Crystal structures of the regulatory subunit of
Thr-sensitive aspartate kinase from Thermus thermophilus
Ayako Yoshida
1
, Takeo Tomita
1
, Hidetoshi Kono
2
, Shinya Fushinobu
3
, Tomohisa Kuzuyama
1
and
Makoto Nishiyama
1,4
1 Biotechnology Research Center, The University of Tokyo, Japan
2 Computational Biology Group, Quantum Beam Science Directorate, Japan Atomic Energy Agency, Kyoto, Japan
3 Department of Biotechnology, The University of Tokyo, Japan
4 RIKEN SPring-8 Center, Hyogo, Japan
Aspartate kinase (AK; EC 2.7.2.4) is an enzyme that
catalyzes the first committed step, the phosphorylation
of the c-carboxyl group of aspartate, of the biosynthetic
pathway of the aspartic acid group amino acids Lys,
Thr, Ile, and Met, in microorganisms and plants. AK
is classified into two groups according to subunit orga-
nization: homo-oligomer or heterotetramer. AK from
Thermus thermophilus (TtAK), AK from C. glutami-
cum (CgAK) and AKII from Bacillus subtilis (BsAKII)
are heterotetramers containing equimolar amounts of
a-subunits and b-subunits [1–3], whereas AKIII from
Escherichia coli (EcAKIII), AKI from Arabidopsis

in the Thr-bound form (TtAKb-Thr) and at 2.98 A
˚
in the
Thr-free form (TtAKb-free). Although both forms are crystallized as
dimers, the contact surface area of the dimer interface in TtAKb-free
(3200 A
˚
2
) is smaller than that of TtAKb-Thr (3890 A
˚
2
). Sedimentation
equilibrium analyzed by ultracentrifugation revealed that TtAKb is present
in equilibrium between a monomer and dimer, and that Thr binding shifts
the equilibrium to dimer formation. In the absence of Thr, an outward
shift of b-strands near the Thr-binding site (site 1) and a concomitant loss
of the electron density of the loop region between b3 and b4 near the Thr-
binding site are observed. The mechanism of regulation by Thr is discussed
on the basis of the crystal structures. TtAKb has higher thermostability
than the regulatory subunit of Corynebacterium glutamicum AK, with a dif-
ference in denaturation temperature (T
m
)of40°C. Comparison of the
crystal structures of TtAKb and the regulatory subunit of C. glutamicum
AK showed that the well-packed hydrophobic core and high Pro content
in loops contribute to the high thermostability of TtAKb.
Abbreviations
AK, aspartate kinase; BsAKII, aspartate kinase II from Bacillus subtilis; CgAK, aspartate kinase from Corynebacterium glutamicum; CgAKb,
regulatory subunit of aspartate kinase from Corynebacterium glutamicum; DSC, differential scanning calorimetry; EcAKIII, aspartate kinase III
from Escherichia coli; MAD, multiwavelength anomalous diffraction; MjAK, aspartate kinase from Methanococcus jannaschii; TtAK, aspartate

threonine deaminase, two ACT domains in a single
peptide are arranged side-by-side to form an Ile ⁄ Val-
binding unit [16,17]. For the ACT domain in AK,
two types of association modes are seen, as reviewed
by Curien et al. [18]: one is found in homo-oligo-
meric AKs [4,6,19] and the other in a
2
b
2
-type CgAK
[20]. In these ACT domains of AK proteins, there
are common structural features: (a) two ACT
domains are arranged in the C-terminal portion of a
single polypeptide; (b) the ACT1 domain is inserted
into the ACT2 domain; and (c) two ACT domains,
each from a different chain, interact to form an effec-
tor-binding unit. The effector-binding unit of the
ACT domain in the b-subunit of CgAK (CgAKb)is
organized differently from those of homo-oligomeric
AKs. In CgAKb, ACT1 and ACT2 from different
chains associate side-by-side to form an eight-
stranded b-sheet, and two eight-stranded b-sheets face
each other perpendicularly. In CgAKb, both eight-
stranded b-sheets are involved in effector binding. On
the other hand, in homo-oligomeric AKs, two ACT1
domains from different chains associate with each
other to form an eight-stranded b-sheet, and two
ACT2 domains from different chains form an addi-
tional eight-stranded b-sheet, although these two
eight-stranded b-sheets are also arranged perpendicu-

