Báo cáo khoa học: Large conformational changes in the Escherichia coli tryptophan synthase b2 subunit upon pyridoxal 5¢-phosphate binding pot

Large conformational changes in the Escherichia coli
tryptophan synthase
b
2
subunit upon pyridoxal
5¢-phosphate binding
Kazuya Nishio
1
, Kyoko Ogasahara
1
, Yukio Morimoto
2,3
, Tomitake Tsukihara
1,4
, Soo Jae Lee
5
and
Katsuhide Yutani
3
1 Institute for Protein Research, Osaka University, Japan
2 Research Reactor Institute, Kyoto University, Japan
3 RIKEN SPring-8 Center, Harima Institute, Japan
4 Department of Life Science, University of Hyogo, Japan
5 College of Pharmacy, Chungbuk National University, Korea
Keywords
apo- and holo-forms; conformational change;
PLP-binding; tryptophan synthase b
2
subunit; X-ray crystal structure
Correspondence
Katsuhide Yutani, RIKEN SPring-8 Center,

Harima Institute, 1-1-1 Kouto, Sayo, Hyogo
679-5148, Japan
Fax: 81-791-58-2917
Tel: 81-791-58-2937
E-mail:
Soo Jae Lee, College of Pharmacy,
Chungbuk National University, Sungbong-ro
410, Cheongju, Chungbuk, Korea
Fax: 82-43-268-2732
Tel: 82-43-261-2816
E-mail:
Database
Structural data are available from the Protein
Data Bank under the accession codes for
the holo- (2DH5) and apo- (2DH6) forms
(Received 16 October 2009, revised 24
February 2010, accepted 1 March 2010)
doi:10.1111/j.1742-4658.2010.07631.x
To understand the basis for the lower activity of the tryptophan synthase b
2
subunit in comparison to the a
2
b
2
complex, we determined the crystal struc-
tures of apo-b
2
and holo-b
2
from Escherichia coli at 3.0 and 2.9 A

˚
resolu-
tions, respectively. To our knowledge, this is the first report of both b
2
subunit structures with and without pyridoxal-5¢-phosphate. The apo-type
molecule retained a dimeric form in solution, as in the case of the holo-b
2
subunit. The subunit structures of both the apo-b
2
and the holo-b
2
forms
consisted of two domains, namely the N domain and the C domain.
Although there were significant structural differences between the apo- and
holo-structures, they could be easily superimposed with a 22° rigid body rota-
tion of the C domain. The pyridoxal-5¢-phosphate-bound holo-form had
multiple interactions between the two domains and a long loop (residues
260–310), which were missing in the apo-form. Comparison of the structures
of holo-Ecb
2
and Stb
2
in the a
2
b
2
complex from Salmonella typhimurium
(Sta
2
b

2
) identified the cause of the lower enzymatic activity of holo-Ecb
2
in
comparison with Sta
2
b
2
. The substrate (indole) gate residues, Tyr279 and
Phe280, block entry of the substrate into the b
2
subunit, although the indole
can directly access the active site as a result of a wider cleft between the N
and C domains in the holo-Ecb
2
subunit. In addition, the structure around
bAsp305 of the holo-Ecb
2
subunit was similar to the open state of Sta
2
b
2
with low activity, resulting in lower activity of holo-Ecb
2
.
Structured digital abstract
l
MINT-7712009: Ecb2 (uniprotkb:P0A879) and Ecb 2 (uniprotkb:P0A879) bind (MI:0407)by
x-ray crystallography (
MI:0114)

l
MINT-7712032: Ecb2 (uniprotkb:P0A879) and Ecb 2 (uniprotkb:P0A879) bind (MI:0407)by
biophysical (
MI:0013)
Abbreviations
DSC, differential scanning calorimetry; Eca, tryptophan synthase a subunit from E. coli; Ecb, tryptophan synthase monomer b subunit from
E. coli; Ecb
2
, tryptophan synthase b
2
subunit from E. coli; PLP, pyridoxal 5¢-phosphate; Sta, tryptophan synthase a subunit from
S. typhimurium; Sta
2
b
2
, tryptophan synthase a
2
b
2
complex from S. typhimurium; Stb, tryptophan synthase monomer b subunit from
S. typhimurium; Stb
2
, tryptophan synthase b
2
subunit from S. typhimurium; T
d
, denaturation temperature; bA, bB, two b subunits in the
same Ecb
2
dimer.

FEBS Journal 277 (2010) 2157–2170 ª 2010 The Authors Journal compilation ª 2010 FEBS 2157
Introduction
Tryptophan synthase (EC 4.1.2.20) catalyzes the final
two steps in the biosynthesis of l-tryptophan. The
bacterial enzyme, a multifunctional a
2
b
2
complex
(M
r
= 143 300), is composed of nonidentical a (M
r
=
28 700) and b (M
r
= 43 000) subunits. The a
2
b
2
complex can be isolated as a monomeric a subunit and
dimeric b
2
subunits in solution. The a and b
2
subunits
catalyze different reactions, namely the a and b reac-
tions (Eqns 1, 2 respectively). The physiologically
important reaction is the ab reaction (Eqn 3), which is
catalyzed by the a

2
b
2
complex.
a reaction:
Indole 3-glycerol phosphate $ indole
þ
D-glyceraldehyde 3-phosphate
ð1Þ
b reaction:
Indole +
L-serine ! L-tryptophan þ H
2
O ð2Þ
ab reaction:
Indole 3-glycerol phosphate +
L-serine ! L-tryptophan
þ
D-glyceraldehyde 3-phosphate þ H
2
O ð3Þ
Combining the a and b
2
subunits to form the a
2
b
2
complex stimulates the enzymatic activity of each
subunit by one to two orders of magnitude [1,2].
This mutual activation of the two subunits is

thought to derive from conformational changes in
the subunits upon formation of the complex [3,4].
Therefore, tryptophan synthase is an excellent model
for using to study the relationship between functional
activation and conformational changes in proteins.
The quaternary structure of the a
2
b
2
complex from
Salmonella typhimurium (Sta
2
b
2
) is an extended linear
abba subunit arrangement. The active sites of the a
and b subunits of Sta
2
b
2
are connected by a 25–30 A
˚
hydrophobic tunnel through which the indole is trans-
ferred from the a subunit to the b subunit [5,6]. Crys-
tal structures of the Sta
2
b
2
complex with allosteric
cations and ⁄ or ligands, and of the Pfa

