The crystal structure of the tryptophan synthase b 2 subunit from the hyperthermophile Pyrococcus furiosus Investigation of stabilization factors Yusaku Hioki 1,2 , Kyoko Ogasahara 1 , Soo Jae Lee 1 , Jichun Ma 1 , Masami Ishida 3 , Yuriko Yamagata 4 , Yoshiki Matsuura 1 , Motonori Ota 5 , Mitsunori Ikeguchi 6 , Seiki Kuramitsu 2 and Katsuhide Yutani 7,8 1 Institute for Protein Research, Osaka University, Japan; 2 Department of Biology, Graduate School of Science, Osaka University, Japan; 3 Tokyo University Marine Science and Technology, Japan; 4 Graduate School of Pharmaceutical Sciences, Kumamoto University, Japan; 5 Global Scientific Information and Computing Center, Tokyo Institute of Technology, Japan; 6 Graduate School of Integrated Science, Yokohama City University, Japan; 7 Kwansei Gakuin University, Graduate School of Sciences, Hyogo, Japan; 8 RIKEN Harima Institute, HTPF, Hyogo, Japan The structure of the tryptophan synthase b 2 subunit (Pfb 2 ) from the hyperthermophile, Pyrococcus furiosus,wasdeter- mined by X-ray crystallographic analysis at 2.2 A ˚ resolu- tion, and its stability was examined by DSC. This is the first report of the X-ray structure of the tryptophan synthase b 2 subunit alone, although the structure of the tryptophan synthase a 2 b 2 complex from Salmonella typhimurium has already been reported. The structure of Pfb 2 was essentially similartothatoftheb 2 subunit (Stb 2 )inthea 2 b 2 complex from S. typhimurium. The sequence alignment with secon- dary structures of Pfb and Stb in monomeric form showed that six residues in the N-terminal region and three residues in the C-terminal region were deleted in Pfb, and one residue at Pro366 of Stb and at Ile63 of Pfb was inserted. The denaturation temperature of Pfb 2 was higher by 35 °Cthan the reported values from mesophiles at  pH 8. On the basis of structural information on both proteins, the analyses of the contributions of each stabilization factor indicate that: (a) the higher stability of Pfb 2 is not caused by either a hydrophobic interaction or an increase in ion pairs; (b) the number of hydrogen bonds involved in the main chains of Pfb is greater by about 10% than that of Stb, indicating that the secondary structures of Pfb aremorestabilizedthan those of Stb and (c) the sequence of Pfb seems to be better fitted to an ideally stable structure than that of Stb,as assessed from X-ray structure data. Keywords: calorimetry; crystal structure; hyperthermophile; tryptophan synthase b 2 subunit; stability. Prokaryotic tryptophan synthase (EC 4.2.1.20) is an a 2 b 2 complex composed of nonidentical a and b subunits [1,2]. The a 2 b 2 complex with an abba arrangement [3] can be isolated as the a monomer and b 2 subunits. The a and b 2 subunits catalyse inherent reactions, termed the a and b reactions (Eqns 1 and 2), respectively. The physiologically important reaction catalysed by the a 2 b 2 complex, termed the ab reaction (Eqn 3), is the sum of the a and b reactions: a reaction indole-3-glycerol phosphate $ indole þ d-glyceraldehyde 3-phosphate ð1Þ b reaction l-serine þ indole ! l-tryptophan þ H 2 O ð2Þ ab reaction l-serine þ indole 3-glycerol phosphate ! l-tryptophan þ d-glyceraldehyde 3-phosphate þ H 2 O ð3Þ When the a and b 2 subunits associate to form the a 2 b 2 complex, the enzymatic activity of each subunit is syn- chronically enhanced by one to two orders of magnitude [2]. The tryptophan synthase is a typical allosteric enzyme whose activity is affected by the ligands [3–6]. Prokaryotic tryptophan synthase has been studied extensively as an excellent model system for investigating protein–protein interaction mechanisms [2,7–10]. In order to elucidate the structural basis of the subunit communication and mutual activation of the functions of each subunit resulting from the formation of the a 2 b 2 complex, it is necessary to determine the three-dimensional Correspondence to K. Yutani, RIKEN Harima Institute, HTPF, Kouto, Mikazuki-cho, Sayo-gun, Hyogo 679-5148, Japan. Fax: +81 791 58 2917, Tel.: +81 791 58 2937, E-mail: yutani@spring8.or.jp Abbreviations: ASA, accessible surface area; Eca, tryptophan synthase a subunit from Escherichia coli; Ecb 2 , tryptophan synthase b 2 subunit from E. coli; Pfa, tryptophan synthase a subunit from Pyrococcus furiosus; Pfb 2 ,tryptophansynthaseb 2 subunit from P. furiosus; Pfb, monomer of tryptophan synthase b 2 subunit from P. furiosus; PLP, pyridoxal 5¢-phosphate; Sta, tryptophan synthase a subunit from Salmonella typhimurium; Stb 2 , tryptophan synthase b 2 subunit from S. typhimurium; Stb, monomer of tryptophan synthase b 2 subunit from S. typhimurium; RMSD, root mean square deviation. Enzymes: prokaryotic tryptophan synthase (EC 4.2.1.20). (Received 21 January 2004, revised 25 March 2004, accepted 28 April 2004) Eur. J. Biochem. 271, 2624–2635 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04191.x structures of the a or b 2 subunits alone as well as that of the complex. The three-dimensional structure of the tryptophan synthase a 2 b 2 complex from Salmonella typhimurium was determined by X-ray analysis in 1988 [3]. However, the determination of the structure of the a or b 2 subunit alone has not yet succeeded, although much effort has expended on obtaining good quality crystals of the subunits from mesophiles. Recently, the structure of the a subunit alone of tryptophan synthase from a hyperthermophile, Pyrococcus furiosus, was determined by X-ray analysis [11]. In this report we describe the crystal structure of the b 2 subunit alone of tryptophan synthase from P. furiosus. Proteins from hyperthermophiles are remarkably stable compared with homologous proteins from mesophiles [12,13]. Three-dimensional structures of many proteins from hyperthermophiles have been analysed to determine the structural bases of unusually high stability [14–17]. Structural features of hyperthermophile proteins compared with their mesophilic homologues vary depending on the individual proteins. Hydrophilic