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 thaliana and AK from Methanococcus jannaschii (MjAK) are homo-oligomers of identical subunits [4–6]. AK of the a 2 b 2 type is encoded by in-frame overlapping genes, so that the amino acid sequence of the b-subunit is identical to about 160 amino acids of the C-terminus of the a-subunit. As seen in other enzymes involved in the first step in amino acid bio- synthesis, AK is regulated through feedback inhibition Keywords ACT domain; allosteric regulation; crystal structure; thermostability; threonine biosynthesis Correspondence M. Nishiyama, Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan Fax: +81 3 5841 8030 Tel: +81 3 5841 3074 E-mail: umanis@mail.ecc.u-tokyo.ac.jp (Received 3 November 2008, revised 10 March 2009, Accepted 31 March 2009) doi:10.1111/j.1742-4658.2009.07030.x Crystal structures of the regulatory subunit of Thr-sensitive aspartate kinase (AK; EC 2.7.2.4) from Thermus thermophilus (TtAKb) were deter- mined at 2.15 A ˚ 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 kinase from Thermus thermophilus; TtAKb, regulatory subunit of aspartate kinase from Thermus thermophilus; TtAKb-free, Thr-free regulatory subunit of aspartate kinase from Thermus thermophilus; TtAKb-Thr, Thr-bound regulatory subunit of aspartate kinase from Thermus thermophilus. 3124 FEBS Journal 276 (2009) 3124–3136 ª 2009 The Authors Journal compilation ª 2009 FEBS by end-products. CgAK is regulated through concerted inhibition by Lys and Thr [7], whereas TtAK, which is involved in the biosynthesis of Thr and Met but not of Lys, because T. thermophilus synthesizes Lys through a-aminoadipate as an intermediate [8,9], is Thr-sensitive [1]. In a 2 b 2 -type AK, the N-terminal regions of the a-subunits serves as catalytic domains, and the C-terminal regions of the a-subunits and the b-subunits act as regulatory domains [10,11]. The regulatory domains of AK contain conserved motifs named ACT domains that are found among many allosteric enzymes involved in amino acid and purine biosynthesis [12,13]. The motif has a babbab fold, and serves as a small molecule-binding domain for allosteric regulation. Several crystal structures have been determined for enzymes containing ACT domains; however, the mode of association between ACT domains is quite different among the enzymes, as summarized by Grant [14]. For example, the archetypical ACT domain association with two side- by-side domains, each from a different chain, is found in 3-phosphoglycerate dehydrogenase [15]. In 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- larly and face-to-face, as in CgAKb. In homo- oligomeric AKs, only one of the eight-stranded b-sheets is involved in effector binding. Determination of the crystal structure of the regulatory subunit of TtAK (TtAKb) would provide information not only on the catalytic mechanism but also on the structural features common to a 2 b 2 -type AKs. As T. thermophilus is an extremely thermophilic bac- terium, proteins produced by T. thermophilus have high thermostability. Previously, we found that chime- ric AK, named BTT, which is composed of a catalytic domain from BsAKII and regulatory domains (a regulatory domain in the a-subunit, and a b-subunit with the same sequence as the regulatory domain) from TtAK, improved thermostability as much as wild-type TtAK [11]. This result indicated that the regulatory domain of TtAK contributes not only to catalytic regulation, but also the thermostability of TtAK. Comparison of the crystal structures of TtAKb and CgAKb was expected to elucidate the mechanism of the elevated thermostability of TtAKb. In this article, we describe the crystal structures of TtAKb in two forms, Thr-bound and Thr-free, and discuss the regulatory mechanism of Thr and the struc- tural features responsible for the high thermostability of TtAKb. 