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

Large conformational changes in the Escherichia coli tryptophan synthase b2 subunit upon pyridoxal 5¢-phosphate binding Kazuya Nishio1, Kyoko Ogasahara1, Yukio Morimoto2,3, Tomitake Tsukihara1,4, Soo Jae Lee5 and Katsuhide Yutani3

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 b2 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: yutani@spring8.or.jp 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: sjlee@chungbuk.ac.kr

Database Structural data are available from the Protein Data Bank under the accession codes for the holo- (2DH5) and apo- (2DH6) forms

To understand the basis for the lower activity of the tryptophan synthase b2 subunit in comparison to the a2b2 complex, we determined the crystal struc- tures of apo-b2 and holo-b2 from Escherichia coli at 3.0 and 2.9 A˚ resolu- tions, respectively. To our knowledge, this is the ﬁrst report of both b2 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-b2 subunit. The subunit structures of both the apo-b2 and the holo-b2 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(cid:2) 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-Ecb2 and Stb2 in the a2b2 complex from Salmonella typhimurium (Sta2b2) identiﬁed the cause of the lower enzymatic activity of holo-Ecb2 in comparison with Sta2b2. The substrate (indole) gate residues, Tyr279 and Phe280, block entry of the substrate into the b2 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-Ecb2 subunit. In addition, the structure around bAsp305 of the holo-Ecb2 subunit was similar to the open state of Sta2b2 with low activity, resulting in lower activity of holo-Ecb2.

(Received 16 October 2009, revised 24 February 2010, accepted 1 March 2010)

Structured digital abstract l MINT-7712009: Ecb2 (uniprotkb:P0A879) and Ecb2 (uniprotkb:P0A879) bind (MI:0407) by

doi:10.1111/j.1742-4658.2010.07631.x

x-ray crystallography (MI:0114)

l MINT-7712032: Ecb2 (uniprotkb:P0A879) and Ecb2 (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; Ecb2, tryptophan synthase b2 subunit from E. coli; PLP, pyridoxal 5¢-phosphate; Sta, tryptophan synthase a subunit from S. typhimurium; Sta2b2, tryptophan synthase a2b2 complex from S. typhimurium; Stb, tryptophan synthase monomer b subunit from S. typhimurium; Stb2, tryptophan synthase b2 subunit from S. typhimurium; Td, denaturation temperature; bA, bB, two b subunits in the same Ecb2 dimer.

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Introduction

reason for a low enzymatic activity of the b2 subunit in the absence of the a subunit, it is necessary to solve the structures of the b2 subunit from E. coli (Ecb2) with and without its cofactor, pyridoxal-5¢-phosphate (PLP).

Tryptophan synthase (EC 4.1.2.20) catalyzes the ﬁnal two steps in the biosynthesis of l-tryptophan. The bacterial enzyme, a multifunctional a2b2 complex (Mr = 143 300), is composed of nonidentical a (Mr = 28 700) and b (Mr = 43 000) subunits. The a2b2 complex can be isolated as a monomeric a subunit and dimeric b2 subunits in solution. The a and b2 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 a2b2 complex.

a reaction:

ð1Þ

Indole 3-glycerol phosphate $ indole þ D-glyceraldehyde 3-phosphate

b reaction:

ð2Þ

Indole + L-serine ! L-tryptophan þ H2O

ab reaction:

ð3Þ

Indole 3-glycerol phosphate + L-serine ! L-tryptophan þ D-glyceraldehyde 3-phosphate þ H2O

the

two subunits

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 b2 subunit belongs to the b family, members of which catalyze b-replace- ment or b-elimination reactions. This family has been classiﬁed into ﬁve-fold-types based on distinctly sequence and structural features [21]. Fold-type II enzymes in the b family include the tryptophan syn- thase b2 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 isolated apo- and holo-types of serine dehydratase, 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-b2 subunit and nonco- operatively to the a2apo-b2 complex in E. coli [28,29]. Therefore, it is important to determine the b2 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 Ecb2. The structures of the apo-b2 and holo-b2 subunits were solved at 3.0 and 2.9 A˚ resolutions, respectively. We evaluated the thermal stabilities of the holo-Ecb2 and apo-Ecb2 forms using differential scanning calorimetry (DSC). In this communication we will discuss the role of PLP in the stabilization of Ecb2, the mechanism of PLP binding to apo-Ecb2 and the structural basis for lower enzymatic activity of Ecb2 in the absence of the a subunit, from a compari- son of the apo-Ecb2, holo-Ecb2 and Sta2b2 complex structures.