factors such as ion pairs and hydrogen bonds are superior in some proteins [11,14,18–23], and hydrophobic interaction is favoured in others [24,25]. The internal cavity decreases in hyperthermo- phile proteins [25]. An entropic effect has been reported to be important for enhanced stability [11]. However, the cause of the extremely high stabilization of proteins from hyperthermophiles still remains unclear. Elucidating the structural basis of the ultra-thermostability of proteins is an important for understanding protein folding problems, aspects of biotechnological applications, and progress in structural genomics. Using mutant human lysozymes Funahashi et al. [26,27] have proposed the parameters of various stabilization factors estimated by a unique equation, considering the relationship between stability and conformational changes due to the mutations. Using these parameters, the stabilization mechanism of pyrrolidone carboxyl peptidase from P. furiosus has been elucidated on the basis of its X-ray structure [17]. In this report, the stabilization mechanism of the hyperthermophilc b 2 subunit will be discussed on the basis of the crystal structures, compared with the structural features of the hyperthermophile and mesophile proteins. Materials and methods Purification of proteins The b 2 subunit of tryptophan synthase from P. furiosus (Pfb 2 ) was overproduced in Escherichia coli strain JM109 (pb1837) [28]. Pfb 2 and the a-subunit of tryptophan synthase from P. furiosus (Pfa) were purified as described [29,11]. The equivalent subunits from E. coli (Eca, Ecb 2 ) were purified also [10,30,31]. All of the purified proteins showedasinglebandonSDS/PAGE. The protein concentrations were determined from the absorbance at 278.5 nm using A 1% 1cm ¼ 6.92 for Pfa and 10.18 for Pfb 2 [29], 4.4 for Eca [32] and 6.5 for Ecb 2 [33]. Enzymatic activity assay The b activity was measured by the disappearance of indole using a phenol reagent [1] instead of the direct spectropho- tometric assay ordinarily used [33], because temperature control of the spectrophotometer was difficult above 80 °C. The assay was carried out in the presence of a 3 : 1 molar excess of the a subunit over the b subunit monomer. One unit of activity is defined by the formation of 0.1 lmol of product in 20 min at the indicated temperature [33]. DSC DSC was carried out using an adiabatic differential microcalorimeter, VP-DSC (Microcal) at a scan rate of 1 °CÆmin )1 . Before making measurements, the protein solution was dialysed against buffer with the composition 10 m M Gly/KOH, 1 m M EDTA, 0.02 m M pyridoxal 5¢-phosphate (PLP) (as described in Fig. 1). The dialysed sample was filtered through a 0.22-lm pore size membrane and then degassed in a vacuum. Protein concentrations during the measurements were 0.5–1.5 mgÆmL )1 . Protein crystallization and data collection The crystals were grown by a hanging drop vapour diffusion at 10 °C, by mixing 2 lL of the protein solution with 2 lL of a reservoir solution containing 12% (w/v) PEG 20 000 and 100 m M Mes, pH 6.5. The concentration of Pfb 2 was 10–12 mgÆmL )1 in 20 m M Tris/HCl pH 8.5 containing 100 l M dithioerythritol and 20 l M PLP. Diffraction experiments with the Pfb 2 crystal were performed at the beam line, BL44XU and BL411XU at SPring8. The crystal belonged to the orthorhombic space group of P2 1 2 1 2 1 with unit cell dimensions of a ¼ 84.8, b ¼ 110.5, c ¼ 160.0 A ˚ . The value of the Matthews coefficient is 2.2 A ˚ 3 ÆDa )1 for two Pfb 2 per asymmetric unit, correspond- ing to a solvent content of 44.0%. The crystals were flash- cooled in a cold nitrogen gas stream immediately after cryoprotection by addition of the reservoir solution con- taining 25% (w/v) glycerol to the crystallization buffer at Fig. 1. pH dependence of the denaturation temperature of Pf b 2 . The denaturation temperature, T d , represents the peak temperatures of DSC curves observed at a scan rate of 1 °CÆmin )1 . d, s and m represent Pfb 2 , Ecb 2 ,andStb 2 , respectively. The buffer conditions were 10 m M Gly-KOH with 1 m M EDTA and 0.02 m M PLP. The pH indicates the values after DSC measurements. The data for Ecb 2 and Stb 2 are those reported in [29] and [35] respectively. Ó FEBS 2004 Crystal structure of tryptophan synthase b 2 subunit alone (Eur. J. Biochem. 271) 2625 100 K. This crystal diffracted to a maximum of 2.2 A ˚ and was suitable for structure determination. The data collected were processed and integrated by DENZO andscaledby SCALEPACK [34]. Data collection statistics are summarized in Table 1. Structure determination and refinement The dimeric structure (Stb 2 )oftheb subunit in the tryptophan synthase a 2 b 2 complex (Sta 2 b 2 )from S. typhimurium (1BKS) [3] provided the initial model for molecular replacement solutions using AMORE . The cross- rotation function showed two peaks for the two-dimer molecules. The model was subjected to cycles of rigid body refinement using noncrystallographic symmetry (NCS): the four b subunit molecules in the asymmetric unit were refined using NCS restraints. The experimental map at 2.2 A ˚ was of high quality and allowed unambiguous modelling of all residues 1–388. The model was built using O and refined by energy minimization, simulated annealing and restrained B-factor refinement procedures with NCS. Successive refinement with temperature factors and addition of solvents resulted in an R-value of 22.0% and an R free of 26.4% for all reflections in the resolution range 100–2.2 A ˚ . R free was calculated with 10% of the reflections. The current model consists of four chains of residues 1–388 of Pfb and 193 water molecules per asymmetric unit. All residues are within the most favoured (89.7%) and additional allowed regions (10.3%) of the Ramachandran plot. Refinement statistics are summarized in Table 1. The final coordinates have been deposited in the Protein Data Bank (PDB accession no. 1V8Z). Results Thermal stability and enzymatic activity of Pf b 2 Figure 