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 and C, and residues 1–5 in chains E–G) and C-termi- nal (residues 158–161 in chains A–F, and residues 159–161 in chain B) portions of the structure are not 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 3125 seen on the map. The electron densities of the sections (residues 54–56 in chains A and D, residues 53–59 in chain B, residues 55–56 in chain C, residues 56–57 in chain E, and residues 53–56 in chain F) of the loop between b3 and b4 are not observed in every chain. The overall geometry of the model is good, with 89.0% of the residues in the most favored regions, 10.0% in allowed regions, 0.7% in generously allowed regions and 0.3% in disallowed regions from pro- check. Table 1 summarizes the refinement statistics. Overall structure The crystal structure of TtAKb-Thr was determined as a homodimer (Fig. 1A). As TtAK is a heterotetramer with an a 2 b 2 configuration, where the b-subunit is identical to the C-terminal portion of the a-subunit, as in CgAK, the dimeric structure revealed in this study represents the structure of the regulatory region of an ab-heterodimer. The rmsd is 0.50 A ˚ between two monomers in the asymmetric unit. Structural differ- 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 domains forming an effector-binding unit of the front of the dimer are indicated. Site 1 and site 2 of the effector-binding unit on the front are indicated by solid and dotted cir- cles. (B) Superposition of two monomers in a dimer of TtAKb-Thr. ACT domains are shown by dotted circles. Regions showing structural differences between monomers are indicated by solid circles. Fig. 2. Overall structure of TtAKb-free. (A) Three TtAKb-free dimers in the asymmetric unit. A chain, magenta; B chain, yellow; C chain, cyan; D chain, green; E chain, brown; F chain, blue. (B) EF chain dimer. Crystal structures of regulatory subunit of Thr-sensitive aspartate kinase A. Yoshida et al. 3126 FEBS Journal 276 (2009) 3124–3136 ª 2009 The Authors Journal compilation ª 2009 FEBS effector-binding unit binds two effectors. The other possible effector-binding sites (site 2) in the structural unit are vacant in the TtAKb-Thr structure. The structure of TtAKb-free is also determined to be a homodimer (Fig. 2), although TtAKb was eluted in volumes close to that of a molecular mass of mono- mer in the absence of Thr in gel filtration chromatog- raphy, as described below. The TtAKb-free crystal contains three dimers in the asymmetric unit, and rmsd values for Ca among the three dimers are 0.58 A ˚ between AB and CD dimers, 0.63 A ˚ between CD and 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; asterisks denote residues from another chain), and Table 1. Data collection and refinement statistics. SeMet-TtAKb-Thr Native Peak Edge Remote TtAKb-Thr TtAKb-free Data collection X-ray source PF-NW12 PF-NW12 PF-NW12 PF-NW12 PF-NW12 Wavelength (A ˚ ) 0.9792 0.9794 0.9630 1.000 1.000 Space group P4 3 32 P4 3 32 P4 3 32 P4 3 32 P3 1 Resolution (A ˚ ) a 2.40 (2.49–2.40) 2.40 (2.49–2.40) 2.40 (2.49–2.40) 2.15 (2.19–2.15) 2.98 (3.09–2.98) Reflections (total ⁄ unique) 364 163 ⁄ 19 507 365 251 ⁄ 19 539 364 474 ⁄ 19 539 578 149 ⁄ 27 145 133 032 ⁄ 22 878 R sym b (%) 9.5 (46.5) 9.5 (46.3) 9.7 (51.5) 6.7 (33.2) 9.2 (37.1) I ⁄ r(I) 30.8 (6.0) 30.2 (5.8) 29.5 (5.5) 59.9 (11.1) 21.0 (3.2) 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 Additionally favored (%) 4.6 10.0 Generously allowed (%) 0 0.7 Disallowed (%) 0 0.3 a Values in parentheses are data of the highest-resolution shell. b R sym ¼ RjI i À <I>j=R<I>. c Figure of merit (FOM) was calculated with the SOLVE program. d R - factor ¼ R hkl F o jjÀF c jjjj=R hkl F o jj. e Calculated using PROCHECK. 