Download Crystallization and X-Ray Crystallographic Studies of Wild

Mol. Cells, Vol. 19, No. 2, pp. 219-222
Molecules
and
Cells
KSMCB 2005
Crystallization and X-Ray Crystallographic Studies of Wild-Type
and Mutant Tryptophan Synthase α-Subunits from Escherichia
coli
Mi Suk Jeong and Se Bok Jang*
Korea Nanobiotechnology Center, Pusan National University, Busan 609-735, Korea.
(Received October 14, 2004; Accepted December 27, 2004)
The α-subunit of Escherichia coli tryptophan synthase
(αTS), a component of the tryptophan synthase α2β2
complex, is a monomeric 268-residues protein (Mr =
28,600). αTS by itself catalyzes the cleavage of indole3-glycerol phosphate to glyceraldehyde-3-phosphate
and indole, which is converted to tryptophan in tryptophan biosynthesis. Wild-type and P28L/Y173F double mutant α-subunits were overexpressed in E. coli
and crystallized at 298 K by the hanging-drop vapordiffusion method. X-ray diffraction data were collected to 2.5 Å resolution from the wild-type crystals
and to 1.8 Å from the crystals of the double mutant,
since the latter produced better quality diffraction
data. The wild-type crystals belonged to the monoclinic space group C2 (a = 155.64 Å, b = 44.54 Å, c =
71.53 Å and β = 96.39°) and the P28L/Y173F crystals
to the monoclinic space group P21 (a = 71.09 Å, b =
52.70, c = 71.52 Å, and β = 91.49°). The asymmetric
unit of both structures contained two molecules of αTS.
Crystal volume per protein mass (Vm) and solvent content were 2.15 Å3 Da−1 and 42.95% for the wild-type
and 2.34 Å3 Da−1 and 47.52% for the double mutant.
Keywords: α-Subunit of Tryptophan Synthase (αTS);
Escherichia coli; P28L/Y173F Double Mutant; WildType; X-Ray Crystallographic Analysis.
Introduction
The structure of the tryptophan synthase α2β2 complex of
Salmonella typhimurium has been determined by X-ray
analysis (Hyde et al., 1988). Although crystallization of
the separate subunits of tryptophan synthase from S. ty* To whom correspondence should be addressed.
Tel: 82-51-510-2523; Fax: 82-51-581-2546
E-mail: [email protected]
phimurium and E. coli has been attempted for many years,
there have been no reports of X-ray structures. The residues involved in substrate binding and catalysis at the
active site of the α-subunit have been studied. The α2β2
complex exists as an equilibrium between a low-activity
‘open’ and a high-activity ‘closed’ state that is affected by
allosteric ligands and monovalent cations (Fan et al.,
2000). The basis of the conformational transitions involved has been examined by X-ray analysis of a number
of enzyme-ligand complexes (Rhee et al., 1997). In the
crystal structures of the complexes with indole-3acetylglycine and indole-3-acetyl-L-aspartic acid, ligand
binding leads to closure of loop αL6 of the α-subunit,
which is consistent with the allosteric effect (Weyand et
al., 1999).
The native conformation of a protein is maintained by
non-covalent interactions involving hydrogen bonds, salt
bridges and hydrophobic effects. The last have been extensively analyzed by site-directed mutagenesis that has
shown that hydrophobic residues in the interior of a protein contribute to conformational stability (Kellis et al.,
1988; Shortle et al., 1990). Residues 28 and 173 of the αsubunit interact with the carboxyl-terminal folding domain, and when they are substituted the rate of folding of
the enzyme and the stabilities of folding intermediates
change (Jeong, 2003). Pro28 may contribute to the formation of a folding nucleus that facilitates interaction between helices in the two folding domains, and the 173
region may form another folding nucleus which suppresses defective helix interaction resulting from overrapid folding.