1 shows the pH dependence of the denaturation temperatures of Pfb 2 measured by DSC. The heat denatur- ation of Pfb 2 was not reversible. The peak temperatures of the DSC curves above pH 6.5 were around 115 °C independent of pH, which were higher by about 35 °Cthan those reported for mesophilic proteins [29,35]. DSC meas- urements could not be carried out between pH 6 and 4, because the protein became turbid on heating. Below pH 4, the denaturation temperatures decreased markedly. Ultra- centrifugation analysis of Pfb 2 indicates that the apparent molecular weight of the protein, which exists in a dimeric form in solution around pH 7, decreases with decreasing pH below 4.0, resulting in dissociation to a monomer at pH 3.0 [29]. This suggests that the decreased denaturation temperature below pH 4.0 is correlated with the dissociation from a dimer to a monomer. The mesophilic protein of E. coli (Ecb 2 )wasdenaturedintheacidicregion. The enzymatic activities of Pfb 2 and Ecb 2 were measured at various temperatures in the presence of excess a subunit from P. furiosus or E. coli (Fig. 2B). The activity for Ecb 2 rapidly decreased at temperatures above 55 °C. This decrease might be due to thermal denaturation of Eca in a 2 b 2 complex, because the denaturation temperature of Eca is around 55 °C, although Ecb 2 denatures at 80 °C [29]. It has also been reported that Sta in the complex is inactivated by 50% at 55 °C, whereas 50% inactivation of Stb 2 occurs at 80 °C [4]. The activity of Pfb 2 at the physiological temperature of mesophiles was negligible, although the specific activity for Pfb 2 around 90 °C was comparable with that of Ecb 2 around 50 °C. This was in marked contrast to the result with a hyperthermophilic pyrrolidone carboxyl peptidase from P. furiosus, which exhibits higher specific activity over a broad range of temperature than the corresponding mesophilic protein [13]. The Arrhenius plots of the activity for Pfb 2 were clearly divided into two lines at a boundary around 45 °C (Fig. 2A). The low-temperature portion showed a much higher slope than the high-temperature portion. The Arrhenius activation energies (Ea) of Pfb 2 calculated from the slopes were 215.4 and 54.6 kJÆmol )1 for the low- and high-temperature portions, respectively. The Ea values of Pfb 2 , especially in the low-temperature portion, were higher than that for Ecb 2 (135.7 and 43.0 kJÆmol )1 , respectively) which also showed biphasic Arrhenius plots (Fig. 2A). The Ea values for Ecb 2 were similar to those of b activity of tryptophan synthase (Sta 2 b 2 )fromS. typhimurium reported [5,36]. Based on the effect of temperature on the catalytic properties for Sta 2 b 2 and Stb 2 in the presence of monova- lent cations and an allosteric ligand, Fan et al. have shown that biphasic Arrhenius plots are caused by a temperature- dependent conformational change from a low-activity ÔopenÕ conformation to a high-activity ÔclosedÕ conformation [36].ItseemsthatthePfb 2 is also converted from a low activity conformation to a highly active one by increasing temperature. Table 1. Data collection and refinement statistics of the tryptophan synthase b subunit from P. furiosus. Characteristics of the crystals Space group P2 1 2 1 2 1 Cell parameters a(A ˚ ) 84.8 b(A ˚ ) 110.5 c(A ˚ ) 160.0 Z16 V m (A ˚ 3 ÆD a )1 ) 2.2 Solvent content (%) 44 Data collection Resolution (A ˚ ) 2.2 No. of unique reflections 75 098 Average redundancy 9.2 R merge (%) a,b 5.3 (27.6) Completeness (%) a 98.7 (97.0) Refinement statics Resolution (A ˚ ) 67–2.2 No. of reflections 75 065 R factor (%) c 20.8 R free (%) d 26.3 RMSDs RMSD lengths (A ˚ ) 0.006 RMSD angles (°) 1.3 a Values within parentheses are for the last shell of data. b R merge ¼ S h S i |(I h – I hi )|/S h S i I hi * 100. c R factor ¼ S||F obs |–|F calc ||/S|F obs |* 100. d R free ¼ S||F obs |–|F calc ||/S|F obs | * 100 where |F obs | are test set amplitudes (10%) not used in refinement. 2626 Y. Hioki et al. (Eur. J. Biochem. 271) Ó FEBS 2004 Amino acid composition of the b subunit from P. furiosus Table 2 shows the amino acid compositions of both b monomers of Pfb 2 and Stb 2 (Pfb and Stb, respectively). Pfb consists of 388 residues, but Stb has 397. The content (%) of hydophobic residues for Pfb was similar to that for Stb, although the number of hydrophobic amino acid residues of Pfb was slightly lowered. The number of hydrophilic residues increased from 110 (27.71%) to 121 (31.19%) in Pfb, compared with that of Stb. The number of neutral residues of Pfb was largely reduced from 73 (18.39%) to 57 (14.69%). In the case of the a subunit of tryptophan synthase from P. furiosus, hydrophobic residues were remarkably reduced from 58.58% to 53.93%, compared with those from S. typhimurium [11]. Overall structure of the b subunit from P. furiosus The structure of the b subunit from P. furiosus was observed as a dimeric form in which the two b subunits are tightly associated over a broad surface. The buried surface at the interface between the two subunits was estimated to be 3945 A ˚ 2 (Table 3). The dimer structure is depicted by the ribbon drawing in Fig. 3A. The subunit structure consists of two domains, N (residues 1–46, 81–200) and C (residues 47–80, 201–388) domains of almost equal size. The N-terminal (1–200), and the C-terminal (201–388) residues are coloured red and blue, respectively. The core of the N domain is formed from four strands which are surrounded by seven helices. The core of the C domain constitutes six strands with five parallel strands and one antiparallel strand. A short piece (residues 47–80) of the N-terminal residues intrudes into the C domain, forming the first two strands of a b-sheet at the centre of the C domain. A helical structure (residues 58–64) between the first two strands is clearly observed in Pfb although it is not reported in Stb. Arrows in Fig. 3A point to the first two strands and one helical structure (residue 58–64) that intrude into the C domain. The coenzyme PLP is located in the deep cleft between the two domains. PLP forms a Shiff base with the e-amino group of Lys82 in Pfb, corresponding to Lys87 in an active site of Stb. The overall topology of Pfb was equivalent to the b subunit monomer in the Sta 2 b 2 complex reported by Hyde et al.