2
b
2
complex
from the hyperthermophile Pyrococcus furiosus, have
been described [7–16]. These structures provide valu-
able information to help us understand the allosteric
mechanism of the tryptophan synthase. Recently,
structures have been solved of the tryptophan synthase
a subunit from Escherichia coli (Eca) [17] and of the
tryptophan synthase a [18], and b
2
[19] subunits from
P. furiosus. To obtain a clear understanding of the
reason for a low enzymatic activity of the b
2
subunit
in the absence of the a subunit, it is necessary to solve
the structures of the b
2
subunit from E. coli (Ecb
2
)
with and without its cofactor, pyridoxal-5¢-phosphate
(PLP).
PLP-dependent enzymes catalyze multiple reactions
during the metabolism of amino acids. These enzymes
have been classified into a, b and c families based on
the chemical characteristics of the enzymatic reactions
[20]. The tryptophan synthase b

2
subunit belongs to
the b family, members of which catalyze b-replace-
ment or b-elimination reactions. This family has been
distinctly classified into five-fold-types based on
sequence and structural features [21]. Fold-type II
enzymes in the b family include the tryptophan syn-
thase b
2
subunit, O-acetylserine sulfhydrylase [22] and
serine dehydratase [23]. Several crystal structures of
the apo- and holo-forms of PLP-binding enzymes
exhibit only minor re-arrangements in the positions of
residues lining the active site between the apo- and
holo-enzymes [24,25]. Other PLP-binding enzymes,
however, display significant conformational changes
between the apo- and holo-forms [23,26,27]. Both the
apo- and holo-types of serine dehydratase, isolated
from the rat liver, form a homodimer. In the apo-
serine dehydratase dimeric form, a small domain
inserts into the catalytic cleft of the partner subunit
so that the active site is closed when inactive. Trypto-
phan synthase, however, is a unique PLP-binding
protein because the b reaction mediated by the b
subunit is regulated via an allosteric mechanism trig-
gered by association with the cognate a subunit. PLP
binds cooperatively to the apo-b
2
subunit and nonco-
operatively to the a

2
apo-b
2
complex in E. coli [28,29].
Therefore, it is important to determine the b
2
subunit
structure of the apo-type without PLP, as well as that
of the holo-type, to elucidate the mechanism of PLP
binding.
We obtained crystals of the apo-form in the
absence of PLP, as well as the holo-type bearing PLP,
for Ecb
2
. The structures of the apo-b
2
and holo-b
2
subunits were solved at 3.0 and 2.9 A
˚
resolutions,
respectively. We evaluated the thermal stabilities of
the holo-Ecb
2
and apo-Ecb
2
forms using differential
scanning calorimetry (DSC). In this communication
we will discuss the role of PLP in the stabilization of
Ecb

2
, the mechanism of PLP binding to apo-Ecb
2
and
the structural basis for lower enzymatic activity of
Ecb
2
in the absence of the a subunit, from a compari-
son of the apo-Ecb
2
, holo-Ecb
2
and Sta
2
b
2
complex
structures.
Apo- and holo-tryptophan synthase b
2
subunits K. Nishio et al.
2158 FEBS Journal 277 (2010) 2157–2170 ª 2010 The Authors Journal compilation ª 2010 FEBS
Results and Discussion
Contribution of PLP to the stabilization of the
b
2
subunit
Using the holo-Ecb
2
, the apo-Ecb

2
and the reconsti-
tuted holo-Ecb
2
proteins, we confirmed the contribu-
tion of PLP to the stabilization of the b
2
subunits.
Holo-Ecb
2
bound to PLP demonstrated an absorption
spectrum bearing a peak at 413 nm, characteristic for
a PLP internal aldimine bound to a Lys in the b
2
sub-
unit [30], and a CD spectrum with a peak around
420 nm (data not shown). Dissociation of PLP from
the b
2
subunit by dialysis against 0.1 m Mes buffer
(pH 6.5) containing 0.1 m Li
2
SO
4
could be confirmed
by the disappearance of these peaks from repeat analy-
ses. Ultracentrifugation analysis indicated that both
apo-Ecb
2
and holo-Ecb

2
remain in dimeric forms in
solution. Reconstitution of holo-Ecb
2
from apo-Ecb
2
by dialysis against 50 mm potassium phosphate buffer
(pH 7.0) containing 0.2 mm PLP for 1 day at 4 °C was
also confirmed by the characteristic changes seen on
absorption and CD spectra, and DSC curves.
Figure 1 displays the pH dependence of holo-Ecb
2
and apo-Ecb
2
stabilities; the denaturation temperature,
T
d
, of the holo-protein increases by nearly 25 °Cat
approximately pH 9 as a result of PLP binding. The
difference in the T
d
values between the holo- and apo-
proteins decreased with decreasing pH (Fig. 1), sug-
gesting that the binding constant of PLP decreases at
pH 6.5. The denaturation temperature of the reconsti-
tuted holo-Ecb
2
was similar to that of the native
holo-Ecb
2

bound to PLP (Fig. 1), indicating that the
dissociation of PLP from holo-Ecb
2
is reversible. These
results confirmed that PLP plays an important role in
stabilizing the b
2
subunit of the tryptophan synthase.
For the b
2
subunit from S. typhimurium (Stb
2
), PLP
dissociation also decreased the thermal stability [31].
PLP binding to proteins has been reported to play an
important role in protein stabilization [32,33]. From
the structural differences between the apo- and holo-
Ecb
2
forms, the stabilization of Ecb
2
by PLP binding
appears to be caused by increases in the number of
hydrogen bonds, salt bridges and hydrophobic interac-
tions, as described below.
Overall structures of the tryptophan synthase
b
2
subunit from E. coli
The crystal structures of both apo- and holo-Ecb