We have studied the roles of these amino acids by mutation of tyrosine 173 to phenylalanine (Y173F) and of
proline 28 to leucine (P28L). Proline is unique among
amino acids in its effect on protein stability and folding
(Kim et al., 1990; Wu and Matthews, 2002). The structural information obtained should provide information
about the stabilization mechanisms involved in intersub-
220
Crystallographic Studies of αTS
unit communication. The purified wild-type and P28L/
Y173F α-subunits formed suitable crystals for X-ray crystallographic analysis. We report here preliminary X-ray
crystallographic studies of these subunits (Jeong et al.,
2004).
A
Materials and Methods
Protein expression and purification Plasmid ptactrpAMK-M13
containing the trpA gene was used as an expression vector
(Sarker and Hardman, 1995) and E. coli RB797 was used as the
host strain for αTS expression vectors (Lim et al., 1991). The
P28L/Y173F double-mutant was constructed by cloning from
the P28L and Y173F mutants whose construction was described
previously (Jeong, 2003). The P28L and Y173F plasmids were
digested with BssHII and SalI, and the BssHII/SalI fragment,
containing Phe at residue 173 was subcloned into the P28L vector as the expression vector ptactrpA containing the mutated
codon for inclusion body formation (Lim et al., 1989). The cells
were grown to OD600 0.6 at 37°C and expression of the αTS
protein was induced with 1% lactose. The cultured cells were
harvested after 24 h and the αTS purified as described (Sarker
and Hardman, 1995). The protein was concentrated with a
Vivaspin 20 Polyethersulfone membrane to about 10 mg ml−1
and was at least 95% pure as judged by gel electrophoresis.
Crystallization of P28L/Y173L αTS Crystals of the wild-type
and P28L/Y173L αTS were obtained by the hanging-drop vapor-diffusion method at 298 K using 24-well Linbro plates
(Hampton Research). Hanging drops were prepared by mixing
equal volumes (1.0 µl each) of the protein solution and the reservoir solution. Each hanging drop was placed over a 0.5 ml
reservoir solution, and the initial crystallization conditions were
established by sparse-matrix sampling. The wild-type αTS crystallized as clusters of plate-shaped crystals using a precipitant
containing 0.5 M ammonium sulfate, 0.1 M trisodium citrate
dihydrate, and 1.0 M lithium sulfate monohydrate (pH 5.6).
Under these conditions, crystals appeared after 7−10 days and
the crystals grew to maximum size within two weeks. They
formed extended plates or lathes either in the form of single
small crystals or of clusters. The thinness of the crystal made
them fragile and difficult to handle. Truncated crystals were
transferred to the same buffer in order to pick up a single crystal
with a mounted cryoloop for data collection (Lee et al., 2003).
The crystals obtained were thin plates of typical dimensions
0.05−1 × 0.2−0.4 × 0.3−0.5 mm (Fig. 1A). The crystals of the
P28L/Y173F mutant were grown from 0.1 M HEPES-Na pH 7.5,
10% iso-propanol, 20% polyethylene glycol 4000 within 5 d.
They were long tetragonal sticks with typical dimensions 0.6 ×
0.3 × 0.3 mm (Fig. 1B). Crystals of the single mutants P28L and
Y173F were not obtained.
Data collection and analysis Wild-type and P28L/Y173F data
were collected from flash-cooled crystals at 100 K with a Mac-
B
Fig. 1. A. Truncated plate crystal forms of the wild-type tryptophan synthase α-subunit of E. coli. B. Tetragonal stick crystals
of the P28L/Y173F tryptophan synthase α-subunit.