[3]. Structural comparison of Pf b and St b Fig. 4 shows the secondary structure-based sequence align- ment using the secondary structure elements assigned by Fig. 2. Temperature dependence of the specific enzymatic activities of the b reaction for the Pfb 2 subunit (d) and the Ecb 2 subunit (s) at pH 7.0. Activities for Pfb 2 and Ecb 2 were measured in the presence of an excess of the a subunit from P. furiosus and E. coli, respectively. (A) Arrhenius plots of the data from (B). (B) Comparison of the activities of Pfb 2 and Ecb 2 . Table 2. Comparison of the amino acid compositions of the tryptophan synthase b subunit monomers from P. furiosus and S. typhimurium. Values within parentheses are for the percentage of residue per total number of residues. Residue Pfb a Stb b D(Pfb–Stb) Residue number Residue number Differences in residue number Hydrophobic 210 (54.12) 214 (53.90) )4() 0.22) Gly 42 (10.82) 43 (10.83) 1 ()0.01) Ala 38 (9.79) 43 (10.83) )5() 1.04) Val 30 (7.73) 19 (4.79) 11 (2.94) Leu 35 (9.02) 38 (9.57) )3()0.55) Ile 23 (5.93) 24 (6.05) )1()0.12) Met 11 (2.84) 15 (3.78) )4()0.94) Phe 13 (3.35) 13 (3.27) 0 (0.08) Trp 3 (0.77) 1 (0.25) 2 (0.52) Pro 15 (3.87) 18 (4.53) )3()0.66) Neutral 57 (14.69) 73 (18.39) )16 ()3.70) Ser 15 (3.87) 19 (4.79) )4()0.92) Thr 16 (4.12) 21 (5.29) )5()1.17) Asn 13 (3.35) 11 (2.77) 2 (0.58) Gln 12 (3.09) 17 (4.28) )5()1.19) Cys 1 (0.26) 5 (1.26) )4()1.00) Hydrophilic 121 (31.19) 110 (27.71) 11 (3.48) Asp 17 (4.38) 18 (4.53) )1()0.15) Glu 30 (7.73) 28 (7.05) 2 (0.68) Lys 30 (7.73) 19 (4.79) 11 (2.94) His 11 (2.84) 14 (3.53) )3()0.69) Arg 16 (4.12) 19 (4.79) )3()0.67) Tyr 17 (4.38) 12 (3.02) 5 (1.36) Total number of residues 388 397 )9 Amino acid compositions were taken from: a Ishida et al. [28] and b Hyde et al. [3]. Ó FEBS 2004 Crystal structure of tryptophan synthase b 2 subunit alone (Eur. J. Biochem. 271) 2627 DSSP [37]. The sequence homology between Pf b and Stb is 58.5%. The alignment indicates that six residues in the N-terminal domain and three residues in the C-terminal domain were deleted in Pfb. Pro366 of Stb and Ile63 of Pfb were inserted in each protein. Fig. 5 shows a schematic stereo view of superimposed b monomer structures of the tryptophan synthase b 2 from P. furiosus and S. typhimu- rium. The most different part is an a-helical structure around position 60 of Pfb in place of a turn structure in Stb (anarrowinFig.5). Structures of N and C domains. The structures of Pfb and Stb (1BKS) could be superimposed with a root mean square deviation (RMSD) of 1.181 A ˚ between 385 equivalent Ca atoms in both monomers (Fig. 6). The RMSD values of only the N domain (168 residues) and C domain (185 residues) were 0.596 and 1.003 A ˚ , respectively. These results indicate that the structures of both b monomers show a smaller deviation compared with that of the Pfa subunit (RMSD ¼ 2.82 A ˚ )[11], especially for the N domain of the b subunits, because of higher sequence identity. The sequence identities between Pfb and Stb in the N and C domains are 64.5 and 54.1, respectively, while that of Pfa and Sta is 31.5%. As shown in Fig. 6, two large deviations are found in peaks II and IV. In the case of peak II, Ile63 is inserted in Pfb and the region from Lys57 to Ile63 of Pfb clearly forms the a-helix, although the corresponding region of Stb is judged to be in a turn. There is no sequence identity except for one residue (Thr) in this region (Fig. 4). At peak IV, one residue of Pfb at Pro366 of Stb is deleted in a turn region, and there is also no sequence identity between residues 360 and 367 of Pfb (Fig. 4). The deviations of the other two peaks I and III are not great, less than about 3 A ˚ . These regions are slightly decreased in sequence identity compared with the others. The core region of the N domains of Stb has been reported to have a conformation similar to that of the C domain [3]. To estimate the structural similarity between the N and C domains in Pfb, the RMSD values of the structurally homologous region of the two domains were calculated using 73 Ca pairs corresponding to the residues of Stb, which are reported to deviate by less than 4.0 A ˚ between both domains. The values were 2.7 and 2.4 A ˚ for Pfb and Stb, respectively. That for Stb was quite similar to that reported (2.2 A ˚ )[3].AsshowninFig.3B,theoverall topology of the N and C domains in Pfb is similar, and especially, a four-stranded b-sheet structure is well super- imposed. In order to superimpose the C domain on the N domain, the C domain had to be rotated 165.2° about an axis and moved by 26.0 A ˚ between the centroids of the two domains for Pfb and 160.5° and 26.6 A ˚ for Stb, respectively. This slight difference might be due to the differences in the structures of the b 2 subunit alone and the a 2 b 2 complex, although the complex structure from P. furiosus has not yet been solved. Active site. The X-ray crystal structure of the Sta 2 b 2 complex indicates the presence of a 25-A ˚ long hydrophobic tunnel connecting a and b active sites through which the metabolic intermediate of the a reaction, indole, would be transferred from the a subunit to the b subunit. The residues Table 3. Estimate of the difference in stability between tryptophan synthase b subunits from P. furiosus and S. typhimurium on the basis of structural information. ASA values were calculated for Pfb and Stb without PLP. DDG HP, DDG HB, DDG CAV, and DDG ENT represent the difference of DG values between Pfb and Stb, due to hydrophobic interaction, hydrogen bond, cavity volume, and entropic effect, respectively. ÔMonomer/dimerÕ represents the values calculated using monomer and dimer forms of b subunit, respectively. The positive value of DG means