Stabilization of a ba8-barrel protein by an engineered disulfide bridge Andreas Ivens1, Olga Mayans2, Halina Szadkowski3, Catharina Ju¨rgens1, Matthias Wilmanns2 and Kasper Kirschner3 1
Trang 1Stabilization of a (ba)8-barrel protein by an engineered disulfide bridge
Andreas Ivens1, Olga Mayans2, Halina Szadkowski3, Catharina Ju¨rgens1, Matthias Wilmanns2
and Kasper Kirschner3
1
Universita¨t zu Ko¨ln, Institut fu¨r Biochemie, Ko¨ln, Germany;2EMBL c/o DESY, Hamburg, Germany;
3
Biozentrum, Universita¨t Basel, Basel, Switzerland
The aim of this study was to increase the stability of the
thermolabile (ba)8-barrel enzyme indoleglycerol phosphate
synthase from Escherichia coli by the introduction of
disul-fide bridges For the design of such variants, we selected two
out of 12 candidates, in which newly introduced cysteines
potentially form optimal disulfide bonds These variants
avoid short-range connections, substitutions near catalytic
residues, and crosslinks between the new and the three
parental cysteines The variant linking residues 3 and 189
fastens the N-terminus to the (ba)8-barrel The rate of
ther-mal inactivation at 50°C of this variant with a closed
disulfide bridge is 65-fold slower than that of the reference
dithiol form, but only 13-fold slower than that of the
parental protein The near-ultraviolet CD spectrum, the reactivity of parental buried cysteines with Ellman’s reagent
as well as the decreased turnover number indicate that the protein structure is rigidified To confirm these data, we have solved the X-ray structure to 2.1-A˚ resolution The second variant was designed to crosslink the terminal modules ba1 and ba8 However, not even the dithiol form acquired the native fold, possibly because one of the targeted residues is solvent-inaccessible in the parental protein
Keywords: indoleglycerol phosphate synthase; (b/a)8-barrel proteins; stabilizing disulfide bonds; protein engineering
Indoleglycerol phosphate synthase (IGPS) is a (ba)8-barrel
protein with an N-terminal extension of 48 residues In
Escherichia coli,IGPS (eIGPS) is the N-terminal domain of
a monomeric, bifunctional enzyme, where the C-terminal
domain is phosphoribosyl anthranilate isomerase (ePRAI),
folded into another (ba)8-barrel [1] The catalytic efficiencies
of the engineered separated domains are virtually identical
to those in the bifunctional enzyme [2] eIGPS is, however,
more labile than ePRAI The catalytic activity of eIGPS
decays at 55°C with a half-life of 0.5 min [3] In contrast,
ePRAI activity decays at 60°C with a half-life of 100 min
(R Sterner, Institut fu¨r Biochemie, Universita¨t zu Ko¨ln,
Germany, personal communication) The eIGPS domain,
in turn, is also more labile than eIGPS in the native
bifunctional protein [4,5,6]
In contrast to eIGPS [1], the IGP synthases from the
hyperthermophiles Sulfolobus solfataricus (sIGPS [7]) and
Thermotoga maritima(tIGPS [3]), are thermostable, mono-functional monomers The comparison of the three high resolution crystal structures suggests that an increased number of salt bridges over that in eIGPS decreases the rates of irreversible thermal inactivation of both sIGPS and tIGPS In support of this proposal, mutational disruption of salt bridge that crosslinks its terminal ba1 and ba8 modules, significantly destabilized the variants by comparison to the parental enzyme [3], in support of analogous findings reported previously [8]
The aim of this work was to stabilize the labile eIGPS domain by introducing new disulfide bonds rather than new salt bridges Disulfide bonds can stabilize proteins under-going reversible unfolding by decreasing the main chain entropy of their unfolded states [9–11] For example, the most thermostable single-disulfide variants of the mono-meric xylanase were crosslinked between the N- and C-termini [12] Similar observations have been made with another monomeric (ba)8-barrel protein, PRAI from yeast (yPRAI [13]) Disulfide bonds can also stabilize irreversibly unfolding proteins by decreasing the unfolding rate [14,15] This investigation focuses on two variants of the eIGPS domain that are predicted to form geometrically favourable single, new, long-range disulfide bonds, far removed from the active site and the parental cysteines They either clamp the N-terminus to the core of the (ba)8-barrel fold or crosslink the ba1 and ba8 modules
M A T E R I A L S A N D M E T H O D S
DNA manipulations and sequence analysis Preparation of DNA samples, digestion with restriction endonucleases, agarose gel electrophoresis, and DNA
Correspondence to A Ivens, Universita¨t zu Ko¨ln, Institut fu¨r
Biochemie, Otto-Fischer-Str 12–14, D-50674 Ko¨ln, Germany.