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 3127 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- free. Residues in blue are in the E chain, and residues in orange are in the F chain. (D) Structure-based sequence alignment of TtAKb and CgAKb. Alignment was performed with CLUSTALW [42], and alignment with secondary structures of TtAKb and CgAKb was performed with ESPRIPT [43]. Regions for ACT1 and ACT2 are shown by solid and broken divergent arrows, respectively. Crystal structures of regulatory subunit of Thr-sensitive aspartate kinase A. Yoshida et al. 3128 FEBS Journal 276 (2009) 3124–3136 ª 2009 The Authors Journal compilation ª 2009 FEBS sedimentation equilibrium by analytical ultracentri- fugation, and gel filtration chromatography. In gel filtration, TtAKb was eluted in a volume corresponding to a molecular mass of 21.7 kDa in the absence of Thr (Fig. 4A). The molecular mass, esti- mated by gel filtration, is a little larger than the calcu- lated mass of a monomer (17.7 kDa). When gel filtration was performed in the presence of 5 mm Thr, elution profiles gave an estimated molecular mass of 30.9 kDa. To further confirm that Thr affects monomer–dimer equilibrium even in TtAKb, we also analyzed the sub- unit arrangement by sedimentation equilibrium. The data fitted well with the monomer–dimer equilibrium, with equilibrium constants of 5.6 · 10 )3 m )1 (goodness of fit = 4.0 · 10 )4 ) and 9.1 · 10 )4 m )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 between a-subunits and b-subunits is stronger in TtAK than in CgAK; however, even TtAK showed sharp and broad elution profiles in gel filtration in the pres- ence and absence of Thr, respectively. SDS⁄ PAGE of the fractions in gel filtration showed that a-subunits and b-subunits are eluted in the same volumes from the column in the presence of Thr, whereas b-subunits are eluted from the column later than a-subunits in the absence of Thr (Fig. 4B–E). From these results, we conclude that b-subunits can interact with the regula- tory domains of a-subunits even without Thr, but the interaction is tighter in the presence of Thr. Fig. 4. Stabilization of oligomer formation of TtAK and TtAKb by Thr. (A) Elution profiles of TtAKb in the presence and absence of 5m M Thr. The solid line with circles and the dotted line with squares indicate profiles in the presence and absence of 5 m M Thr, respectively. Elution volumes for BSA (67 kDa), chymotrypsinogen A (43 kDa), ovalbumin (25 kDa) and ribonuclease A (13 kDa) are indicated by a, b, c, and d, respectively. (B, C) SDS ⁄ PAGE of each frac- tion by gel filtration for TtAK in the presence (B) and absence (C) of 5 m M Thr. Elution profiles of TtAK were quantitated by IMAGEJ [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 manner by both Lys and Thr binds two Thr molecules at site 1, like TtAKb-Thr, and easily dissociates into monomers in the absence of Thr [20]. Therefore, a sim- ilar conformational change is expected for these two enzymes, depending on the presence or absence of Thr. In CgAK, mutations of the residues in the b3–b4 loop close to site 1 induced resistance to Lys or a Lys ana- log, S-2-aminoethyl-l-cysteine [20]. As Lys is bound to a vacant effector-binding site (site 2) in the effector- binding unit composed of the ACT1 and ACT2 Fig. 5. Comparison of a single effector-binding unit between TtAKb-Thr and TtAKb-free. (A) Superposition of the effector-binding units of TtAKb-Thr and TtAKb-free. ACT1 displays the Ca models of the B chain (residues 15–93) from TtAKb-Thr and the F chain (residues 15–93) from TtAKb-free, and ACT2 shows Ca models of the A chain (residues 5–14 and 94–157) from TtAKb-Thr and the E chain (residues 6–14 and 94–157) from TtAKb-free. TtAKb-Thr and TtAKb-free are in blue and red, respectively. The Thr molecule is shown as a stick model. The loop between b4 and a2 corresponding to the latch loop in EcAKIII is shown as a dotted oval. (B) Movement of Ca atoms caused by Thr binding mapped on the effector-binding