that the protein from P. furiosus is more stable than the other. Pfb StbD(Pfb–Stb) Total number of residues 388 397 )9 ASA value (N-state) C/S atoms (monomer/dimer) 8095/13 700 A ˚ 2 7788/13 208 A ˚ 2 307/419 A ˚ 2 N/O atoms (monomer/dimer) 7404/13 353 A ˚ 2 7259/13 266 A ˚ 2 145/87 A ˚ 2 ASA value ( D -state) C/S atoms (monomer) 33 838 A ˚ 2 34 093 A ˚ 2 )255 A ˚ 2 N/O atoms (monomer) 19 935 A ˚ 2 20 161 A ˚ 2 )226 A ˚ 2 DASA value (D–N) C/S atoms (monomer/dimer) 25 743/53 976 A ˚ 2 26 305/54 905 A ˚ 2 )562/)929 A ˚ 2 N/O atoms (monomer/dimer) 12 531/26 517 A ˚ 2 12 902/27 056 A ˚ 2 )371/)539 A ˚ 2 Surface area buried at b/b interface C/S atoms 2490 A ˚ 2 2295 A ˚ 2 195 A ˚ 2 N/O atoms 1455 A ˚ 2 1252 A ˚ 2 203 A ˚ 2 Cavity volume (monomer/dimer) 292/595 A ˚ 3 343/734 A ˚ 3 )51/)139 A ˚ 3 Secondary structure content (a-helix/b-sheet) 44.3/19.6% 40.3/19.4% 4.0/0.2% Contribution of various factors to the stability DG HP (monomer/dimer) )76.9/)129.1 kJ mol )1 DDG HP b/b interface 24.7 kJ mol )1 DDG HB (monomer) 291.0 kJ mol )1 DDG CAV (monomer/dimer) 2.7/7.2 kJ mol )1 DDG ENT (–TDS) (25/100 °C) 101.7/127.3 kJ mol )1 2628 Y. Hioki et al. (Eur. J. Biochem. 271) Ó FEBS 2004 Tyr279 and Phe280 in Stb, having a gating function in the tunnel are substituted by Phe274 and His275 in Pfb. Theactivesite(Lys82)ofPfb corresponding to Lys87 in Stb is located at a flexible region between the two topologically similar domains of the b subunit (Fig. 3A). The phosphate group of PLP covalently bonded with the e- amino group of Lys82 in Pfb is highly ligated through hydrogen bonds with the peptide backbone atoms of residues Gly227, Gly228, Gly229, Ser230, and Ala232 and with the side chains of Ser230 and Asn231, which are likely binding sites for the substrate, L -serine [3]. These residues are completely conserved in Stb, and also the GGGSN sequence is conserved in all the b subunits of the tryptophan synthase reported (protein sequence data bank in a SWISS PLOT ). The distances between the Ca atom of Lys82 and those of the above residues interacted with PLP were calculated and compared with the distance between the corresponding residues of Stb. They agreed within 0.1 A ˚ , indicating that the conformation of the active site in Pfb alone is the same as that in Stb in the a 2 b 2 complex. Complex formation of the b 2 subunit with an a sub- unit. Four residues of Lys167, Asn171, Arg175, and Ser178 in the region of Stb, which interact with the a subunit, correspond to Lys162, Asn166, Arg170, and Val173 in Pfb, respectively. The substitution with Val for Ser might not affect a hydrogen bond forming between the a and b subunits because a peptide backbone atom of Ser178 forms a hydrogen bond with the N atom of Gly181 in Sta.From titration calorimetry [38], it has been reported that the formation of the a 2 b 2 complex from subunits of E. coli follows local folding coupled to the subunit association, corresponding to an Ôinduced fitÕ with a large conforma- tional change. However, in the case of P. furiosus,the conformational change coupled to the subunit association is slight, resembling a rigid body association [29]. These results suggestthattheb subunit from P. furiosus in the complex form might be similar to the structure of the b 2 subunit solved in this study. On the other hand, the a and/or b 2 subunits from S. typhimurium, which have not yet been solved, might have a much more flexible region than the structure of the a and/or b subunits in the complex form reported. Discussion Structure of Pf b 2 and mutual activation Since the first report in 1988 [3], the crystal structures of the a 2 b 2 complex from S. typhimurium have been determined for several forms with bound allosteric ligands [39–45]. These results provide important information for under- standing the allosteric mechanism of the tryptophan synthase complex. The structure of the isolated subunit alone should be determined in order to understand the structural basis of the subunit communication and the allosteric mechanism. In the case of tryptophan synthase from P. furiosus, the structure of the a 2 b 2 complex is not determined yet, although the a [11] and b 2 (present work) subunits alone have already been solved. Therefore, we compared the crystal structures of Pfa and Pfb 2 alone with those of Sta and Stb 2 in the Sta 2 b 2 complex, respectively. The overall structures were quite similar, suggesting that the stimulation effects of enzymatic activities due to a 2 b 2 complex formation are not involved with drastic conform- ational changes. From the isothermal titration calorimetry, the number of residues of local folding coupled to the subunit association in tryptophan synthase from P. furiosus is postulated to be slight, although large conformational changes occur coupled to the subunit association in tryptophan synthase from E. coli [29]. This agrees with present structural results. However, the mechanism of the mutual activation of tryptophan synthase complexes from hyperthermophile and mesophiles could not be understood in detail without the complete set of structures for the two subunits alone and the complex. Fig. 3. Crystal structure of b 2 subunit alone of tryptophan synthase from P. furiosus. (A) The overall structure of the tryptophan synthase b 2 dimer from P. furiosus. The N-terminal (1–200) and the C-terminal (201–388) residues are coloured red and blue, respectively. Arrows point to the first two strands and one helical structure (residue 58–64) that intrude into the C domain. The PLP molecule is represented as a CPK model, coloured gold. Drawings were prepared using MOLSCRIPT [71]. (B) Two similar N and C domains of Pfb were superimposed using69Ca pairs fitted well among the 73 residues of Stb,whichare reported to deviate by less than 4.0 A ˚ betweenbothdomains[3].TheN and C domains are depicted in gold and green, respectively. Fitting used program LSQKAB [72]. Ó FEBS 2004 Crystal structure of tryptophan synthase b 2 subunit alone (Eur. J. Biochem. 