Results and Discussion
Model quality
The crystal structure of the Thr-bound form of TtAKb
(TtAKb-Thr) was determined at 2.15 A
˚
resolution,
using multiwavelength anomalous diffraction (MAD)
phases derived from selenomethionine (SeMet)-substi-
tuted TtAKb. TtAKb-Thr is a dimer containing two
Thr molecules (Fig. 1A), acetate molecules, which are
derived from the crystallization buffer, and 153 water
molecules in an asymmetric unit. The electron densities
of the N-terminal (residues 1–4 in chains A and B)
and C-terminal (residues 158–161 in chains A and B)
sections of the structure are not seen on the map,
probably owing to disorder of these regions. The over-
all geometry of the model according to the procheck
program [21] is of good quality, with 95.4% of the res-
idues in the most favored regions and 4.6% in allowed
regions of the Ramachandran plot.
The crystal structure of the Thr-free form of TtAKb
(TtAKb-free) was determined at 2.98 A
˚
resolution by
molecular replacement using the structure of TtAKb-
Thr as a search model. The TtAKb-free crystal
contains three dimers (Fig. 2A), each composed of AB,
CD and EF chains, and 79 water molecules in an
asymmetric unit. The electron densities of the N-termi-
nal (residues 1–3 in chain A, residues 1–4 in chains B

ences between monomers are found in regions 84–88,
94–95, and 102–104 (Fig. 1B). A single chain of
TtAKb contains two ACT domains, ACT1 (N-termi-
nal domain) and ACT2 (C-terminal domain) domains.
The ACT domain organization of TtAKb-Thr is
similar to that of CgAKb [20] but not to those of
homo-oligomeric AKs. ACT1 and an ACT2, each
from different chains, are arranged side-by-side to
form an effector (Thr)-binding unit, an eight-stranded
antiparallel b-sheet with four a-helices on one side. We
assume that this characteristic dimer organization of
the regulatory domain of AK is a feature limited to
a
2
b
2
-type AKs, because, in homo-oligomeric AKs, two
equivalent ACT domains from different chains are jux-
taposed to form a structural unit, and two structural
units, each composed of two equivalent ACT
domains, are not equivalent to each other. Owing to
the difference in the ACT domain arrangement,
TtAKb binds two Thr molecules per dimer at two sites
(site 1), each in an equivalent effector-binding unit
(Fig. 1A), whereas in homo-oligomeric AKs, a single
Fig. 1. Overall structure of TtAKb-Thr. (A)
Overall structure of TtAKb-Thr. The A chain
and the B chain are shown in purple and
green, respectively. Thr molecules are
shown as an orange stick model. Both ACT

EF dimers, and 0.58 A
˚
between AB and EF dimers.
Moreover, the rmsd values of Ca between the mono-
mers in the dimers are 0.55 A
˚
between the A and B
chains, 0.96 A
˚
between the C and D chains, and
0.51 A
˚
between the E and F chains. The main differ-
ence between TtAKb-free and TtAKb-Thr is that the
residues in the loop region between b3 and b4 near the
Thr-binding site are disordered in TtAKb-free, as
described later.
Thr-binding site
In TtAK b-Thr, the electron density of one Thr mole-
cule is observed at site 1 between ACT1 and ACT2,
each from different chains (Fig. 3A,B). The structure
of TtAKb-Thr is quite similar to that of Thr-bound
CgAKb (rmsd value of Ca is 1.73 A
˚
). Bound Thr mol-
ecules are stabilized by ionic bonds (Asp26-Od2 for the
amino group), hydrogen bonds (Gln50-Oe1 and
Ile126*-O for the side chain hydroxyl group; Asn125*-
Od1 and Ile126*-O for the amino group; Ile30-N,
Ile126-N and Asn125*-Od1 for the carboxyl group;