2
form
dimers with a crystallographic two-fold axis (Fig. 2).
The apo- and holo-Ecb
2
subunit structures are each com-
posed of two domains, namely an N domain (residues 1–
206) and a C domain (residues 207–397); one stretch of
the N-terminal sequence (residues 53–84), however,
crosses over into the C domain. A wide cleft in the inter-
face between the two domains of holo-Ecb
2
corresponds
to the indole tunnel. The holo-Ecb C domain contains a
long stretched loop (residues 260–310) with a B-factor of
82.0 A
˚
2
for the main chains, which includes a short helix
and two strands. By contrast, no electron density was
observed for the long loop (residues 259–308) in apo-Ecb
(Fig. 3). The long loop forms seven salt bridges with
residues in the N (Arg275-Glu11, Lys283-Glu11,
Glu296-Lys167 and Asp305-Lys167) and C (His260-
Glu266, His260-Asp329 and Lys272-Asp323) domains
Fig. 1. pH dependence of the thermal stability (T
d
) of holo-Ecb
2
and apo-Ecb

2
. The T
d
value represents the peak temperature on
the DSC curve measured at a scan rate of 1 °CÆmin
)1
. Closed
circles, open circles, and open triangles indicate the T
d
values for the
native holo-, reconstituted holo- and apo-b
2
subunits, respectively.
Buffers used were 50 m
M potassium phosphate supplemented
with 1 m
M EDTA at a pH of <8.5 and 50 mM Gly supplemented
with 1 m
M EDTA at pH 9.
Fig. 2. Schematic view of the holo-Ecb
2
dimeric structure. The two
holo-Ecb enzymes form a dimer relative to a crystallographic two-
fold axis. The symmetry-related molecule is colorless. Magenta,
blue and green regions represent the N domain, the C domain and
the long loop (residues 260–310), respectively. The PLP molecule is
inserted in the CPK representation. Figures displaying protein struc-
tures were prepared using
MOLSCRIPT software [58].
K. Nishio et al. Apo- and holo-tryptophan synthase b

2
subunits
FEBS Journal 277 (2010) 2157–2170 ª 2010 The Authors Journal compilation ª 2010 FEBS 2159
in addition to many hydrogen bonds: no such salt
bridges or hydrogen bonds were observed in the apo-
form. These results suggest that the long loop interacts
with the N and C domains only upon PLP binding,
which contributes to the stabilization of the holo-form.
Alignment of the secondary structures with the amino
acid sequences of the apo- and holo-Ecb subunits and
the b subunit of Sta
2
b
2
(Stb) is shown in Fig. 4. The
secondary structures were assigned with numbering as
referenced from Stb [5].
The overall topologies of the apo- and holo-Ecb
structures were equivalent to that of the Sta
2
b
2
b sub-
unit [5]. Equivalent Ca atoms of the apo- and holo-
Ecb structures could be superimposed with an rmsd of
3.3 A
˚
, a remarkably large value in comparison to the
rmsd value of 0.5 A
˚

for the superposition of holo-Ecb
and Stb (PDB code 1BKS). To investigate whether
this significant difference was caused by rigid move-
ment of the domains or induced-fitting, we systemati-
cally analyzed the domain movement caused by PLP
binding using D ynDom software [34]. This analysis
indicated that holo-Ecb can be divided into an N-ter-
minal domain (residues 1–52 and 85–206) and a C-ter-
minal domain (residues 53–84 and 207–397)
corresponding to the N and C domains of Stb, respec-
tively. A rotation of 21.8° combined with a transition
of just 0.2 A
˚
from the centroid of apo-Ecb permitted
good superposition of the C domain of the apo-form
with that of the holo-form. This result indicated that
the conformational re-arrangement upon PLP binding
is predominantly a rigid body rotation of the C
domain with local conformational fittings of several
residues that accommodate PLP. The structure of
apo-Ecb is an open form in which the N and C
domains are separated in comparison to holo-Ecb,
which is a closed form (Fig. 3). PLP binding induces
the conformational change through a rigid body rota-
tion, inducing a transition from the open to the closed
form at the deep cleft between the two domains of
Ecb.
Difference in structures between apo-Ec b
2
and

holo-Ec b
2
The differences in structures surrounding the active
sites in apo-Ecb
2
and holo-Ecb (shown in Fig. 5A,B
respectively) illustrate the conformational changes
directly related to PLP binding. The PLP coenzyme,
located in a deep cleft between the two domains, forms
a Schiff base with Lys87 in holo-Ecb and is shielded
from the solvent by the residues in the long loop (resi-
dues 260–310) (Fig. 5B). The residues interacting with
PLP via hydrogen bonding are His86 and Thr190 in
the N domain and Gly232, Gly234, Ser235, Asn236
and Ser377 in the C domain. To explore the conforma-
tional changes caused by PLP binding, we determined
the rmsd values (A
˚
) of the Ca atoms for each N or C
domain between apo-Ecb and holo-Ecb from the
superposition of the respective domains. Five signifi-
cant conformational changes (peaks 1–5) were
observed to result from PLP binding. Three peaks –
peak 2 (near Gly84), peak 3 (near Gly234) and peak 5
(near Arg379) – are close to the active site, while peaks
1 (near Tyr52) and 4 (near Thr319) are far from the
active site. Nine hydrogen bonds were observed
between these peak regions and PLP in the holo-form
that are absent in the apo-form. Three sets of hinge
residues are located at the sites connecting the N and