unit of TtAKb-Thr. Cyan, < 1 A ˚ ; green, < 2 A ˚ ; yellow, < 3 A ˚ ; orange, < 4 A ˚ ; red, > 4 A ˚ . Regions showing larger movement are marked as A–E. The loop between b4 and a2 corresponding to the latch loop in EcAKIII is shown as a dotted oval. (C) Ca distance between TtAKb-Thr and TtAKb-free. Blue indicates the distance between the A chain from TtAKb-Thr and the E chain from TtAKb-free, and red indicates the distance between the B chain from TtAKb-Thr and the F chain from TtAKb-free. The regions shown in A–E are: A, 42–45; B, 46–50; C, 51–58; D, 102–110; E, 131–134. (D) Domain motion in TtAKb caused by Thr binding. The structures of 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 observations suggest that the Pro-Gly motif functions as a hinge to facilitate conformational change upon effector binding in a 2 b 2 -type AKs. In TtAKb, on the other hand, the corresponding section (positions 109 and 110) has a Pro-Glu sequence (Fig. 3D). Although both dihedral angles shown by Gly29 in the presence and absence of Thr are within the permissible range, an allowed or generously allowed region on the Rama- chandran plot, for Glu in general, such a marked change upon effector binding would not be expected for Glu. At present, we cannot judge whether the sec- ond Thr is bound to site 2 for catalytic control of TtAK. In order to further clarify the regulatory mech- anism of TtAK by Thr, the crystal structure of full- length TtAK in the a 2 b 2 form is obviously required. It should be noted that, on comparison of the amino acid sequences of CgAK and TtAK, CgAK had an extra 11 residues at the C-terminus, forming b9, con- sisting of a b-sheet with a b1-strand at the N-terminus (Fig. 5D). As TtAK, which is only inhibited by Thr, does not have this extra b-strand, the b-strand may be involved in a process of concerted inhibition by Thr 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 (Fig. 5A,B), similar to Thr-sensitive MjAK. In MjAK, Thr binding causes the entire regulatory domain to rotate 6.5 away from the fixed kinase domain, resulting in opening of the catalytic site. In this case, the entire domain moves as a rigid body with no significant change in the interaction between ACT domains [22]. In contrast, Thr binding causes the ACT2 domain to rotate by 12° from the fixed ACT1 domain (Fig. 5D), which is presumed to be the motion that closes the active site of TtAK. Thus, the direction of domain motion is different between TtAK and MjAK, suggesting a different inhibitory mecha- nism in TtAK. Isothermal titration calorimetry suggests that MjAK has not only Thr-binding sites with high affinity in the regulatory domain, but also five weak Thr-binding sites per dimer, which may include a Thr bound near the Asp-binding site and those bound on the protein surface nonspecifically [22]. As we have not yet determined the crystal structure of TtAK in the a 2 b 2 form, we do not know whether TtAK also contains weak Thr-binding sites. However, it should be noted that TtAK has a K i value of less than 10 lm for Thr, which is markedly lower than that of MjAK (0.3 mm) [19]. The low K 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 dimer formation caused by Thr. Similar protein stabil- ization via oligomer formation has been shown for a thermostable homoisocitrate dehydrogenase [23]. In the crystal, the contact surface area in the dimer inter- face is larger in TtAKb-Thr (3890 A ˚ 2 ) than in TtAKb- free (3200 A ˚ 2 ), indicating that Thr binding tightens the interaction of the two chains. Many factors are involved in protein stability, such as hydrophobic interactions [24], hydrogen and ionic bonds [25], cavity volume [26], and other entropic fac- tors [27]. Proteins are generally stabilized by a combi- nation of these factors [28]. Among them, an increased number of hydrogen bonds (ionic interactions) and