The crystal structure of the tryptophan synthase b2 subunitInvestigation of stabilization factors Yusaku Hioki1,2, Kyoko Ogasahara1, Soo Jae Lee1, Jichun Ma1, Masami Ishida3, Yuriko Yama
Trang 1The crystal structure of the tryptophan synthase b2 subunit
Investigation of stabilization factors
Yusaku Hioki1,2, Kyoko Ogasahara1, Soo Jae Lee1, Jichun Ma1, Masami Ishida3, Yuriko Yamagata4, Yoshiki Matsuura1, Motonori Ota5, Mitsunori Ikeguchi6, Seiki Kuramitsu2and Katsuhide Yutani7,8
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;7Kwansei Gakuin University, Graduate School of Sciences, Hyogo, Japan;
8
RIKEN Harima Institute, HTPF, Hyogo, Japan
The structure of the tryptophan synthase b2subunit (Pfb2)
from the hyperthermophile, Pyrococcus furiosus, was
deter-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 b2
subunit alone, although the structure of the tryptophan
synthase a2b2 complex from Salmonella typhimurium has
already been reported The structure of Pfb2was essentially
similar to that of the b2subunit (Stb2) in the a2b2complex
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 Pfb2was higher by 35C than
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 Pfb2 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 are more stabilized than 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 b2subunit; stability
Prokaryotic tryptophan synthase (EC 4.2.1.20) is an a2b2
complex composed of nonidentical a and b subunits [1,2]
The a2b2 complex with an abba arrangement [3] can be
isolated as the a monomer and b2subunits The a and b2
subunits catalyse inherent reactions, termed the a and b
reactions (Eqns 1 and 2), respectively The physiologically
important reaction catalysed by the a2b2complex, 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-serineþ indole ! l-tryptophan þ H2O ð2Þ
ab reaction
l-serineþ indole 3-glycerol phosphate !
l-tryptophanþ d-glyceraldehyde 3-phosphate þ H2O
ð3Þ When the a and b2 subunits associate to form the a2b2 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 a2b2 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 , tryptophan synthase b 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)
Trang 2structures of the a or b2subunits alone as well as that of the
complex The three-dimensional structure of the tryptophan
synthase a2b2 complex from Salmonella typhimurium was
determined by X-ray analysis in 1988 [3] However, the
determination of the structure of the a or b2subunit 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 b2 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
b2 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 b2 subunit of tryptophan synthase from P furiosus
(Pfb2) was overproduced in Escherichia coli strain JM109
(pb1837) [28] Pfb2 and the a-subunit of tryptophan
synthase from P furiosus (Pfa) were purified as described
[29,11] The equivalent subunits from E coli (Eca, Ecb2)
were purified also [10,30,31] All of the purified proteins
showed a single band on SDS/PAGE
The protein concentrations were determined from the
absorbance at 278.5 nm using A1%1 cm¼ 6.92 for Pfa and
10.18 for Pfb2[29], 4.4 for Eca [32] and 6.5 for Ecb2[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 80C 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
1CÆmin)1 Before making measurements, the protein solution was dialysed against buffer with the composition
10 mM Gly/KOH, 1 mM EDTA, 0.02 mM 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 10C, by mixing 2 lL of the protein solution with 2 lL
of a reservoir solution containing 12% (w/v) PEG 20 000 and 100 mMMes, pH 6.5 The concentration of Pfb2was 10–12 mgÆmL)1 in 20 mM Tris/HCl pH 8.5 containing
100 lMdithioerythritol and 20 lMPLP
Diffraction experiments with the Pfb2 crystal were performed at the beam line, BL44XU and BL411XU at SPring8 The crystal belonged to the orthorhombic space group of P212121with unit cell dimensions of a¼ 84.8, b ¼ 110.5, c¼ 160.0 A˚ The value of the Matthews coefficient is 2.2 A˚3ÆDa)1for two Pfb2per 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 , and Stb 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 are those reported in [29] and [35] respectively.