Fax: +49 221 4706 731, Tel.: +49 221 470539,
E-mail: andreas.ivens@uni-koeln.de
Abbreviations: IGP, indoleglycerol phosphate; CdRP,
1-(o-carboxy-phenylamino)-1-deoxy- D -ribulose-5-phosphate; PRA,
N-phos-phoribosyl anthranilate; ePRAI, PRA isomerase domain from
Escherichia coli; eIGPS, IGP synthase domain from Escherichia coli;
eIGPS-PRAI, indoleglycerol phosphate
synthase–phosphoribosyl-anthranilate isomerase bifunctional protein from Escherichia coli;
etrpC, gene encoding eIGPS; sIGPS, IGP synthase from Sulfolobus
solfataricus; tIGPS, IGP synthase from Thermotoga maritima; Nbs 2 ,
5,5¢-dithiobis(2-nitrobenzoic acid); ASA, accessible surface area.
(Received 30 August 2001, revised 30 November 2001, accepted 17
December 2001)
Trang 2ligation were performed as described by Sambrook et al.
[16] Pulsed liquid-phase sequencing was carried out on a
Applied Biosystems 477 A sequencer according to the
manufacturer’s specifications PCRs were performed in
the Trio-block from Biometra (Go¨ttingen, Germany), using
thermostable Pyrococcus furiosus DNA-polymerase
(Stratagene, Heidelberg, Germany) Oligonucleotides were
purchased from Microsyn (Windisch, Switzerland)
Strains and plasmids
Protein was expressed in E coli BL21(DE3) [F–ompT gal
[dcm] [Ion] hsdSB (rB–mB–)], genetic manipulation and
mutagenesis was carried out with E coli JM109 [F¢, traD36
lacIqD(lacZ)M15 proA+B+/e14–(McrA–) D(lac-proAB) thi
gyrA96(Nalr) endA1 hsdR17 (rK–mK+) relA1 supE44
recA1] The expression vector pET21a(+), where protein
production is under control of the T7-RNA-polymerase
promoter [17], was used for expression of etrpC mutants
Oligonucleotides
The following PCR primers were used to amplify the etrpC
gene from the vector pMc-C/F, which contains the
bifunc-tional eIGPS:ePRAI gene [2] The 5¢ primer was used as a
mutagenic primer for replacing Thr3 by Cys (bold letters
indicate the mutated codon) T3C 5¢ primer, 5¢-CGAGGG
TAACATATGCAATGCGTTTTAGCGAA-3¢; etrpC 3¢
primer, 5¢-CCACGCGTCAAGCTTCATACTTTATTC-3¢
The NdeI and HindIII restriction sites for ligation into
vector pET21a(+) are underlined, respectively
The following primers were used for replacing Arg189 by
Cys: T189C 5¢ primer, 5¢-AACCGTGATCTGTGCGATT
TGTCGATT-3¢; R189C 3¢ primer, 5¢-AATCGACAAATC
GCACAGATCACGGTT-3¢ The mutated codons are
shown in bold letters
The following four primers were used for replacing both
Ile64 and Met240 by Cys: I64C 5¢ primer, 5¢-AAAG
GCGTGTGTCGTGATGATTTCGATCCA-3¢; I64C 3¢
primer, 5¢-GAAATCATCACGACACACGCCTTTTGA 3¢;
M240C 5¢ primer, 5¢-GCGTTGTGTGCCCATGACGAT
TTG-3¢; M240C 3¢ primer, 5¢-ATCGTCATGGGCAC
ACAACGCCGAACCAAT-3¢ The mutated codons are
shown in bold letters The 5¢ primer, which introduced
an NdeI site (underlined) into the etrpC gene for eIGPS
(64–240) was: etrpC 5¢ primer, 5¢-ACGAGGGTAACATA
TGCAAACCGTTTTAGC-3¢
Universal T7 promoter and terminator primers were used
for sequencing (Novagen) Both primers anneal to the T7
promoter and terminator sequences in pET21a(+), up- and
downstream of the etrpC gene
PCR and site-directed mutagenesis
The vector pMc2-trpC/F [2] was used as the template for the
production of the double mutant eIGPS(3–189) and
eIGPS(64–240) Site-directed mutagenesis was performed
by PCR with the overlap-extension method [18,19] The
PCR mix contained 250 lM of each nucleotide
triphos-phate, 20 pmol of each primer, 0.1 lg of template, 4 lL of
10 x Pfu reaction buffer and 2.5 U of Pfu-DNA polymerase
(Stratagene) in a total volume of 40 lL The amplification
protocol for the production of megaprimers consisted of
3 min at 95°C, followed by 35 cycles of 1 min at 95 °C,
2 min at 55°C and 3 min at 72 °C The megaprimers were purified by electrophoresis on a 0.8% agarose gel They were used as templates in various dilutions and at a reduced annealing temperature of 50°C in a second PCR reaction with the 5¢ and 3¢ primers to yield the full-length mutated gene The resulting etrpC fragment was purified by electro-phoresis on a 0.8% agarose gel, digested with NdeI and HindIII and purified again The fragment was then ligated into a NdeI–HindIII digested and dephosphorylated pET21a(+) vector, yielding the vector pET21a(+)-etrpC After transformation of E coli BL21(DE3) with pET21a(+)-etrpC, transformants were grown overnight in