C domains: hinge 1 (residues 48–56), hinge 2 (residues
82–87) and hinge 3 (residues 206–207) (green boxes in
Fig. 4). Hinges 1 and 2 correspond to peaks 1 and 2,
respectively, indicating that PLP binding also induces
changes in the regions connecting the N and C
domains.
AB
Fig. 3. Schematic views of (A) the apo-Ecb
structure and (B) the holo-Ecb structure.
Magenta, blue and green regions represent
the N domain, the C domain and the long
loop, respectively. The PLP molecule is
depicted by a CPK model. The arrow in (A)
denotes the motion of the C domain follow-
ing PLP binding. Figures displaying protein
structures were prepared using
MOLSCRIPT
software [58].
Apo- and holo-tryptophan synthase b
2
subunits K. Nishio et al.
2160 FEBS Journal 277 (2010) 2157–2170 ª 2010 The Authors Journal compilation ª 2010 FEBS
Changes in the intersubunit interface of the b
2
dimer as a result of PLP binding
Dimerization of the apo-Ecb and holo-Ecb subunits
occurs along a twofold crystallographic axis (Fig. 2).
Apo-Ecb
2
was confirmed by analytical ultracentrifuga-

tion, at pH 7.0 (as described earlier), to be a dimer in
solution. The total areas buried in the subunit interface
for apo-Ecb
2
and holo-Ecb
2
were estimated at 1548
and 1711 A
˚
2
, respectively. The buried area of the b
2
subunit within Sta
2
b
2
(PDB code 1BKS) is 1619 A
˚
2
.
The hydrophobicity of the contact area at the apo-
Ecb
2
and holo-Ecb subunit interfaces was estimated at
165 and 180 kJÆmol
)1
, respectively, using the human
lysozyme parameters obtained by Funahashi et al. [35].
This result indicates that the hydrophobic interaction
at the subunit interface of the dimer increases with

PLP binding. Upon binding of PLP to the active site
of one b subunit (bA), the side chain of bA-Glu350
moves in the direction of the subunit interface from
the active site (Fig. 6). By contrast, the side chain of
bA-Lys382 moves from the subunit interface to the
active site to form a salt bridge with bA-Glu350. The
side chain of bA-Arg379 moves to the subunit interface
as a result of repulsion by the bA-Glu350 side chain.
The side chain of bA-Arg379, in concert with
bB-Arg379 from another subunit (bB), creates an
arginine–arginine short-range interaction, which in
Fig. 4. Sequence alignments of apo-Ecb and holo-Ecb forms with Stb. The first line represents the alias of the secondary segments as
named by Hyde et al. [5]. The red bars, blue arrows and lines, which denote the a helices, b strands and the others, respectively, comprise
the secondary structural elements of Stb (PDB code 1BKS), holo-Ecb and apo-Ecb, based on the definitions established by
PROCHECK soft-
ware [53]. The red letters in the sequences indicate identical residues between Stb and Ecb. The cyan squares in the third line represent
hydrogen bonds between Stb and Sta, while the green squares in the fourth line indicate the hinge region in Ecb. The hinge regions, includ-
ing hinges 1 (residues 48–56), 2 (82–87) and 3 (206–207), were defined using D
YNDOM software. The asterisks in the sixth and seventh lines
represent those residues binding PLP via hydrogen bonds (blue), hydrophobic interactions (black) or covalent bonds (red) in Ecb.
K. Nishio et al. Apo- and holo-tryptophan synthase b
2
subunits
FEBS Journal 277 (2010) 2157–2170 ª 2010 The Authors Journal compilation ª 2010 FEBS 2161
protein–protein interactions is often an important fac-
tor in stabilization and recognition [36]. This interac-
tion induces large conformational changes in the
empty bB subunit, facilitating the subsequent binding
of PLP. The dissociation constant of PLP for the iso-
lated b

2
subunit is 8.7 · 10
)6
m for the first (bA) site
and 2.3 · 10
)7
m for the second (bB) site [28]. The
more efficient binding of the second PLP indicates that
this interaction occurs under different structural cir-
cumstances from the first. The side chain of the shifted
bA-Arg379 contacts the bB-Asp381 side chain in
another subunit (data not shown). Each His82 turns to
the external surface of the molecule, forming a salt
bridge with Asp79 in the peak 2 region of the paired b
subunits. The extent of the hydrophobic interaction,
and the number of hydrogen bonds and salt bridges
between the two subunits, are greater in the holo-Ecb
2
when compared with the apo-Ecb
2
. These results indi-
cate that the two b subunits of Ecb
2
, bA and bB, are
more tightly associated in holo-Ecb
2
than in apo-Ecb
2
;
the stronger subunit–subunit association for holo-Ecb

2
results from local conformational changes caused by
PLP binding.
While the subunit–subunit interface did not change
drastically between the crystal structures of dimeric
apo-Ecb
2
and holo-Ecb
2
, the crystal structures of sev-
eral PLP-dependent b-elimination enzymes exhibit dif-
ferent intersubunit interfaces for the apo-dimeric and
holo-dimeric structures [23,27]. In the apo-dimeric
structure of serine dehydratase, the small domain
rotates to open the active site cleft, allowing the small
domain of the partner subunit to enter the opened
cleft. While bound PLP and potassium readily dissoci-
ate from serine dehydratase by simple dialysis in the
presence of cysteine [37], the binding of PLP to the
tryptophan synthase b
2
subunit is very tight (K
d
of
approximately 10
)7
m).
Mechanism of PLP binding
Five regions (peaks 1–5) exhibited significant conforma-
tional changes following PLP binding. The N terminus

K87
S377
T190
S235
N236
H86
G232
SO4
2-
SO4
2-
SO4
2-
K87
S377
T190
S235
G234
N236
H86
G232
SO4
2-
SO4
2-
SO4
2-
Helix-9
Helix-8
Helix-7

Helix-12
Helix-9
Helix-8
Helix-7
Helix-12
PLP
long-loop
K87
S377
T190
S235
G234
N236
H86
P
OP4
OP2
OP3
OP1
G232
NZ
C4A
K87
P
OP4
OP2
OP3
OP1
NZ
C4A

SO4
2-
PLP
long-loop
K87
S377
T190
S235
G234
N236
H86
P
N1
N1
OP4
OP2
OP3
OP1
G232
NZ
C4A
K87
P
OP4
OP2
OP3
OP1
NZ
C4A
SO4