Trang 3100 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 and scaled by SCALEPACK [34] Data collection
statistics are summarized in Table 1
Structure determination and refinement
The dimeric structure (Stb2) of the b subunit in the
tryptophan synthase a2b2 complex (Sta2b2) from
S typhimurium (1BKS) [3] provided the initial model for
molecular replacement solutions usingAMORE 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 Rfreeof
26.4% for all reflections in the resolution range 100–2.2 A˚
Rfreewas 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 ofPf b2 Figure 1 shows the pH dependence of the denaturation temperatures of Pfb2measured by DSC The heat denatur-ation of Pfb2was not reversible The peak temperatures of the DSC curves above pH 6.5 were around 115C independent of pH, which were higher by about 35C than 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 Pfb2indicates 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(Ecb2) was denatured in the acidic region
The enzymatic activities of Pfb2and Ecb2were measured
at various temperatures in the presence of excess a subunit from P furiosus or E coli (Fig 2B) The activity for Ecb2 rapidly decreased at temperatures above 55C This decrease might be due to thermal denaturation of Eca in
a2b2complex, because the denaturation temperature of Eca
is around 55C, although Ecb2denatures at 80C [29] It has also been reported that Sta in the complex is inactivated
by 50% at 55C, whereas 50% inactivation of Stb2occurs
at 80C [4] The activity of Pfb2 at the physiological temperature of mesophiles was negligible, although the specific activity for Pfb2around 90C was comparable with that of Ecb2around 50C 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 Pfb2were clearly divided into two lines at a boundary around 45C (Fig 2A) The low-temperature portion showed a much higher slope than the high-temperature portion The Arrhenius activation energies (Ea) of Pfb2calculated from the slopes were 215.4 and 54.6 kJÆmol)1for the low-and high-temperature portions, respectively The Ea values of Pfb2, especially in the low-temperature portion, were higher than that for Ecb2(135.7 and 43.0 kJÆmol)1, respectively) which also showed biphasic Arrhenius plots (Fig 2A) The
Ea values for Ecb2 were similar to those of b activity of tryptophan synthase (Sta2b2) from S typhimurium reported [5,36] Based on the effect of temperature on the catalytic properties for Sta2b2and Stb2in 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
open conformation to a high-activity closed conformation [36] It seems that the Pfb2 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
Cell parameters
Data collection
R merge (%)a,b 5.3 (27.6)
Refinement statics
RMSDs
a
Values within parentheses are for the last shell of data.bR 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.
Trang 4Amino acid composition of the b subunit fromP furiosus
Table 2 shows the amino acid compositions of both b
monomers of Pfb2and Stb2(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 fromP 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 Sta2b2complex reported by Hyde et al [3]
Structural comparison ofPf b and St b Fig 4 shows the secondary structure-based sequence align-ment using the secondary structure elealign-ments 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
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)
Neutral 57 (14.69) 73 (18.39) )16 ()3.70)
Hydrophilic 121 (31.19) 110 (27.71) 11 (3.48)
Total number
of residues
Amino acid compositions were taken from: a Ishida et al [28] and
b
Hyde et al [3].
Trang 5DSSP [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 b2 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
(an arrow in Fig 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 (RMSD¼ 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] As shown in Fig 3B, the overall 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 b2 subunit alone and the a2b2 complex, although the complex structure from P furiosus has not yet been solved
Active site The X-ray crystal structure of the Sta2b2 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 Monomer/dimer 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.