Combining the a and b2 subunits to form the a2b2 complex stimulates the enzymatic activity of each subunit by one to two orders of magnitude [1,2]. This mutual activation of is to derive from conformational changes in thought the complex [3,4]. the subunits upon formation of 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 a2b2 complex from Salmonella typhimurium (Sta2b2) is an extended linear abba subunit arrangement. The active sites of the a and b subunits of Sta2b2 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 Sta2b2 complex with allosteric cations and ⁄ or ligands, and of the Pfa2b2 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 b2 [19] subunits from P. furiosus. To obtain a clear understanding of the

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Results and Discussion

Contribution of PLP to the stabilization of the b2 subunit

results conﬁrmed that PLP plays an important role in stabilizing the b2 subunit of the tryptophan synthase. For the b2 subunit from S. typhimurium (Stb2), 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- Ecb2 forms, the stabilization of Ecb2 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 b2 subunit from E. coli

Using the holo-Ecb2, the apo-Ecb2 and the reconsti- tuted holo-Ecb2 proteins, we conﬁrmed the contribu- tion of PLP to the stabilization of the b2 subunits. Holo-Ecb2 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 b2 sub- unit [30], and a CD spectrum with a peak around 420 nm (data not shown). Dissociation of PLP from the b2 subunit by dialysis against 0.1 m Mes buffer (pH 6.5) containing 0.1 m Li2SO4 could be conﬁrmed by the disappearance of these peaks from repeat analy- ses. Ultracentrifugation analysis indicated that both apo-Ecb2 and holo-Ecb2 remain in dimeric forms in solution. Reconstitution of holo-Ecb2 from apo-Ecb2 by dialysis against 50 mm potassium phosphate buffer (pH 7.0) containing 0.2 mm PLP for 1 day at 4 (cid:2)C was also conﬁrmed by the characteristic changes seen on absorption and CD spectra, and DSC curves.

The crystal structures of both apo- and holo-Ecb2 form dimers with a crystallographic two-fold axis (Fig. 2). The apo- and holo-Ecb2 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-Ecb2 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 in the N (Arg275-Glu11, Lys283-Glu11, residues Glu296-Lys167 and Asp305-Lys167) and C (His260- Glu266, His260-Asp329 and Lys272-Asp323) domains

Figure 1 displays the pH dependence of holo-Ecb2 and apo-Ecb2 stabilities; the denaturation temperature, Td, of the holo-protein increases by nearly 25 (cid:2)C at approximately pH 9 as a result of PLP binding. The difference in the Td 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-Ecb2 was similar to that of the native holo-Ecb2 bound to PLP (Fig. 1), indicating that the dissociation of PLP from holo-Ecb2 is reversible. These

Fig. 2. Schematic view of the holo-Ecb2 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].

Fig. 1. pH dependence of the thermal stability (Td) of holo-Ecb2 and apo-Ecb2. The Td value represents the peak temperature on the DSC curve measured at a scan rate of 1 (cid:2)CÆmin)1. Closed circles, open circles, and open triangles indicate the Td values for the native holo-, reconstituted holo- and apo-b2 subunits, respectively. Buffers used were 50 mM potassium phosphate supplemented with 1 mM EDTA at a pH of <8.5 and 50 mM Gly supplemented with 1 mM EDTA at pH 9.

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A

B

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].

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 b2 and holo-Ec b2

illustrate

conformational

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 Sta2b2 (Stb) is shown in Fig. 4. The secondary structures were assigned with numbering as referenced from Stb [5].

conformational

(peaks

(residues

domain

53–84

and

The overall topologies of the apo- and holo-Ecb structures were equivalent to that of the Sta2b2 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 DynDom 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 207–397) corresponding to the N and C domains of Stb, respec- tively. A rotation of 21.8(cid:2) 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 the C is predominantly a rigid body rotation of 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-

The differences in structures surrounding the active sites in apo-Ecb2 and holo-Ecb (shown in Fig. 5A,B changes the respectively) 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 1–5) were changes 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.