271) 2629 Stabilization mechanism of Pf b 2 on the basis of the structure In order to elucidate the stabilization mechanism, the structure of a protein should be analysed in detail, because the conformation of a protein is marginally maintained by many positive and negative factors for stabilization. Using mutant human lysozymes with systematic and comprehen- sive substitutions, changes in stabilities and structures due to mutations have been analysed by DSC and X-ray crystal structures, respectively. It has been proposed that changes in the stability of each mutant human lysozyme are represen- ted by a unique equation, considering the conformational changes due to the mutations [26,27]. The obtained parameters of the relationship between changes in stability and structure should be useful in elucidating the stabiliza- tion mechanism of Pfb on the basis of structural differences between Pfb and Stb. Hydrophobic interaction. A hydrophobic effect is one of the most important stabilizing forces of a folded structure. The change in unfolding Gibbs energy (DG) due to a hydrophobic effect between the wild-type and mutant proteins (DDG HP ) can be expressed as follows: DDG HP ¼ aDDASA nonpolar þ bDDASA polar ð4Þ where, DDASA nonpolar and DDASA polar represent the differ- ence in the change in accessible surface area (ASA) of nonpolar and polar atoms of all residues in a protein, respectively, upon denaturation between the wild-type and mutant proteins. The parameters a and b have been determined to be 0.154 and )0.026 kJÆmol )1 ÆA ˚ )2 , respect- ively, using the stability/structure database upon denatur- ation of mutant human lysozymes [27]. For calculation of the ASA value, carbon and sulfur atoms in the residues were assigned to ASA nonpolar , and nitrogen and oxygen atoms to ASA polar . The contribution of hydrophobic interaction in Pfb and Stb to stabilization was estimated using Eqn 4. The ASA values in the native state were calculated by the procedure of Connolly [46] using the X-ray structures of the two proteins. The values in the denatured forms were estimated using extended structures of each protein, which were generated from the native structures using INSIGHT II . As shown in Table 3, the DG HP values due to hydrophobic interaction of Pfb were less than those of Stb, and the differences between them (DDG HP )were)76.9 and )129.1 kJÆmol )1 in Fig. 4. Sequence alignments based on secondary structures of the b monomers of tryptophan synthase from P. furiosus and S. typhimurium. The first and sixth lines shown residue numbers of Stb and Pfb, respectively. The second and fifth lines represent secondary structural elements of the Stb subunit (1BKS) and the Pfb subunit, respectively, as judged from the secondary structure definition established by DSSP [37]. H, E, B, G, T, and S in the secondary structure elements represent the a-helix, b-strand, b-bridge, 3-helix, turn, and bend, respectively. The third and fourth lines represent the amino acid sequences of Stb and Pfb, respectively. 2630 Y. Hioki et al. (Eur. J. Biochem. 271) Ó FEBS 2004 a monomeric form and a dimeric form, respectively. This means that the higher stability of Pfb is not caused by the hydrophobic interaction. The number of hydrophobic aminoacidresiduesofPfb was slightly decreased compared with that of Stb (Table 2). This trend has been observed in the comparison of the a subunit of tryptophan synthase from P. furiosus with that from S. typhimurium [11]. The hydrophobic effects at the interface of the b/b interaction were also examined. DDG HP at the interface between Pfb 2 and Stb 2 was 24.7 kJÆmol )1 , indicating that the subunit interaction of Pfb is more stabilized due to hydrophobic interaction compared with that of Stb. It has been reported that subunit–subunit interaction and higher order organ- ization contribute to the enhanced stability of hyperthermo- phile proteins [13,47]. Ion pairs (salt bridges) and hydrogen bonds. Ion pairs (salt bridges) seem to play important roles in the stabiliza- tion of hyperthermophile proteins because they occur frequently in hyperthermophile proteins [11,14,17–19,22, 48–53]. Table 4 lists the numbers of ion pairs for Pfb 2 and Stb 2 . The number of ion pairs in Pfb was less than that in Fig. 5. Schematic stereo view of the superimposed b monomer structures of the tryptophan synthase b 2 from P. f uriosus and S. typh imurium. Blue and red lines represent the coordinates of Pfb and Stb (1BKS), respectively. Drawings were prepared using MOLSCRIPT [71]. Residual numbers are shown with an increase of 10 for the Pfb. An arrow indicates the most different part between the proteins around position 60 of Pfb. Fig. 6. RMSDs in Ca atoms between Pf b and Stb after a least-squares fit of the corresponding Ca atoms. The residue number represents the value for Pfb. I to IV represents a discrimination mark for large differences. Ó FEBS 2004 Crystal structure of tryptophan synthase b 2 subunit alone (Eur. J. Biochem. 271) 2631 Stb, although the number of the charged (hydrophilic) residues in Pfb was higher than that in Stb (Table 2). The number of ion pairs at the b/b interface was also less in Pfb than in Stb. These results suggest that the higher stability of Pfb 2 is not caused by the increase in charged residues. Many studies of mutant proteins connected with hydro- gen bonds have shown that hydrogen bonds contribute to stabilizing the conformation of a protein [54–57]. The number of hydrogen bonds involved in the main chains of Pfb was greater by about 10% than that of Stb (Table 4). This increase in Pfb mainly comes from the extra a-helix (Helix 2¢) from Lys57 to Ile63 and the extension of the a-helix in Leu344–Ser346 (Fig. 4). The net contribution of intramolecular hydrogen bonds has been estimated to be 8.56 kJÆmol )1 for a 3 A ˚ hydrogen bond [27]. Using this parameter, the contribution due to hydrogen bonds (of the main and side chains; Table 4) to the stability of Pfb was estimatedtobegreaterby291kJÆmol )1 than that of Stb (Table 3). This suggests that hydrogen bonds remarkably contribute to enhancing the stability of Pfb. Further extensive analyses of the electrostatic interaction, i.e. solving the Poisson–Boltzmann equation may be promising [58]. However the results appear to be very sensitive to the dielectric constant, other parameters used and the assumed denatured state. Hence we should leave this for the future work. Cavity volume. Changes in the cavity size in the interior of a protein affect the conformational stability [59]. Therefore, the cavity volume was determined by attempting to insert a probe sphere of radius 1.4 A ˚ (assuming a water molecule) [46]. In the case of Pfb 2 in a dimer state, 19 cavities were found and the total volume was 595 A ˚ 3 (Table 3). These cavities with a small volume were distributed throughout the molecule. In the case of Stb 2 , 14 cavities were found with a total volume of 734 A ˚ 3 which included two newly intro- duced cavities (total: 48 A ˚ 3 ) when associated. The cavity volumes for the monomer and the dimer were lower in Pfb 2 than in Stb 2 , suggesting a more rigid packing of the Pfb 2 molecule. The energy term for protein stability (DG) due to changes in the cavity size can be expressed in terms of the cavity volume (52 JÆmol )1 ÆA ˚ )3 ) [27]. Using this parameter, the increment in stabilization of the Pfb in a dimer state due to the decrease in cavity volume could be calculated to be 7.2 kJÆmol )1 (Table 3), compared with that of the Stb. Entropic effect. An entropic effect is one of the important stabilizing factors (DG ¼ DH ) TDS). When the conform- ational entropy of a protein is decreased in the denatured state due to substitution(s) or deletion(s) of an amino acid residue, the stability is increased. We can calculate the entropic effects of denaturation from the amino acid compositions using thermodynamic parameters proposed by Oobatake and Ooi [60]: the denaturation entropies for Pfb and Stb were 1.00 and 1.34 kJÆmol )1 ÆK )1 , respectively. This indicates that Pfb is stabilized by 101.7 kJÆmol )1 at 25 °C and 127.3 kJÆmol )1 at 100 °C due to its entropic effect (Table 3). Aromatic–aromatic interaction. Aromatic–aromatic inter- action of the side chains of Phe, Tyr, or Trp has been reported to contribute to the conformational stability of a protein [61]. In the case of the small ribonuclease from Bacillus amyloliquefacience, the edge of the aromatic ring of Tyr17 interacts with the face of that of Tyr13, and the interaction energy is estimated to be )5.4 kJÆmol )1 using the double-mutant cycle analysis [62]. As shown in Table 2, the numbers of aromatic residues of Pfb increase by two residues for Trp and by five for Tyr compared with those of Stb. However, there seem to be no aromatic–aromatic interaction with suitable angles to contribute to the stabilization in Pfb. Analysis by knowledge-based potential. Using the know- ledge-based potential derived from PDB, several methods have been developed to estimate the stability of mutant proteins [63–65]. These methods are computationally rapid and look very robust for the parameters of use. Correlations between the experiment and calculations are expected within 0.5–0.9 depending on the samples. A method developed by Ota et al. [65] estimates the changes in conformational stability due to all of the single amino acid substitutions and represents them in SPMP (Stability Profiles of Mutant Protein). A pseudo-energy potential (DDG SPMP ) consisting of four elements is used: side-chain packing (DDG SP ), hydration (DDG Hyd ), local structure (DDG LC ) and backbone side-chain repulsion (DDG BR ) [66]: DDG SPMP ¼ DDG SP þ DDG Hyd þ DDG LC þ DDG BR ð5Þ This method had been applied to the mutants of several proteins, e.g. Ribonuclease HI [65], human lysozyme [67,68], as well as the evaluating structure–sequence com- patibility, i.e. threading [69]. Recently, locating the func- tional sites of enzymes to identify the structurally destabilizing residues was included in the method [70]. The conformational stabilities of both b subunit struc- tures were analysed by SPMP. The individual stability scores for four terms are summarized in Table 5. The total score of the b subunit from the hyperthermophile clearly shows higher stability than that from the mesophile. All of the score terms contribute to the higher stabilization of Pfb in a monomer state. The scores for a dimeric form in both Table 4. Number of ion pair and hydrogen bond in the tryptophan synthase b 2 subunit from P. furiosus and S. typhimurium. Pfb StbD(Pfb–Stb) Number of ion pairs (monomer)  3A ˚ 17 18 )1  4A ˚ 42 46 )4  5A ˚ 73. 72 1 Number of ion pairs at b/b interface  3A ˚ 02)2  4A ˚ 862  5A ˚ 14 16 )2 Number of hydrogen bonds within 3.2 A ˚ Main chain (monomer) 284 256 28 Main chain and side chain (monomer) 405 371 34 Number of hydrogen bonds at b/b interface 614)8 2632 Y. Hioki et al. (Eur. J. Biochem. 271) Ó FEBS 2004 proteins were higher than those in a monomeric form, coinciding with experimental results showing that both proteins stably exist as a dimeric form in solution. SPMP provides stability scores for each residue at every site of an amino acid sequence. In the case of Pfb (388 residues solved by X-ray analysis), the DDG value for 388 · 19 mutants can be predicted by SPMP using the crystal structure, resulting in ranking of the native residues of the Pfb. The average ranking of all native residues among 20 amino acids for Pfb and Stb was 5.47 and 5.88, respectively, in a monomeric form, and 5.33 and 5.76, respectively, in a dimeric form (Table 6), indicating that the hyperthermophile protein in both monomer and dimeric forms adopts (selects) the residues with lower ranking. The average ranking among 56 rotamers [66] including side- chain conformations of both proteins also showed the same trend (Table 6). These results indicate that the conformation of a hyperthermophile protein (Pfb)ismorefittedtoan ideal structure (lower