2 mL Luria–Bertani medium [16], containing 0.1 lg amp-icillinÆmL)1 (Luria–Bertani/amp medium) The plasmids were isolated and digested with NdeI and HindIII to screen for clones with inserts One positive clone was confirmed by complete DNA sequencing
Expression and purification ofE coli eIGPS(3–189) The protein was expressed in E coli BL21(DE3) Single colonies harbouring the plasmid pET21a(+)-etrpC (3–189) were grown overnight in Luria–Bertani medium [16], supplemented with 100 lgÆmL)1 ampicillin at 37°C On the following day, 15 L Luria–Bertani/amp medium in conical flasks were inoculated with 15 mL of the overnight culture The cells were allowed to grow for three days at
22°C After 24 and 48 h, 50 lgÆmL)1ampicillin was again added The cells were harvested after 64 h and suspended in
50 mMpotassium phosphate, pH 7.8, with 1 mMEDTA For breakage of the cells, the suspension was sonified in a Branson sonifier (2· 2 min, level 5, 60% pulse, ice cooled) DNase and RNase were added to a final concentration of
5 lgÆmL)1for digestion of nucleic acids The homogenate was centrifuged twice at 10 000 g, 4°C and the supernatant was diluted with deionized water to yield a conductivity of 1.27 mSÆcm)1
The crude extract was loaded on a DEAE–Sepharose fast-flow column (5· 25 cm, 510 mL) with a flow rate of
205 mLÆh)1, equilibrated with 10 mMpotassium phosphate,
pH 7.5 After washing with equilibration buffer for 1.5 col-umn vol., the colcol-umn was eluted with a linear gradient from
10 to 300 mM potassium phosphate buffer pH 7.5, 1 mM
EDTA eIGPS(3–189) eluted, as determined by activity and SDS/PAGE, at a phosphate concentration of 150 mM Fractions containing eIGPS(3–189) were pooled and dialyzed overnight against 5 mM potassium phosphate buffer pH 6.8, 100 mMKCl
The dialysate was loaded onto a hydroxylapatite column (2.5· 25 cm, 122 mL) with 34.2 mLÆh)1, that had been equilibrated with 5 mMpotassium phosphate buffer pH 6.8,
100 mMKCl, washed with 1 column vol at 68.2 mLÆh)1and eluted with a linear gradient from 5 to 300 mMpotassium phosphate buffer pH 6.8, 100 mM KCl At the same flow rate, eIGPS(3–189) eluted at 100 mMpotassium phosphate
pH 6.8, 100 mMKCl Fractions containing eIGPS(3–189) were pooled and concentrated by ultrafiltration to
10 mgÆmL)1for a gel permeation chromatography run The concentrated protein solution was adjusted to a final concentration of 300 mM NaCl, 3% sucrose (v/v) The solution was loaded on a Sephacryl S-200 column (2.5· 90 cm, 440 mL) equilibrated with 50 m potassium
Trang 3phosphate buffer pH 7.5, 300 mM NaCl, and eluted with
equilibration buffer at a flow rate of 34.2 mLÆh)1 Fractions
with pure eIGPS(3–189) were concentrated by ultrafiltration
and stored at)70 °C after dripping into liquid nitrogen
Enzymatic assay for indoleglycerol phosphate synthase
Indoleglycerol phosphate synthase activity was assayed at
25°C in 50 mMTris/HCl pH 7.5, 1 mMEDTA, with 40–
70 nMeIGPS and 3–5 lMCdRP The reaction was started
by addition of the nonfluorescent substrate CdRP [20]
Appearance of IGP was measured continuously by its
fluorescence excited at 280 nm and emitted at 350 nm
Because IGP accumulates, the progress curves were fitted to
the integrated Michaelis–Menten equation that takes
com-petitive product inhibition into account [21] The formation
of 1 lmol IGP per minute at 25°C was defined as one unit
of activity (Table 1)
SDS/PAGE
SDS/PAGE was carried out according to the method of
Laemmli [22] The stacking gel and separation gel contained
6 and 12.5% acrylamide, respectively The protein samples
were mixed with 1 vol of 2 x SDS-sample buffer (100 mM
Tris, pH 6.8, 1% SDS, 20% glycerol, 0.01% bromphenol
blue) and heated to 100°C for 5 min before loading The
gels were run with constant current of 30 mA for 1–2 h,
stained with Coomassie Brilliant Blue solution (0.1%
Coomassie blue, 20% acetic acid, 40% methanol) and
destained by boiling in water for 5 min in a microwave