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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 DYNDOM 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.

Changes in the intersubunit interface of the b2 dimer as a result of PLP binding

side

Dimerization of the apo-Ecb and holo-Ecb subunits occurs along a twofold crystallographic axis (Fig. 2). Apo-Ecb2 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-Ecb2 and holo-Ecb2 were estimated at 1548 and 1711 A˚ 2, respectively. The buried area of the b2 subunit within Sta2b2 (PDB code 1BKS) is 1619 A˚ 2. The hydrophobicity of the contact area at the apo- Ecb2 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. in concert with chain of bA-Arg379, The bB-Arg379 from another subunit (bB), creates an arginine–arginine short-range interaction, which in

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A

SO42-

T190

K87

Helix-7

SO42-

N236

G234 N236

G234

H86

G232

S235

S377

Helix-9

SO42-

Helix-8

Helix-12

B

Helix-7

T190

SO42-

S235

K87 K87

NZ NZ

H86

OP3 OP3

G234

C4A C4A

OP4 OP4

P P

PLP

OP1 OP1

OP2 OP2

G232

N1N1

N1

S377

Helix-9

N236 Helix-8

long-loop

Helix-12

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].

results from local conformational changes caused by PLP binding.

large conformational changes

[23,27].

While the subunit–subunit interface did not change drastically between the crystal structures of dimeric apo-Ecb2 and holo-Ecb2, the crystal structures of sev- eral PLP-dependent b-elimination enzymes exhibit dif- ferent intersubunit interfaces for the apo-dimeric and In the apo-dimeric holo-dimeric structures 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 b2 subunit is very tight (Kd 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

protein–protein interactions is often an important fac- tor in stabilization and recognition [36]. This interac- tion induces in the empty bB subunit, facilitating the subsequent binding of PLP. The dissociation constant of PLP for the iso- lated b2 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 the bB-Asp381 side chain in bA-Arg379 contacts 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-Ecb2 when compared with the apo-Ecb2. These results indi- cate that the two b subunits of Ecb2, bA and bB, are more tightly associated in holo-Ecb2 than in apo-Ecb2; the stronger subunit–subunit association for holo-Ecb2

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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 Sta2b2 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 the holo-b2 subunit. PLP also forms a closure of 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.

Fig. 6. Stereo view of the conformational changes in the intersub- unit interface of the b2 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].

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 Ecb2 with both active sites bound to PLP. Kinetic and calorimetric studies of PLP binding to the apo-b2 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 b2 subunit of tryptophan synthase caused by formation of the a2b2 complex

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-Ecb2 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 Sta2b2 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.

We compared the structures of holo-Ecb2 with the b2 subunits of Sta2b2 by superimposition of equivalent Ca atoms (Fig. 7). The rmsd values for a comparison between holo-Ecb2 and Sta2b2 without ligands bound to either subunit active site (PDB code 1BKS), between holo-Ecb2 and Sta2b2 with a subunit ligands only (1A50), and between holo-Ecb2 and Sta2b2 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-Ecb2 superimposed well on that of the b2 subunit from an unliganded Sta2b2 complex (1BKS). The structures of both b2 subunits were simi- lar despite the absence of residues 291–292 from the Ecb2 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) of Ecb2 was higher than that of the entire molecule (59.06 A˚ 2). By con-

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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].

especially

trast, the B-factor of the long loop (26.50 A˚ 2) in the Sta2b2 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 a2b2 complex when compared with the b2 dimer as a result of the formation of several hydrogen bonds between the a and b subunits at their interface in the a2b2 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.

the lack of

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-Ecb2, around bAsp305 (Fig. 8). The side chains of bLys167 and bAsp305, in the unliganded conformers which are double Sta2b2, exhibit different conformations in the struc- tures bearing different ligands (Fig. 8A). The structure around bAsp305 of holo-Ecb2 is similar to the low- activity open state of Sta2b2. Holo-Ecb2 cannot trans- form into the higher-activity ‘closed-state’ from the ‘open-state’, resulting in a low-activity enzyme because 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