energy level) than that of a mesophilic protein (Stb). Dimeric form of Pfb.Pfb has been reported to exist in a dimeric form in solution like prokaryotic tryptophan synthase b subunit from mesophiles [29]. The surface areas buried at the b/b interface of Pfb for C/S and N/O atoms were increased by 195 and 203 A ˚ 2 , respectively, com- paredwiththatofStb, indicating that the b/b interface of Pfb is more stabilized by 24.7 kJÆmol )1 due to hydrophobic interaction (Table 3). As shown in Table 3, the decrease in the cavity volume at the b/b interface contributes to the stabilization of the dimeric form of Pfb. Stability profiles of mutant protein analyses also suggest that the dimeric forms of both proteins are more stable than the monomeric forms (Table 5). Only the contributions of hydrogen bonding and ion pairs were comparable. As shown in Fig. 1, the denaturation temperatures of Ecb 2 and Stb 2 from meso- philes were considerably high, 80 °C around pH 8.0, although that of Pfb 2 is higher, 115 °C. The dimeric forms strongly contribute to the higher thermal stability in the case of the b subunits from both mesophiles and hypertherm- ophiles. The unusual stability of Pfb 2 might be caused by the contribution of the intensive b/b subunit interaction in addition to the enhanced stability in a monomeric form. Conclusions The structure of the tryptophan synthase b 2 subunit (Pfb 2 ) from the hyperthermophile, Pyrococcus furiosus,wasdeter- mined by X-ray crystallographic analysis at 2.2 A ˚ resolu- tion, which was the first report of the X-ray structure of the tryptophan synthase b 2 subunit alone, although the struc- ture of the tryptophan synthase a 2 b 2 complex from Salmonella typhimurium has already been reported. The structure of Pfb 2 was essentially similar to that of the b 2 subunit in the a 2 b 2 complex from S. typhimurium. Stability was examined by DSC. Denaturation temper- atures above pH 6.5 are around 115 °C independent of pH; this is about 35 °C higher than those reported for mesophilic proteins. On the basis of structural information on Pfb and Stb, it could be concluded that: (a) the higher stability of Pfb is not caused by either a hydrophobic interaction or an increase in ion pairs; (b) the number of hydrogen bonds involved in the main chains of Pfb (monomeric form) is greater by about 10% than that of Stb, and the contribution due to hydrogen bonds (of the main and side chains) to the stability of Pfb was estimated to be greater by 291 kJÆmol )1 than that of Stb, suggesting that hydrogen bonds remarkably contribute to enhancing the stability of Pfb; (c) the dimeric form of Pfb is stabilized due to hydrophobic interaction and a decrease in cavity volume at the b/b interface; and (d) in total, the sequence of Pfb seems to be more fitted to an ideally stable structure (lower energy level) than that of Stb, as judged from X-ray structure data. References 1. Yanofsky, C. & Crawford, I.P. (1972) Tryptophan synthase. In The Enzymes (Boyer, P.D., ed.), 3rd edn, pp. 1–31. Academic Press, New York. 2. Miles, E.W. (1995) Tryptophan synthase. Structure, function, and protein engineering. Subcell. Biochem. 24, 207–254. 3. Hyde, C.C., Ahmed, S.A., Padlan, E.A., Miles, E.W. & Davies, D.R. (1988) Three-dimensional structure of the tryptophan synthase a 2 b 2 multienzyme complexfrom Salmonella typhimurium. J. Biol. Chem. 263, 17857–17871. 4. Ruvinov, S.B. & Miles, E.W. (1994) Thermal inactivation of tryptophan synthase: Stabilization by protein–protein interaction and protein–ligand interaction. J. Biol. Chem. 269, 11703–11706. 5. Fan, Y X., McPhie, P. & Miles, E.W. (2000) Thermal repair of tryptophan synthase mutations in a regulatory intersubunit salt bridge. J. Biol. Chem. 275, 20302–20307. 6. Pan, P., Woehl, E. & Dunn, M.F. (1997) Protein architecture, dynamics and allosteryin tryptophan synthase channeling. TIBS 22, 22–27. Table 6. SPMP stability scores of a monomer and a dimer of the b subunit from P. furiosus and S. typhimurium: average ranking order. 20 aa and 56 rotamers mean the ranking order out of 20 amino acids and 56 kinds of rotamers, respectively. Monomer Dimer 20 aa 56 rotamers 20 aa 56 rotamers pf b 5.47 9.2 5.33 8.82 Stb 5.88 10.17 5.76 9.85 Difference(Pf–St) )0.41 )0.97 )0.43 )1.03 Table 5. SPMP stability scores of a monomer and a dimer of the b subunit from P. furiosus and S. ty phimurium: stability scores. Units are kJÆmol )1 . Positive values show stabilization in Pfb and Pfb 2 .Total,SP, Hyd, LC, and BR represent the value of DDG SPMP , DDG SP , DDG Hyd , DDG LC ,andDDG BR , respectively, for each subunit. Total SP Hyd LC BR Monomer Pfb 728.6 584.6 55.7 206.8 )118.6 Stb 670.1 535.0 52.0 204.9 )121.4 Difference(Pf-St) 58.5 49.6 3.7 1.9 2.8 Dimer Pfb 2 765.0 621.1 63.7 206.8 )126.9 Stb 2 698.0 559.8 58.9 204.9 )125.5 Difference(Pf-St) 67.0 61.3 4.8 1.9 )1.4 Ó FEBS 2004 Crystal structure of tryptophan synthase b 2 subunit alone (Eur. J. Biochem. 271) 2633 [...]... (1984) The mechanism of self-assembly of the multi-enzyme complex tryptophan synthase from Escherichia coli EMBO J 3, 279–287 10 Ogasahara, K., Hiraga, K., Ito, W., Miles, E.W & Yutani, K (1992) Origin of the mutual activation of the a and b2 subunits in the a 2b2 complex of tryptophansynthase Effect of alanine or glycine substitutions at proline residues in the a subunit J Biol Chem 267, 5222–5228 11... 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The crystal structure of the tryptophan synthase b 2 subunit from the hyperthermophile Pyrococcus furiosus Investigation of stabilization factors Yusaku Hioki 1,2 ,. examined by DSC. This is the first report of the X-ray structure of the tryptophan synthase b 2 subunit alone, although the structure of the tryptophan synthase a 2 b 2 complex from Salmonella typhimurium. Eca, tryptophan synthase a subunit from Escherichia coli; Ecb 2 , tryptophan synthase b 2 subunit from E. coli; Pfa, tryptophan synthase a subunit from Pyrococcus furiosus; Pfb 2 ,tryptophansynthaseb 2 subunit