Science DIP2030b imaging plate at beamline 6B1 of a Pohang
Light Source, Pohang, Korea. Prior to data collection, the crystal was soaked briefly in a cryoprotectant solution, consisting of
the precipitant solution plus 20% glycerol. Diffraction data (Table 1) were obtained and processed using the programs DENZO
and SCALEPACK (Otwinowski, 1993). The wild-type data were
collected to 2.5 Å resolution at 100 K. A total of 567,474 measured reflections were merged into 17,264 unique reflections
with an Rmerge (on intensity) of 7.2%. The merged data set is
92.2% complete to 2.5 Å resolution. The crystals belong to the
monoclinic space group C2 with unit cell dimensions shown in
Table 1. The double mutant data were collected to 1.8 Å resolution at 100 K. A total of 387,319 measured reflections were
merged into 48,396 unique reflections with an Rmerge (on intensity) of 5.4%. The merged data set is 91.9% complete to 1.8 Å
resolution. The crystals belong to the monoclinic space group
P21 with unit cell dimensions (Table 1), and gave a better defined diffraction pattern than the wild-type crystals. The asymmetric unit contained two molecules of αTS, giving a crystal
volume per protein mass (Vm) of 2.15 Å3 Da−1 and a solvent
content of 42.95% for the wild-type. The P28L/Y173F crystals
give a Vm of 2.34 Å3 Da−1 and a solvent content of 47.52%. The
complete data are summarized in Table 1. The difference in the
gross morphology of the wild-type and double mutant crystals is
consistent with observed differences in cell symmetry. Although
the unitcell parameters differ, implying some degrees of relatedness, the crystals of the double mutant belong to the monoclinic space group P21, whereas the data for wild-type crystals
can be reduced to the monoclinic space group C2. The differences in space group often observed in different crystalline en-
Mi Suk Jeong & Se Bok Jang
Table 1. Crystal information and data collection statistics.
Data
Space group
Unit cell parameters
Resolution (Å)
Completeness (%)
Observed reflections
Unique reflections
I/σ(I)
Rmerge(%)a
Wild-type
P28L/Y173F
C2
a = 155.68
b = 44.54
c = 71.53
β = 96.39°
30.0-2.5
92.2 (91.3)
567,474
17,264
33.6 (6.5)
7.2 (38.9)
P21
71.09 Å
52.70 Å
71.52 Å
91.49°
30.0-1.8
91.9 (79.7)
387,319
48,396
24.8 (2.8)
5.4 (28.9)
Numbers in parentheses indicate values for the highest resolution
bin (2.59-2.50 Å for wild-type data and 1.86−1.80 Å for wildtype data).
a
Rmerge = ∑h∑i|I(h,i) - <I(h)>|/∑h∑iI(h,i), where I(h,i) is the intensity of the ith measurement of reflection h and <I(h)> is the mean
value of I(h,i) for all i measurements.
Fig. 2. Sequence alignment of αTS from E. coli and S. typhimurium. Different residues in the two species are indicated by
black underlines. Successive 20 residues are indicated by stars (*).
vironments can reflect the conformational flexibility of loops
and domains, and even different oligomeric states (Lee et al.,
1996; Mancheno et al., 2003; Zhang et al., 1994).
αTS consists of a single polypeptide chain of 268 residues
(Mr = 28,600). It has 85% sequence identity with S. typhimurium αTS (Fig. 2). The residues of the α-subunit are disordered when it is bound to the β-subunit to form the mature tryptophan synthase, or when the structure is regulated allosterically
by ligand binding (Weyand et al., 2002; Wu and Mattews, 2003).
The polypeptide chains of the wild-type and double αTS molecules have simple TIM 8-fold α/β barrel topologies, one of the
most common protein folds. The highly mobile residues are
221
found at the interface of the α- and β-subunits, loops, and substrate binding regions. These highly mobile residues may play
an important role in inter-subunit communication.
The determination of the structures of the wild-type and double
mutant αTS should contribute to the understanding of the folding
mechanism of the protein. Understanding the molecular origin of
the conformational stability of E. coli αTS should also provide
valuable insights into the basis of protein stability and folding
behavior, and facilitate approaches to protein engineering.
Acknowledgments This study made use of the beamline 6B1 at
the Pohang Accelerator Laboratory, Pohang, Korea. MSJ was
supported by grant No. R03-2002-000-00007-0 from the Basic
Research Program of the Korea Science and Engineering Foundation.
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