2-
Helix-9
Helix-9
Helix-8
Helix-7
Helix-12
Helix-8
Helix-7
Helix-12
N1
G234
A
B
Fig. 5. Schematic stereo view of the con-
formations surrounding the active sites of
(A) apo-Ecb and (B) holo-Ecb, shown from
the same orientation. Magenta, blue and
green regions represent the N domain, the
C domain and the long loop, respectively.
The residues forming hydrogen bonds with
PLP (yellow) are indicated by the ball-and-
stick model, labeled in holo-Ecb (B). Figures
displaying protein structures were prepared
using
MOLSCRIPT software [58].
Apo- and holo-tryptophan synthase b
2
subunits K. Nishio et al.
2162 FEBS Journal 277 (2010) 2157–2170 ª 2010 The Authors Journal compilation ª 2010 FEBS
of Helix 9 (peak 3 region) is structurally similar to a

region found in several other PLP-binding enzymes,
called the ‘anchoring alpha-helix’ [38]. Therefore, the
first binding site of PLP must be the peak 3 region (resi-
dues 230–236). This region binds to the phosphate
group of PLP via multiple hydrogen bonds as well as by
the electrostatic effects of the Helix 9 dipole (Figs 4 and
5B). These interactions may rapidly induce the rigid
body rotation of the C domain, resulting in PLP binding
to holo-Ecb
2
via 10 hydrogen bonds, of which nine are
with the PLP phosphate group (Fig. 5B). PLP binding
is also additionally stabilized by seven hydrogen bonds
and one hydrophobic interaction from the peak 3
region. Schiff-base formation with Lys87 is not required
for the binding of PLP, as the K87T mutant of the
Sta
2
b
2
b subunit [8] and the K41A mutant of S. ty-
phimurium O-acetylserine sulfhydrylase both retained
the ability to bind PLP in the active site [39].
The peak 5 region near Arg379 also interacts with
the phosphate group of PLP. Hydrogen bonds between
OG of Ser377 and N1 of PLP (Figs 4 and 5B) may
cause the conformational changes in this region upon
PLP binding. The conformational changes in the peak
4 region near Thr319 are also affected by alterations
transmitted from the peak 3 region. The C terminus of

the long-loop and the N terminus of Helix 10 are
pulled into the interior of the molecule by hydrogen
bonds between the N of Gly310 and the O of Gly233
and between the NE2 of His313 and the O of Gly234.
The His86 side chain, which is located in the peak 2
region next to the Lys87 of the PLP-binding residue,
shifted in location near the PLP upon PLP binding.
The H86L mutant of the Sta
2
b
2
b subunit exhibited a
PLP-binding ability that was reduced by approxi-
mately 20-fold [40], indicating the importance of His86
in PLP binding. The conformational changes in His86
upon PLP binding may correlate directly with interac-
tions between the N and C domains, leading to the
closure of the holo-b
2
subunit. PLP also forms a
hydrogen bond with Thr190 in the N domain, located
in a loop between Sheet 6 and Helix 7 (Figs 4 and 5).
The peak 4 region near Thr319 does not interact with
PLP, but the conformation appears to be affected by
the conformational changes in the peak 3 region.
The formation of these hydrogen bonds and the
molecular rotation facilitate Schiff-base formation by
bringing Lys87 close to PLP. After the Schiff-base for-
mation, residues in the long loop of the C domain
form salt bridges and hydrogen bonds with residues in

the N domain. PLP binding to one b subunit (bA)
induces a simultaneous conformational change in the
other b subunit (bB). This conformational change in
the bA subunit results in more efficient binding of PLP
to the bB subunit [28], generating an Ecb
2
with both
active sites bound to PLP. Kinetic and calorimetric
studies of PLP binding to the apo-b
2
subunit of trypto-
phan synthase from E. coli have suggested four bind-
ing processes [29,41], which are consistent with these
structural studies.
Conformational changes in the b
2
subunit of
tryptophan synthase caused by formation of the
a
2
b
2
complex
We compared the structures of holo-Ecb
2
with the b
2
subunits of Sta
2
b

2
by superimposition of equivalent
Ca atoms (Fig. 7). The rmsd values for a comparison
between holo-Ecb
2
and Sta
2
b
2
without ligands bound
to either subunit active site (PDB code 1BKS),
between holo-Ecb
2
and Sta
2
b
2
with a subunit ligands
only (1A50), and between holo-Ecb
2
and Sta
2
b
2
with
ligands bound to both the a and the b subunits
(2TRS) were 0.5, 0.7 and 1.2 A
˚
, respectively. The
structure of holo-Ecb

2
superimposed well on that of
the b
2
subunit from an unliganded Sta
2
b
2
complex
(1BKS). The structures of both b
2
subunits were simi-
lar despite the absence of residues 291–292 from the
Ecb
2
structure and differences between the long loop
(residues 260–310) and the COMM (residues 102–189)
domain. The average B-factor value of the main-chain
atoms for the long loop (82.00 A
˚
2
)ofEcb
2
was higher
than that of the entire molecule (59.06 A
˚
2
). By con-
Fig. 6. Stereo view of the conformational changes in the intersub-
unit interface of the b

2
dimer following PLP binding. The C domains
of apo-Ecb and holo-Ecb were superimposed. The blue and light-
blue domains represent apo-Ecb and its pair subunit, respectively,
while the red and pink domains represent holo-Ecb and its pair sub-
unit, respectively. Stick models are used to display the side chains
of the residues with conformational changes between the apo-
(cyan) and holo- (pink) b forms. Black arrows denote the movement
of the residues following PLP binding. Figures displaying protein
structures were prepared using
PYMOL software [59].
K. Nishio et al. Apo- and holo-tryptophan synthase b
2
subunits
FEBS Journal 277 (2010) 2157–2170 ª 2010 The Authors Journal compilation ª 2010 FEBS 2163
trast, the B-factor of the long loop (26.50 A
˚
2
) in the
Sta
2
b
2
lacking ligands was similar to that for the entire
molecule (28.30 A
˚
2
). This result suggests that the flexi-
ble long loop becomes more rigid in the a
2

b
2
complex
when compared with the b
2
dimer as a result of the
formation of several hydrogen bonds between the a
and b subunits at their interface in the a
2
b
2
complex.
The long loop and the COMM domain constitute the
interface of the N and C domains that forms the wall
of the hydrophobic tunnel.
Indole, the product of the a subunit, is transferred
via a 25–30 A
˚
hydrophobic tunnel from the active site
of the a subunit to that of the b subunit [5,6]. The
width of the holo-Ecb
2
tunnel was larger by 2–3 A
˚
and
3–4 A
˚
than those of Sta
2
b