better internal packing are reported to be the most important protein-stabilizing factors [29,30]. To under- stand the difference in thermostability between CgAKb and TtAKb, we compared the crystal structures of the two proteins. When the numbers of ionic bonds and hydrogen bonds are compared, unexpectedly, both numbers are larger in CgAKb than in TtAKb (Table 3). In contrast, when cavity volumes were calcu- lated from the crystal structure of TtAKb and CgAKb, the volume of TtAKb was smaller than that of CgAKb (Table 4), suggesting that TtAKb is more tightly 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 Table 2. Denaturation temperatures of TtAKb and CgAKb. T m (°C) No additive 5 m M Thr CgAKb 50.9 55.2 TtAKb 91.7 96.1 Table 3. Thermostabilization factors. Numbers of hydrogen bonds and ionic bonds. Data in parentheses are number of bonds between subunits. TtAKb CgAKb Hydrogen bonds 237 (12) 262 (11) Ionic bonds 22 (2) 78 (16) <3A ˚ 3 (0) 14 (2) <4A ˚ 8 (1) 27 (5) <5A ˚ 11 (1) 37 (9) Table 4. Thermostabilization factors. Accessible surface area and cavity. Data in parentheses are values per amino acid residue. TtAKb CgAKbD(Tt)Cg) Difference in monomer ASA value (D)N) Hydrophobic (A ˚ 2 ) 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 collection for SeMet-substituted TtAKb-Thr has been previously reported [20,31]. Before data collection for TtAKb-free, a crystal was soaked briefly in cryoprotec- tant solution of 25% (v ⁄ v) glycerol in reservoir solution, flash-cooled in a nitrogen gas stream at 95 K, and stored in liquid nitrogen. Diffraction data were collected with a CCD camera on the beamline NW12 of the Photon Factory AR [High Energy Accelerator Research Organi- zation (KEK), Tsukuba, Japan]. Data on TtAKb-free crystals were recorded at 2.98 A ˚ resolution. Diffraction data were indexed, integrated and scaled using the hkl2000 program suite [32]. Structure determination and refinement The structure of TtAKb-Thr was determined by the MAD phasing method. The detailed structure determination and refinement of TtAKb-Thr were as previously described [20]. TtAKb-free crystals have three dimers per asymmetric unit, and belong to the space group P3 1 , with unit cell parame- ters of a = b = 107.2 A ˚ , c = 87.22 A ˚ , a = b =90°, and c = 120°. The structure determination for TtAKb-free by molecular replacement was performed by molrep [33] in the ccp4 program suite [34], using the model of TtAKb- 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 Phe 5 3.11 4 2.33 Trp 0 0 1 0.58 Pro 9 5.59 3 1.74 Neutral 21 13.0 34 19.8 Ser 6 3.73 10 5.81 Thr 5 3.11 11 6.40 Asn 1 0.62 6 3.49 Gln 9 5.59 6 3.49 Cys 0 0 1 0.58 Hydrophilic 38 23.6 50 29.1 Asp 9 5.59 14 8.14 Glu 13 8.07 15 8.72 Lys 7 4.35 8 4.65 His 3 1.86 2 1.16 Arg 5 3.11 9 5.23 Tyr 1 0.62 2 1.16 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 3133 [...]... mechanism of TtAKb, we investigated the factors contributing to the stabilization of two proteins, TtAKb-Thr and Thr-bound CgAKb, using crystal structures The cavity volumes of the proteins were calculated by the voidoo program [38] The hbplus program [39] was used to analyze hydrogen bonds in the crystal structures To estimate the changes in Gibbs free energy (DG) of TtAKb and CgAKb, we calculated the solvent-accessible... Analysis of protein conformational characteristics related to thermostability Protein Eng 9, 265–271 30 Vogt G & Argos P (1997) Protein thermal stability: hydrogen bonds or internal packing? 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Thermus thermophilus; TtAKb-free, Thr-free regulatory subunit of aspartate kinase from Thermus thermophilus; TtAKb-Thr, Thr-bound regulatory subunit of aspartate. 2009) doi:10.1111/j.1742-4658.2009.07030.x Crystal structures of the regulatory subunit of Thr-sensitive aspartate kinase (AK; EC 2.7.2.4) from Thermus thermophilus (TtAKb)