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-Ecb2 tunnel was larger by 2–3 A˚ and 3–4 A˚ than those of Sta2b2 in the absence of ligands (1BKS) and a-, b-liganded Sta2b2 (2TRS), respectively. The tunnel gating residues bTyr279 and bPhe280, which block the untimely passage of indole, were in a closed conformation in the holo-Ecb2 and the unli- ganded Sta2b2 (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 b2 subunit. In the holo-Ecb2, indole, the substrate, can directly approach the active site because the width of the cleft between the N and C domains in holo-Ecb2 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

The crystal structures of apo- and holo-Ecb2 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-Ecb2 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-Ecb2.

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A

B

Comparison of the apo- and holo-Ecb2 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(cid:2), 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-Ecb2, as determined by the DSC measurements, that was 25.3 (cid:2)C greater than that of apo-Ecb2 at pH 9.0.

C

Comparison of the holo-Ecb2 alone and Stb2 in Sta2b2 structures revealed why the holo-Ecb2 enzyme has lower b enzymatic activity in the absence of the a subunit than Eca2b2. (a) The indole gating residues, Tyr279 and Phe280, block entry of the substrate in the b2 subunit alone, a situation similar to that seen for the unliganded Sta2b2 complexed with Na+. (b) The structure around bAsp305 of holo-Ecb2 alone is similar to the low-activity open state of Sta2b2. Holo-Ecb2 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 Ecb2 tunnel was larger, by about 2–3 A˚ , than that of unliganded Stb2. Furthermore, the width of the cleft between the N and C domains in Ecb2 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-Ecb2

Fig. 8. Stereo views of the structures surrounding the allosteric residue, bAsp305, in holo-Ecb2 and the Sta2b2 complex. The sky- blue dotted lines in the ﬁgures represent hydrogen bonds. (A) Red, holo-Ecb2; gray, Sta2b2 (1BKS, both the a and b subunits are in a low-activity open conformation state). (B) Red, holo-Ecb2; gray, Sta2b2 (1A50, the a subunit is in a high-activity closed conformation state, while the b subunit is in the open state). (C) Red, holo-Ecb2; gray, Sta2b2 (2TRS, both a and b subunits are in closed states). Figures displaying protein structures were prepared using PYMOL software [59].

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Holo-Ecb2 [44] was puriﬁed as described previously [45]. The puriﬁed protein was visible as a single band on SDS ⁄ PAGE. Apo-Ecb2 was prepared by the dialysis of holo-Ecb2 against 0.1 m Mes buffer (pH 6.5) containing

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0.1 m Li2SO4, for 3 days at 4 (cid:2)C. The reconstitution of holo-Ecb2 was performed by dialyzing the apo-Ecb2 solu- tion against 50 mm potassium phosphate buffer (pH 7.0) containing 0.2 mm PLP, for 1 day at 4 (cid:2)C.

bration of a mixture containing 1 lL of protein and 1 lL of crystallization reagent (0.25 m Li2SO4, 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.

Physicochemical properties of holo-Ecb2 and apo-Ecb2, 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.

Apo-protein crystals were soaked in cryoprotectant (0.1 m Mes buffer, pH 6.5, containing 0.4 m Li2SO4 and 30% glyc- erol), while holo-protein crystals were soaked in 0.1 m Tris buffer (pH 8.0) containing 0.8 m (NH4)2SO4 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 P4322, 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 (VM) of 3.7 A˚ 3 Da)1 [49]. The crystal of the holo-form belonged to space group P6522, 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 (VM) of 4.1 A˚ 3 Da)1. The statistics for data collection are summarized in Table 1. DSC was performed using a differential scanning micro- calorimeter, VP-DSC (Microcal, Inc., Northampton, MA, USA), at a scan rate of 1 (cid:2)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.