2
in the absence of ligands
(1BKS) and a-, b-liganded Sta
2
b
2
(2TRS), respectively.
The tunnel gating residues bTyr279 and bPhe280,
which block the untimely passage of indole, were in a
closed conformation in the holo-Ecb
2
and the unli-
ganded Sta
2
b
2
(1BKS). As a hydrogen bond between
aAsp56 and bTyr279 seems to be essential for opening
the indole gate [16], it is likely that the tunnel gating
residues are also in a closed conformation in the
b
2
subunit. In the holo-Ecb
2
, indole, the substrate, can
directly approach the active site because the width of
the cleft between the N and C domains in holo- Ecb
2
is
wide despite the indole gate being closed.

An allosteric signal is transmitted between the a
and b subunits of tryptophan synthase via the salt
bridge-type interactions of bLys167 with bAsp305 and
aAsp56. The bLys167–aAsp56 salt bridge is more
important for the transmission of the allosteric signal
than the bLys167– bAsp305 salt bridge [42]. The b
activities of bK167T, aD56A and bD305N mutants
are very low [43]. These allosteric signals regulate the
transformation of the bienzyme complex from an
open conformational state of low activity to a closed
conformation state of high activity, which is triggered
by ligand bound to the a and ⁄ or b active site. Struc-
tures of the unliganded wild-type (1BKS: a, open
state; b, open state), IPP (indole propanol phosphate)
bound wild-type (1A50: a, closed state; b, open state)
and l-Ser-binding bK87T mutant (2TRS: a, closed
state; b, closed state) proteins were compared with
those of holo-Ecb
2
, especially around bAsp305
(Fig. 8). The side chains of bLys167 and bAsp305,
which are double conformers in the unliganded
Sta
2
b
2
, exhibit different conformations in the struc-
tures bearing different ligands (Fig. 8A). The structure
around bAsp305 of holo-Ecb
2

is similar to the low-
activity open state of Sta
2
b
2
. Holo-Ecb
2
cannot trans-
form into the higher-activity ‘closed-state’ from the
‘open-state’, resulting in a low-activity enzyme because
of the lack of interaction between bLys167 and
aAsp56. Furthermore, the gate of the indole tunnel is
blocked in the absence of any interaction with
aAsp56. Therefore, aAsp56 appears to be one of the
key residues for the activation of enzymatic activity
upon complex formation.
Conclusions
The crystal structures of apo- and holo-Ecb
2
dimers
were determined using X-ray crystallographic analysis
at 3.0 and 2.9A
˚
, respectively. This is the first report of
b subunit structures with and without the PLP cofac-
tor. Holo-Ecb
2
consists of two domains, the N and C
domains, with a long loop in the C domain. The long
loop is not observed as a result of high flexibility in

apo-Ecb
2
.
Fig. 7. Stereo view of the superimposed backbone structures of holo-Ecb and the ligand-unbound (1BKS) and ligand-bound (2TRS) Stb su-
bunits. Red, cyan, blue and yellow regions represent holo-Ecb, 1BKS, 2TRS and PLP, respectively. This figure demonstrates that a cleft near
the PLP-binding site between the N and C domains of holo-Ecb is wider than those seen in the other Stb subunits. The COMM domain con-
sists of the region from Gly93 to Gly189, which has few interactions with the rest of the protein, and plays an important role in allosteric
communication between the a and b sites [11]. Figures displaying protein structures were prepared using
MOLSCRIPT software [58].
Apo- and holo-tryptophan synthase b
2
subunits K. Nishio et al.
2164 FEBS Journal 277 (2010) 2157–2170 ª 2010 The Authors Journal compilation ª 2010 FEBS
Comparison of the apo- and holo-Ecb
2
structures
revealed a large conformational change in the C domain
between these two structures. This conformational
change consisted of a rotation of the C domain by
approximately 22°, which was triggered by the local
conformational changes induced by PLP binding to
the active site. PLP binding to the apo-Ecb induced
the following major conformational changes. (a) PLP
bound to the N terminus of Helix 9 induces a rigid
body rotation of the C domain. This binding and rota-
tion results in Schiff-base formation involving Lys87.
This interaction triggers a slow local conformational
re-arrangement in other regions, including residues of
the long loop of the C domain. These changes were
primarily observed around the active site and hinge

residues connecting the N and C domains. (b) PLP
binding to one b-subunit (bA) induces a conforma-
tional change in the other b subunit (bB) within a
dimer. (c) PLP binding strengthens the b–b subunit
association as a result of increases in the hydrophobic
interactions, hydrogen bonds and salt bridges, resulting
in stabilization of the holo-form in comparison to the
apo-form, which was reflected in a higher denaturation
temperature of holo-Ecb
2
, as determined by the DSC
measurements, that was 25.3 °C greater than that of
apo-Ecb
2
at pH 9.0.
Comparison of the holo-Ecb
2
alone and Stb
2
in
Sta
2
b
2
structures revealed why the holo-Ecb
2
enzyme
has lower b enzymatic activity in the absence of the a
subunit than Eca
2

b
2
. (a) The indole gating residues,
Tyr279 and Phe280, block entry of the substrate in the
b
2
subunit alone, a situation similar to that seen for
the unliganded Sta
2
b
2
complexed with Na
+
. (b) The
structure around bAsp305 of holo-Ecb
2
alone is similar
to the low-activity open state of Sta
2
b
2
. Holo-Ecb
2
cannot transform into the higher-activity ‘closed-state’
from the ‘open-state’ because of the absence of an
interaction with aAsp56. (c) By contrast, the width of
the Ecb
2
tunnel was larger, by about 2–3 A
˚