Completeness (%) 100.0 (100.0) 100.0 (100.0) 100.0 (100.0) 100.0 (100.0) 99.8 (100.0)
Phasing
Number of Se sites 8
FOM
c
0.40
Refinement
Resolution (A
˚
) 47.2–2.15 46.5–2.98
R-factor
d
(work ⁄ test) (%) 18.8 ⁄ 22.6 24.4 ⁄ 26.6
Number of atoms 2405 6545
Protein atoms 2228 6466
Thr molecules 2
Acetate molecules 2
Water molecules 153 79
Average B-factor
Protein atoms 32.9 56.3
Thr 23.3
Water 34.8 44.4
rmsd values
Bond length (A
˚
) 0.009 0.010
Bond angle (°) 1.50 1.40
Ramachandran plot
e
Most favored (%) 95.4 89.0

hydrophobic interactions (Ile24, Ile30 and Met62 for
the side chain methyl group). Two water molecules
present near the two oxygen atoms contribute to a
hydrogen bond network between the carboxyl group
of Thr, Gly29-N, Ala31-N, Ala32-N, and Phe116-O
from another chain. Most of the residues and water
molecules recognizing the bound Thr in TtAKb are
conserved in CgAKb. As seen in CgAKb, the carboxyl
group of Thr is located near the N-terminal section of
helix a1, suggesting that the positive charge of the
N-terminal helix dipole facilitates recognition of the
carboxyl group. Importantly, Thr is bound between
two chains and is not exposed to the solvent, suggest-
ing that bound Thr plays an important role in stabiliz-
ing the dimeric structure, as in CgAKb.
Monomer–dimer equilibrium
In CgAKb, Thr binding induces the dimerization of
CgAKb [20]. Thr is bound at an effector-binding unit
formed between two chains in TtAKb in a manner
almost identical to that in CgAKb, suggesting that Thr
binding plays a role in stabilizing the dimeric form of
TtAKb. To examine the effect of Thr on dimerization,
we analyzed the oligomeric state of TtAKb in the pres-
ence or absence of Thr, using two different methods:
Fig. 3. Thr-binding site. (A) 2F
o
)F
c
map of bound Thr molecule and two water molecules. The contour level of the map is 1.0r. (B) Thr-bind-
ing site in TtAKb-Thr. Residues in purple are in the A chain, and residues in green are in the B chain. (C) Vacant Thr-binding site in TtAKb-

)1
(goodness of
fit = 2.3 · 10
)4
) in the presence and absence of 5 mm
Thr, respectively. According to the constants, TtAKb
at 1 mgÆmL
)1
is mostly (91%) present as a monomer
in the absence of Thr, whereas at the same protein
concentration, 31% of TtAKb is present in a dimeric
form in solution containing Thr. At 5 mgÆmL
)1
which
is the protein concentration used for crystallization,
58% and 27% are present as dimers in the presence
and absence of Thr, respectively. Thus, TtAKb is in
monomer–dimer equilibrium, which is displaced by
Thr and ⁄ or protein concentrations; therefore, dimer
formation in the crystal structure in the absence of
Thr can be explained by the high concentration of
TtAKb-free under crystallization conditions.
CgAK is easily dissociated into a-subunits and b-sub-
units during purification without Thr. On the other
hand, TtAK is purified in the a
2
b
2
form even without
Thr. This observation suggests that binding affinity