Structure determination and reﬁnement

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 (cid:2)C. Before measurements, the apo- and holo-Ecb2 solutions were dialyzed overnight at 4 (cid:2)C against 50 mm potassium phosphate buffer, pH 7.9, containing 1 mm EDTA, and the holo-Ecb2 solution was dialyzed overnight at 4 (cid:2)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 cm3Æg)1 [46]. Analysis of the sedimentation equilibrium data was per- formed using xlavel software (Beckman version 2.0).

Crystallization and data collection

The apo- and holo-structures were determined by the molecular replacement method using cns software [50]. The b subunit of the Sta2b2 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 b2 subunit from E. coli into the Protein Data Bank.

Analyses of amino acid sequences and protein models

For crystallization, puriﬁed holo-Ecb2 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-Ecb2 was crystallized using the microbatch method with parafﬁn oil at 288K. Crystals were obtained with 0.5 lL of protein solution [0.8 m combined with 0.5 lL of crystallization reagent (NH4)2SO4, 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.

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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-Ecb2 was crystallized using the hanging-drop vapor- diffusion method at 288K. Crystals were obtained by equili-

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Table 1. Data collection and reﬁnement statistics.

Apo-b2 subunit

Holo-b2 subunit

Crystal data statistics

X-ray source Detector Wavelength (A˚ ) Crystal-to-detector distance (mm) Exposure time (s) Data-collection temperature (K) No. of crystals per images Space group Unit-cell parameters, a, b, c (A˚ ) Mosaicity Resolution range (A˚ )a No. of total reﬂectionsa No. of unique reﬂectionsa a Completeness (%)a Rmerge (%)a Redundanciesa No. of molecules per asymmetric unit )1) VM (A˚ 3 Da Wilson plot B factor (A˚ 2)

SPring-8 BL44XU DIP 6040 0.9 520 10 100 2 ⁄ 66 P4322 110.89, 110.89, 102.21 0.47 46.41–3.00 (3.16–3.00) 69 323(10 086) 13 266 (1887) 6.8 (1.9) 99.9 (100.0) 7.4 (38.9) 5.2 (5.3) 1 3.7 85.4

SPring-8 BL44XU DIP 6040 0.9 480 10 100 1 ⁄ 38 P6522 172.52, 172.52, 82.59 0.26 74.40–2.90 (3.06–2.90) 74 984 (10 943) 15 920 (2297) 6.6 (1.9) 96.8 (97.4) 7.6 (39.0) 4.7 (4.8) 1 4.1 86.3

Reﬁnement statistics

42.82–3.00 (3.19–3.00) 13 244 (2159) 20.6 (31.4) 25.4 (36.1) 0.012 1.7

30.33–2.90 (3.08–2.90) 15 901 (2630) 19.6 (28.5) 24.4 (33.0) 0.009 1.6

Resolution range (A˚ )a No. of reﬂectionsa Rcryst (%)a,b Rfree (%)a,c rmsd, bonds (A˚ ) rmsd, angles ((cid:2)) Ramachandran plot (%)

Most favored Additional allowed Generously allowed Disallowed

90.5 9.5 0 0

88.8 11.2 0 0

No. of atoms or molecules

2514 0 3 0 23

2988 1 2 1 9

Protein atoms PLP molecules Sulfate ions Glycerol molecules Water molecules Average B-factor (A˚ 2)

All atoms Protein atoms Protein main-chain atoms PLP Sulfate ions Glycerol molecules Water molecules

62.3 62.2 61.5 – 98.8 – 46

60.3 59.8 59.1 42.7 84 57.2 48.1

a Values in parentheses are for the highest resolution shell. b Rcryst was calculated from the working set (95% of the data). c Rfree was calcu- lated from the test set (5% of the data).

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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].

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Acknowledgements

subunit communication in tryptophan synthase. Biochemistry 37, 5394–5406. 12 Weyand M & Schlichting I (1999) Crystal structure of

wild-type tryptophan synthase complexed with the natu- ral substrate indole-3-glycerol phosphate. Biochemistry 38, 16469–16480. 13 Sachpatzidis A, Dealwis C, Lubetsky JB, Liang PH,

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.).

Anderson KS & Lolis E (1999) Crystallographic studies of phosphonate-based a-reaction transition-state ana- logues complexed to tryptophan synthase. Biochemistry 38, 12665–12674.

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