, than that
of unliganded Stb
2
. Furthermore, the width of the cleft
between the N and C domains in Ecb
2
was wider,
resulting in better accessibility of the substrate (indole)
to the active site.
Experimental procedures
Purification and preparation of holo-, apo- and
reconstituted holo-Ecb
2
Holo-Ecb
2
[44] was purified as described previously [45].
The purified protein was visible as a single band on
SDS ⁄ PAGE. Apo-Ecb
2
was prepared by the dialysis of
holo-Ecb
2
against 0.1 m Mes buffer (pH 6.5) containing
A
B
C
Fig. 8. Stereo views of the structures surrounding the allosteric
residue, bAsp305, in holo-Ecb
2
and the Sta

2
b
2
complex. The sky-
blue dotted lines in the figures represent hydrogen bonds. (A) Red,
holo-Ecb
2
; gray, Sta
2
b
2
(1BKS, both the a and b subunits are in a
low-activity open conformation state). (B) Red, holo-Ecb
2
; gray,
Sta
2
b
2
(1A50, the a subunit is in a high-activity closed conformation
state, while the b subunit is in the open state). (C) Red, holo-Ecb
2
;
gray, Sta
2
b
2
(2TRS, both a and b subunits are in closed states).
Figures displaying protein structures were prepared using
PYMOL

software [59].
K. Nishio et al. Apo- and holo-tryptophan synthase b
2
subunits
FEBS Journal 277 (2010) 2157–2170 ª 2010 The Authors Journal compilation ª 2010 FEBS 2165
0.1 m Li
2
SO
4
, for 3 days at 4 °C. The reconstitution of
holo-Ecb
2
was performed by dialyzing the apo-Ecb
2
solu-
tion against 50 mm potassium phosphate buffer (pH 7.0)
containing 0.2 mm PLP, for 1 day at 4 °C.
Physicochemical properties of holo-Ecb
2
and
apo-Ecb
2
, monitored by CD, DSC and analytical
ultracentrifugation
The CD spectra were recorded using a Jasco J-720 spectro-
polarimeter (JASCO Co., Hachioji, Tokyo, JAPAN). The
far-UV and near-UV ⁄ visible CD spectra were scanned 16
and 36 times, respectively, at a scan rate of 20 nmÆmin
)1
,

using a time constant of 0.25 s. The light path length of the
cell was 1 and 10 mm in the far-UV and near-UV ⁄ visible
regions, respectively. To calculate the mean residue elliptic-
ity [h] the mean residue weight was assumed to be 108.3.
DSC was performed using a differential scanning micro-
calorimeter, VP-DSC (Microcal, Inc., Northampton, MA,
USA), at a scan rate of 1 °CÆmin
)1
. After dialysis against
the desired buffer, the sample was filtered through a mem-
brane with a pore size of 0.22 lm and degassed under a
vacuum. The protein concentrations during measurements
were 0.2–1.4 mgÆmL
)1
.
Sedimentation analysis was performed using a Beckmann
Optima mode XL-A centrifuge (Beckman Instruments, Inc.,
Fullerton, CA, USA). Sedimentation equilibrium experi-
ments used an An-60Ti rotor at a speed of 7658.3–8049.6 g
at 20 °C. Before measurements, the apo- and holo-Ecb
2
solutions were dialyzed overnight at 4 °C against 50 mm
potassium phosphate buffer, pH 7.9, containing 1 mm
EDTA, and the holo-Ecb
2
solution was dialyzed overnight
at 4 °C against 50 mm potassium phosphate buffer, pH 7.9,
containing both 1 mm EDTA and 0.2 mm PLP. Experi-
ments at three different protein concentrations (ranging
from 0.92 to 0.31 mgÆmL

)1
) were run using Beckman four-
sector cells. The partial specific volume was calculated,
from the amino-acid compositions, to be 0.754 cm
3
Æg
)1
[46].
Analysis of the sedimentation equilibrium data was per-
formed using xlavel software (Beckman version 2.0).
Crystallization and data collection
For crystallization, purified holo-Ecb
2
protein was concen-
trated to 20 mgÆmL
)1
in 20 mm potassium phosphate buffer
(pH 7.8) containing 0.1 mm dithiothreitol, 0.2 mm PLP and
a protease inhibitor mixture. Holo-Ecb
2
was crystallized
using the microbatch method with paraffin oil at 288K.
Crystals were obtained with 0.5 lL of protein solution
combined with 0.5 lL of crystallization reagent [0.8 m
(NH
4
)
2
SO
4

, 0.1 m Tris buffer, pH 8.0]. Light-yellow hexag-
onal rod-shaped crystals grew to maximal dimensions of
0.1 · 0.1 · 0.8 mm over 1 month.
Apo-Ecb
2
was crystallized using the hanging-drop vapor-
diffusion method at 288K. Crystals were obtained by equili-
bration of a mixture containing 1 lL of protein and 1 lL
of crystallization reagent (0.25 m Li
2
SO
4
, 0.1 m Mes buffer,
pH 6.5) against 100 lL of the reservoir solution at 288K.
Needle-like clear crystals grew to maximal dimensions of
0.2 · 0.02 · 0.02 mm in approximately 1 month. All crys-
tallizations were performed in the dark.
Apo-protein crystals were soaked in cryoprotectant (0.1 m
Mes buffer, pH 6.5, containing 0.4 m Li
2
SO
4
and 30% glyc-
erol), while holo-protein crystals were soaked in 0.1 m Tris
buffer (pH 8.0) containing 0.8 m (NH
4
)
2
SO
4

and 35% glyc-
erol for several minutes before flash-cooling in liquid nitro-
gen. Data sets were collected at the beamline BL44XU at
SPring-8 in Japan using the DIP6040 imaging plate detector
(Bruker AXS, Inc., Karlsruhe, Germany). Data were pro-
cessed using Mosflm [47] and ccp4 suite software [48]. The
apo-protein crystal belonged to space group P4
3
22, with cell
dimensions of a = b = 110.89, c = 102.21 A
˚
, one molecule
in an asymmetric unit, a solvent content of 66.1% and a
specific volume (V
M
) of 3.7 A
˚
3
Da
)1
[49]. The crystal of the
holo-form belonged to space group P6
5
22, with the cell
dimensions of a = b = 172.52, c = 82.59 A
˚
, one molecule
in an asymmetric unit, a solvent content of 69.9% and a
specific volume (V
M