[44]. (D) Densitometric calibration of TtAK
subunits of SDS ⁄ PAGE in (B). The a-sub-
units and b-subunits are indicated by a solid
line with circles and a dotted line with
squares, respectively. (E) Densitometric cali-
bration of TtAK subunits of SDS ⁄ PAGE in
(C). The a-subunits and b-subunits are indi-
cated by a solid line with circles and a
dotted line with squares, respectively.
A. Yoshida et al. Crystal structures of regulatory subunit of Thr-sensitive aspartate kinase
FEBS Journal 276 (2009) 3124–3136 ª 2009 The Authors Journal compilation ª 2009 FEBS 3129
Conformational change of TtAKb upon Thr
binding and its implications
Unexpectedly, the structural difference between
TtAKb-Thr and TtAKb-free is not so large: the rmsd
for Ca between two structures is about 1.5 A
˚
. This
contrasts with Lys-sensitive EcAKIII, which shows a
larger conformational change of the regulatory domain
dimer upon Lys binding, resulting in the displacement
of several residues responsible for catalytic function in
the catalytic domain. The most distinct difference
between the structures is that the electron density of
the b3–b4 loop around the Thr-binding site is missing
in TtAKb-free, and that b-strands surrounding the
Thr-binding site show outward shifts in the absence of
Thr, with 12° rotation of the ACT2 domain from the
fixed ACT1 domain (Figs 3B,C and 5D). The regula-
tory domain dimer of CgAK inhibited in a concerted

TtAKb-Thr and TtAKb-free are shown in blue and pink. Domain motion was analyzed by
DYNDOM [45]. A broken line indicates hinge axis for
movement.
Crystal structures of regulatory subunit of Thr-sensitive aspartate kinase A. Yoshida et al.
3130 FEBS Journal 276 (2009) 3124–3136 ª 2009 The Authors Journal compilation ª 2009 FEBS
domains (Yoshida et al., unpublished result), the direct
function of the loop in catalytic control is unexpected
in CgAK. In TtAKb, accompanied by an outward
shift of b-strands, especially b2–b4 from ACT1 near
site 1, a significant shift is also found around site 2
(Fig. 5A–C). This result may suggest that two Thr
molecules bound at site 1 induce a conformational
change in site 2, thereby facilitating the binding of
additional Thr molecules at site 2. In addition, TtAKb
has a Pro-Gly sequence in the N-terminal section
(positions 28 and 29) of helix a1, which contribute to
the recognition of the carboxyl group of bound Thr by
a putative helix dipole (Fig. 3B). Interestingly, the
dihedral angle of Gly29 changes upon Thr binding (for
example, / = 89.85°, w = )13.64° in the A chain of
TtAKb-Thr and / = 103.16°, w = )11.53° in the E
chain of TtAKb-free). In CgAKb, which can bind the
Thr molecule at site 1, the Pro-Gly sequence is con-
served at the same position (positions 27 and 28)
(Fig. 3D). In addition to Pro27-Gly28, CgAKb has a
similar Pro-Gly sequence at positions 109–110 in the
N-terminal portion of helix a3 forming site 2
(Fig. 3D). We also found a similar change in the dihe-
dral angle of Gly110 upon binding of Lys at site 2 of
CgAKb (details will be published elsewhere). These

and Lys in CgAK.
Comparison with other AKs
Recently, the crystal structures of Thr-sensitive MjAK
have been determined in three forms [22]: (a) complex
with magnesium adenosine 5¢-(b,c-imido)triphosphate
and Asp; (b) complex with Asp; and (c) complex with
Thr. MjAK has a homotetrameric structure, and
shows high overall structural similarity to the
inhibitory complex of EcAKIII bound to Lys.
Although EcAKIII binds the effector, Lys, at the
binding unit formed between ACT1 domains from
different chains, MjAK binds Thr at the binding sites
formed between ACT2 domains from different chains.
In EcAKIII, transition from the R-state to the
T-state, accompanied by rotational rearrangements to
form a tetramer, occurs through large movement of a
latch loop from the regulatory domains [4]. In MjAK,
however, the loop corresponding to the latch of
EcAKIII is shortened, and shows no conformational
change upon Thr binding. Instead, Thr binding
rotates the regulatory domain away from the kinase
domain. Accompanied by the rotation of the regula-
tory domain, other loops from the catalytic domains
are displaced to orient the residues important for
cofactor and Asp binding in unfavorable positions.
Thus, in spite of their structural similarity, the
regulatory mechanism is different between these
homo-oligomeric AKs. In TtAKb, the loop, b4–a2,
corresponding to the latch in EcAKIII, is short and
shows no structural rearrangement upon Thr binding