ASA value (N-state)
ASA value ( D -state)
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
Secondary structure content
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 CAV (monomer/dimer) 2.7/7.2 kJ mol)1
DDG ENT (–TDS) (25/100 C) 101.7/127.3 kJ mol)1
Trang 6Tyr279 and Phe280 in Stb, having a gating function in the
tunnel are substituted by Phe274 and His275 in Pfb
The active site (Lys82) of Pfb 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
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 a2b2complex Complex formation of the b2 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 a2b2 complex from subunits of E coli follows local folding coupled to the subunit association, corresponding to an induced fit 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 suggest that the b subunit from P furiosus in the complex form might be similar to the structure of the b2 subunit solved in this study On the other hand, the a and/or b2 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 ofPf b2and mutual activation Since the first report in 1988 [3], the crystal structures of the
a2b2 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 a2b2 complex is not determined yet, although the a [11] and b2(present work) subunits alone have already been solved Therefore, we compared the crystal structures of Pfa and Pfb2alone with those of Sta and Stb2in the Sta2b2complex, respectively The overall structures were quite similar, suggesting that the stimulation effects of enzymatic activities due to a2b2 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
using 69 Ca pairs fitted well among the 73 residues of Stb, which are
reported to deviate by less than 4.0 A˚ between both domains [3] The N
and C domains are depicted in gold and green, respectively Fitting
used program LSQKAB [72].
Trang 7Stabilization mechanism ofPf b2on 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 ) can be expressed as follows:
DDGHP¼ aDDASAnonpolarþ bDDASApolar ð4Þ
where, DDASAnonpolarand DDASApolarrepresent 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)1ÆA˚)2, respect-ively, using the stability/structure database upon denatur-ation of mutant human lysozymes [27] For calculdenatur-ation of the ASA value, carbon and sulfur atoms in the residues were assigned to ASAnonpolar, and nitrogen and oxygen atoms to ASApolar
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 usingINSIGHT II As shown in Table 3, the DGHPvalues due to hydrophobic interaction
of Pfb were less than those of Stb, and the differences between them (DDG ) were)76.9 and )129.1 kJÆmol)1in
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.
Trang 8a 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
amino acid residues of Pfb 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 DDGHPat the interface between Pfb2
and Stb2 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 Pfb2and Stb 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 furiosus and S typhimurium 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.
Trang 9Stb, 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
Pfb2is not caused by the increase in charged residues
Many studies of mutant proteins connected with
hydro-gen bonds have shown that hydrohydro-gen 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
estimated to be greater by 291 kJÆmol)1than 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 Pfb2 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 Stb2, 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 Pfb2
than in Stb2, suggesting a more rigid packing of the Pfb2
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)1ÆA˚)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 (DG¼ 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)1ÆK)1, respectively This indicates that Pfb is stabilized by 101.7 kJÆmol)1
at 25C and 127.3 kJÆmol)1at 100C 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 (DDGSPMP) consisting of four elements is used: side-chain packing (DDGSP), hydration (DDGHyd), local structure (DDGLC) and backbone side-chain repulsion (DDGBR) [66]: DDGSPMP¼ DDGSPþ DDGHydþ DDGLCþ DDGBR 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 Stb D(Pfb–Stb) Number of ion pairs (monomer)
Number of ion pairs at b/b interface
Number of hydrogen bonds
within 3.2 A˚
Main chain and side chain
(monomer)
Number of hydrogen bonds
at b/b interface
Trang 10proteins 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) is more fitted to an
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-pared with that of Stb, indicating that the b/b interface of
Pfb is more stabilized by 24.7 kJÆmol)1due 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 Ecb2 and Stb2 from meso-philes were considerably high, 80C around pH 8.0, although that of Pfb2is higher, 115C 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 Pfb2might 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 b2subunit (Pfb2) from the hyperthermophile, Pyrococcus furiosus, was deter-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 b2subunit alone, although the struc-ture of the tryptophan synthase a2b2 complex from Salmonella typhimurium has already been reported The structure of Pfb2 was essentially similar to that of the b2 subunit in the a2b2complex from S typhimurium Stability was examined by DSC Denaturation temper-atures above pH 6.5 are around 115C independent of pH; this is about 35C 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)1than 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.
20 aa 56 rotamers 20 aa 56 rotamers
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 typhimurium: 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 , and DDG BR , respectively, for each subunit.
Monomer
Difference(Pf-St) 58.5 49.6 3.7 1.9 2.8
Dimer
Difference(Pf-St) 67.0 61.3 4.8 1.9 )1.4