.8 A resolution (Quioch~~ et al., 1977a).
laboratory is engaged in the structural analysis of a family of proteins, called binding proteins, which are essential components of high-affinity active transport systems and chemotaxis in gram negative bacteria. We have recently solved the 2.8 A resolution structure of the L-arabinose-binding protein from Escherichia coli B/r (Quiocho et al., 1977a), and published the preliminary crystallographic data for a leucine, isoleucine, valine-binding protein from E. coli K12 (Meador & Quiocho. 1978). Here we present preliminary crystal data for the maltose-binding and ngalactose-binding proteins from E. wli. Maltose-binding protein was isolated from osmotic shock fluid of E. coli K12 (strain LA 3400 glp - l, ma1 + , phoA ; kindly provided by Dr Winfried Boos) using a DE-23 batch column and two subsequent column chromatographic steps with DE-52 caellulose and QAE-Sephadex A-25 (unpublished procedure). This procedure is an extensive modification of the method of Kellermann & Szmelcman (1974). Crystals were obtained from a 6 mg/ml solution of protein in IS:/, (w/v) polyethylene glycol 6000, 10 mnr-sodium citrate (pH 4.2) by the microdiffusion vapor technique. Purified D-galactose-binding protein was a byproduct of the isolation of bhe Larabinose-binding protein from E. coli K12 (Parsons & Hogg, 1974a,6). Crystals were obtained by dialysis of an 8 mg/ml solution of protein in 13% (w/v) polyethylene glycol, 40 mM-N&l, 6 mM-sodium citrate (pH 4.9) against 1474 (w/v) polyethylene glycol 6000, 40 mM-NaC1, 6 mm-sodium citrate (pH 4.9). The crystals are tabular prismatic, with an average size of 1 mm x 0.5 mm x 0.5 mm. For X-ray diffraction experiments, a crystal was mounted in a thin-walled glass capillary (1 mm diam.) in the usual way. Diffraction patterns of the central projections were recorded with a Buerger precession camera (Ni-filtered CuKa radiation from an Elliot rotating anode generator). The crystal-to-film distance is 10 cm. Crystals of both the maltose-binding protein and the u-galactose-binding protein belong to t’he monoclinic crystal system. The unit cell parameters are shown in Table 1. Since only even-order reflections appear on Olco, the space group for both crystals is P2,, with 2n molecules per unit cell, where n is the number of molecules 181 0 1979 Acatlemic Press Inc. (Lonikm) Ltd. Our
weight
B=
t Measured with a carbon $ Crystals obtained using results).
VM (A3/daltons) Density (g/cm3)t Number of molecules/unit cell References
properties
paper
paper
density gradient column calibrated technique with polyethylene glycol
This
2
wit,h droplets aa precipitant
Quiocho
4
2.01 1.24
Ile, Val-binding protein
4 Meador 8: Quiocho (1978)
2.30 1.29
39.68 70.77 116.01
P2,2,2,
E. coli K12 36,000 Leu = Ile > Val Thr > Ala 1 x 10-e
Leu,
proteins
>
:
4
2.17 1.25
40.9 49.6 141.5
P2,2,2,
“0 x 10-s
unln~blished
8s. typhimurium 33,000 Sulfate :. chromate
Sulfate-binding protein
of NaBr solution of known density. (M. Willcutts, H. Isihara & F. A. Quiocho,
ef cd. (1974)
65.8 36.7 61.4 106”17’ 2.22 1.28
57.7 65.1 44.7 100”40’ 2.04 1.23
2 This
P2,2,21
p21
P2, 55.44 71.72 77.64
E. coli S/r 33,100 L-arabinose = ugalactose > fucose 0.1 x 10-G
L-Arabinose-bincling protein
of crystals of binding
E. coli B/r 32,000 D-galactose = glucose >> L-arabinose 0.35 x 10-e
D-Galactose-binding protein
and chemical
E. coli K12 40,500 Maltose = several maltodextrins 1.5 x 10-e
Maltose-binding protein
tet,rachloride/xylene microdiffusion vapor
K, of primary substrate (M) Crystallographic data Space group Unit cell dimensions (A) : (I, E b= c z
Molecular Specificity
Source
Protein
Physical
TABLET
LETTERS
TO
THE
EDITOR
IA3
per asymmetric unit. The value of n was evaluated by comparing values of J’,, the crystal volume per unit cell of protein molecular weight, for values of n within the range (1.68 to 3.53 A3/daltons) normally observed for protein crystals (Matthews. 1968). As indicated in Table 1, only at n = 1 do the values of Vv, for both binding protein crystals fall within this range. We concluded that for crystals of D-galactosc and maltose-binding proteins the asymmetric unit contains one protein molecule. This conclusion and the crystal densities shown in Table 1 yield solvent contents that compare well with values obtained for other proteins (Matthews, 1968). The same number of molecules per asymmetric unit has been determined for the other three! binding protein crystals listed in Table 1. All five binding proteins have similar molecular weights (Table 1). proteins Of the five binding proteins (Table l), maltose and D-gnlactose-binding have been shown to be components of the shock-sensitive high-affinity active transport and of chemotaxis; the others are apparently important only for transport (Boos. 1974). However, similarities between binding proteins have been indicated. For instance, despite the fact that the L-arabinose-binding protein and D-galactosehinding protein biosyntheses in E. coli are controlled by distinctly different genes. nrnC (Hogg & Engelsberg, 1969) and mgZR (Lengeler et aE., 1971), respectively, the cross-reactivity of antibodies for each shows that both proteins share some regions of similar tertiary structures (Parsons & Hogg, 19743). Furthermore. we ha,ve shown t,hat the dye 2’,4’.5’.7’-tetraiodofluorescein, a chromophoric probe for the nucleot’ideIbinding sit,e in a variety of proteins (Wasserman & Lentz, 1971 :