i
value of TtAK may indicate that AK
activity is controlled through the high-affinity Thr site
present in the regulatory domain in TtAK.
A. Yoshida et al. Crystal structures of regulatory subunit of Thr-sensitive aspartate kinase
FEBS Journal 276 (2009) 3124–3136 ª 2009 The Authors Journal compilation ª 2009 FEBS 3131
Potential factors involved in the high
thermostability of TtAKb
In our previous study, we found that chimeric AK,
BTT, which is composed of the catalytic domain of
BsAKII and the regulatory domain and b-subunit of
TtAK, had thermostability as high as that of wild-type
TtAK, suggesting that the regulatory domain of TtAK
is also responsible for the thermal stability of TtAK
[11].
TtAKb and CgAKb show 36% sequence identity,
and Thr-bound crystal structures of these proteins are
very similar. To understand the mechanism of
enhanced stability of TtAKb, we examined the dena-
turation of TtAKb and CgAKb by differential scan-
ning calorimetry (DSC) in the presence and absence,
respectively, of their inhibitors. TtAKb has a denatur-
ation temperature approximately 40 °C higher than
that of CgAKb (Table 2). Both CgAKb and TtAKb
are more stable at 4.3–4.4 °C in the presence of Thr.
Considering that TtAKb and, putatively, CgAKb are
in equilibrium between monomers and dimers, and
bound Thr shifts the equilibrium towards dimer forma-
tion, this observation indicates that the small increase
in stability results from a shift of the equilibrium to

packed than CgAKb. With regard to the amino acid
composition, TtAKb has a higher ratio of hydrophobic
residues than CgAKb. It is also remarkable that
TtAKb contains more proline residues than CgAKb
(Table 5). Considering that most Pro residues are
located at the N-termini or C-termini of loops in
TtAKb (Fig. 6A), the flexibility of the loop conforma-
tion of TtAKb is likely to be suppressed in the dena-
tured state. We therefore suggest that smaller loss of
entropy upon folding contributes to the stabilization
of TtAKb.
We next calculated changes in Gibbs free energy
from the native to the denatured state, which were esti-
mated on the basis of the solvent-accessible surface
area (Table 4). The difference in changes in Gibbs free
energy between TtAKb and CgAKb was 22 kcalÆ
mol
)1
, indicating that TtAKb is more stable than
CgAKb. A more detailed examination showed that the
difference in the solvent-accessible surface area per
hydrophobic amino acid residue between the native
and denatured states was significantly larger in
TtAKb-Thr than in Thr-bound CgAKb, whereas that
of hydrophilic residues did not change, suggesting a
contribution of internal hydrophobic residues to the
stability of TtAKb. In fact, hydrophobicity inside the
molecule was apparently higher in TtAKb-Thr than in
CgAKb (Fig. 6B). From these results, we conclude
that better internal packing, ensured by tight