) of 4.1 A
˚
3
Da
)1
. The statistics for data
collection are summarized in Table 1.
Structure determination and refinement
The apo- and holo-structures were determined by the
molecular replacement method using cns software [50]. The
b subunit of the Sta
2
b
2
complex (PDB code 1BKS) was
used as the initial search model. The holo-Ecb structure
was determined using a full-length search model. The
apo-Ecb structure, however, could be determined using an
omitted search model without a long loop (residues 260–
310). All refinements were performed using cns software.
The sigma-A-weighted composite omit map was calculated
by CNS to reduce model bias. The structure was visualized
and modified using XtalView [51] and O [52] software.
Both the apo- and holo-Ecb refined models consisted of
one molecule in the asymmetric unit. The apo-Ecb model
included residues 17–258, 309–397, three sulfate ions and
23 water molecules. The holo-Ecb model included residues
3–290, 293–397, one PLP molecule, two sulfate ions, one
glycerol molecule and nine water molecules. The refinement
statistics are summarized in Table 1. We have deposited the

final coordinates of the tryptophan synthase b
2
subunit
from E. coli into the Protein Data Bank.
Analyses of amino acid sequences and protein
models
The secondary structures of all coordinates were defined
using procheck software [53]. lsqkab software [54] in the
CCP4 suite was used for superposition of all coordinates.
The potential hydrogen bonds and hydrophobic interactions
Apo- and holo-tryptophan synthase b
2
subunits K. Nishio et al.
2166 FEBS Journal 277 (2010) 2157–2170 ª 2010 The Authors Journal compilation ª 2010 FEBS
were examined using ligplot software [55]. Residues form-
ing salt bridges were determined using Protein Explore
software [56]. Contact distance was evaluated using the
contact program within the CCP4 suite. Rigid domain
movement analysis was examined using dyndom software
[34]. The accessible surface area values for the proteins
were calculated as described by Connoly with a probe
radius of 1.4 A
˚
[57].
Table 1. Data collection and refinement statistics.
Apo-b
2
subunit Holo-b
2
subunit

Crystal data statistics
X-ray source SPring-8 BL44XU SPring-8 BL44XU
Detector DIP 6040 DIP 6040
Wavelength (A
˚
) 0.9 0.9
Crystal-to-detector distance (mm) 520 480
Exposure time (s) 10 10
Data-collection temperature (K) 100 100
No. of crystals per images 2 ⁄ 66 1 ⁄ 38
Space group P4
3
22 P6
5
22
Unit-cell parameters, a, b, c (A
˚
) 110.89, 110.89, 102.21 172.52, 172.52, 82.59
Mosaicity 0.47 0.26
Resolution range (A
˚
)
a
46.41–3.00 (3.16–3.00) 74.40–2.90 (3.06–2.90)
No. of total reflections
a
69 323(10 086) 74 984 (10 943)
No. of unique reflections
a
13 266 (1887) 15 920 (2297)

<I ⁄ r(I)>
a
6.8 (1.9) 6.6 (1.9)
Completeness (%)
a
99.9 (100.0) 96.8 (97.4)
R
merge
(%)
a
7.4 (38.9) 7.6 (39.0)
Redundancies
a
5.2 (5.3) 4.7 (4.8)
No. of molecules per asymmetric unit 1 1
V
M
(A
˚
3
D
a
)1
) 3.7 4.1
Wilson plot B factor (A
˚
2
) 85.4 86.3
Refinement statistics
Resolution range (A

˚
)
a
42.82–3.00 (3.19–3.00) 30.33–2.90 (3.08–2.90)
No. of reflections
a
13 244 (2159) 15 901 (2630)
R
cryst
(%)
a,b
20.6 (31.4) 19.6 (28.5)
R
free
(%)
a,c
25.4 (36.1) 24.4 (33.0)
rmsd, bonds (A
˚
) 0.012 0.009
rmsd, angles (°) 1.7 1.6
Ramachandran plot (%)
Most favored 90.5 88.8
Additional allowed 9.5 11.2
Generously allowed 0 0
Disallowed 0 0
No. of atoms or molecules
Protein atoms 2514 2988
PLP molecules 0 1
Sulfate ions 3 2

Glycerol molecules 0 1
Water molecules 23 9
Average B-factor (A
˚
2
)
All atoms 62.3 60.3
Protein atoms 62.2 59.8
Protein main-chain atoms 61.5 59.1
PLP – 42.7
Sulfate ions 98.8 84
Glycerol molecules – 57.2
Water molecules 46 48.1
a
Values in parentheses are for the highest resolution shell.
b
R
cryst
was calculated from the working set (95% of the data).
c
R
free
was calcu-
lated from the test set (5% of the data).
K. Nishio et al. Apo- and holo-tryptophan synthase b
2
subunits
FEBS Journal 277 (2010) 2157–2170 ª 2010 The Authors Journal compilation ª 2010 FEBS 2167
Acknowledgements
This study was supported by Grants for Scientific

Research (21227003) (to T. T.) and the ‘National Pro-
ject for Protein Structural and Functional Analysis’(to
K. Y.) funded by the Ministry of Education, Culture,
Sports, Science and Technology of Japan, and a
research grant from Chungbuk National University in
2006 and Chungbuk BIT (to S. J. L.).
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