) 21 313 (70) 22 291 (68) )978 (2)
Hydrophilic (A
˚
2
) 6389 (21) 6709 (21) )320 (0)
DG 283 261 22
Cavity volume
(probe 1.4 A
˚
)(A
˚
3
)
41.4 110.1 )68.7
Crystal structures of regulatory subunit of Thr-sensitive aspartate kinase A. Yoshida et al.
3132 FEBS Journal 276 (2009) 3124–3136 ª 2009 The Authors Journal compilation ª 2009 FEBS
hydrophobic interactions in the interior of protein, and
the richness of Pro residues mainly contribute to the
stabilization of TtAK b. This information might be use-
ful for the generation of more thermostable CgAK
variants for industrial use.
Experimental procedures
Enzyme production and crystallization
Gene cloning and the production, purification and crystalli-
zation of TtAKb-Thr were performed as previously
described [31]. TtAKb-free was crystallized by the hanging
drop, vapor diffusion method. Crystals appeared in 0.1 m
sodium acetate (pH 5.0) and 1.2–2.0 m NaCl.
Data collection
The collection of TtAKb-Thr data and MAD data

Thr. Subsequent refinement was conducted using the
program cns1.1 [35], and model correction in the electron
density map was carried out with the xtalview program
suite [36]. Figures were prepared using xfit in the xtal-
view program suite and pymol [DeLano WL, The PyMOL
Molecular Graphics System (2002) at http://www.pymol.
org]. The atomic coordinates and structure factors deter-
mined in this study have been deposited in the Protein Data
Bank (accession numbers 2dt9 and 2zho).
Determination of quaternary structure
The subunit organization of TtAKb was analyzed by analyt-
ical ultracentrifugation and gel filtration chromatography.
Fig. 6. Factors important for thermostabilization of TtAKb. (A) Pro
residues in TtAKb monomer. (B) Pro residues in CgAKb monomer.
(C, D) Cross-sectional views of TtAKb-Thr dimer and Thr-bound
CgAKb dimer, respectively, drawn by
UCSF CHIMERA [46]. The surface
of the molecules is shown in cyan, and inner hydrophobic residues
are shown in pink.
Table 5. Thermostabilization factors. Comparison of the amino acid
composition of TtAKb and CgAKb.
TtAKb CgAKb
Residues (%) Residues (%)
Hydrophobic 102 63.4 88 51.2
Gly 12 7.45 14 8.14
Ala 28 17.4 18 10.5
Val 15 9.32 19 11.1
Leu 11 6.83 14 8.14
Ile 16 9.94 10 5.81
Met 6 3.73 5 2.92

package origin 2.8, provided by Beckman, with the partial
specific volume 0.747 cm
3
Æg
)1
estimated by sednterp soft-
ware [37]. Goodness of fit was also determined by global
analysis in origin 2.8. Gel filtration was performed using a
HiLoad 26 ⁄ 60 Superdex 75 column on an FLPC system
(GE-Healthcare Japan, Tokyo, Japan) at 4 °C. The column
was equilibrated with buffer A (20 mm Tris ⁄ HCl pH 7.5,
150 mm NaCl) or buffer A supplemented with 5 mm Thr.
Protein samples of 5 mg were loaded and eluted at a flow
rate of 2.5 mLÆmin
)1
. Samples of TtAK eluted from the gel
filtration column were collected every 2.5 mL for the
elution volume between 125 and 225 mL. Gel filtration for
TtAK was performed using a HiLoad 26 ⁄ 60 Superdex 200
column under the same elution conditions as described
above.
DSC measurement
Proteins TtAKb and CgAKb were purified by Ni
2+
affinity
with Ni
2+
–nitrilotriacetic acid resin (Novagen, Madison,
WI, USA) and subsequent gel filtration chromatography
with HiLoad 26 ⁄ 60 Superdex 75 equilibrated with buffer B

Culture, Sports, Science and Technology Japan and by
the Noda Institute for Scientific Research. We thank
the staff of the Photon Factory for their assistance
with data collection. This work was approved by the
Photon Factory Program Advisory Committee (Pro-
posal no. 2005G268, 2007G531). We are also grateful
to H. Fukushima and S. Watabe (University of Tokyo)
for their assistance with analysis of protein thermosta-
bility by DSC.
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