oven Proteins used as molecular mass standards were
bovine pancreatic trypsin inhibitor (6.5 kDa), myoglobin
(16.9 kDa), E coli phosphoribosyl anthranilate isomerase
(21.1 kDa), E coli a-tryptophan synthase (28.7 kDa),
indoleglycerol phosphate synthase (31 kDa), E coli
b-tryptophan synthase (43 kDa), BSA (66.3 kDa) and
phosphorylase b (97.4 kDa)
Protein concentrations were determined according to
Bradford [23] with known concentrations of BSA as
standard, as well as with absorbance spectroscopy at
k¼ 280 nm (eeIGPS ¼ 0.81 cm2Æmg)1) in a Hewlett
Packard Diode Array spectrophotometer (model 8452 A),
connected to a HP Vectra ES/12 computer
Protein thermal stability determined by inactivation
kinetics
For stability measurements, the enzymes were incubated in
0.1M potassium phosphate buffer at a given temperature
and irreversibly heat inactivated Aliquots were taken at
certain time points and chilled on ice, until the remaining
activity was determined (in Tris, as described above) and
plotted against the incubation time Kinetic data were obtained as described above Incubation buffer was 100 mM
potassium phosphate, pH 7.5, 1 mMEDTA, 1 mM dithio-threitol Dithiothreitol was omitted in the case of oxidized eIGPS(3–189)
Oxidation and reduction of the engineered disulfide bridge in eIGPS(3–189)
For reduction of the disulfide bridge, the enzyme at a concentration of 1.5 mgÆmL)1was incubated for 6 h at 4°C
in 50 mM potassium phosphate pH 7.5, 300 mM NaCL,
10 mMdithiothreitol For promoting the formation of the disulfide bond, the enzyme was incubated overnight at 4°C
in 50 mM potassium phosphate pH 7.5, 300 mM NaCL, supplemented with 0.5 mM Nbs2 as the oxidizing com-pound To examine whether the thiols are reduced or the disulfide bridge is formed, the protein samples were run on a nonreducing SDS/PAGE
Determination of thiol content The content of free SH groups and cysteines involved in a disulfide bridge was determined according to the reaction of Ellmann [24] Stock solutions of assay buffer were 1 mM
Nbs2in 50 mMNa-phosphate buffer pH 7.5, 1 mMEDTA
or 50 mM Tris pH 7.5, 1 mM EDTA The following extinction coefficients were used: eTNB (440 nm) ¼ 9.22 mM )1Æcm)1, eNbs2 (325 nm) ¼ 17.38 mM )1Æcm)1 Excess amounts of both reducing and oxidizing compounds were removed before the measurements by gel filtration on NAP columns (Pharmacia) A blank run was performed with assay buffer before the protein was added to a final concentration of 10–30 lMin a final volume of 1 mL After various time points the absorption at 440 nm was recorded
CD spectra
CD spectra were monitored with a Jasco model J-720 spectropolarimeter, which was connected to a Philips SX computer The measurements were carried out in 0.05M
Na-phosphate buffer, pH 7.5, 1 mMEDTA in the absence
of oxidizing agent for oxidized forms and in the presence of
1 mM dithiothreitol for reduced forms For all CD measurements, 10 spectra were recorded and averaged X-ray structure solution
Prior to crystallization, the protein buffer was exchanged in NAP 10 columns (Pharmacia) to 50 mMpotassium phos-phate, pH 7.5, 1 mM EDTA The protein was then concentrated to 10 mgÆmL)1in Centriprep and Centricon ultrafiltration units (Amicon) Crystallization was carried
Table 1 Purification of the eIGPS(3–189) disulfide variant from 69 g (wet weight) of transformed E coli cells.
Fraction
Total protein (mg)
Total activity (U)
Specific activity (UÆmg)1)
Yield
%
Trang 4out as reported for the wild-type monomeric eIGPS [25] in
50 mM potassium phosphate, pH 5.0, 1.2M ammonium
sulphate and 5 mMEDTA
Data were collected at the synchrotron radiation beam
line X11 (EMBL c/o DESY, Hamburg) from shock-frozen
crystals at 100 K using 30% (v/v) glycerol as
cryoprotec-tant Data were recorded on a MAR-CCD detector in three
resolution sweeps to a maximum resolution of 2.1 A˚ The
crystals belong to the space group P6322 and are affected by
strong pseudosymmetry A large cell with dimensions a ¼
141.4 A˚2, c ¼ 156.7 A˚ and containing three molecules per
asymmetric unit coexists with a subcell of dimensions
a ¼ b ¼ 81.6 A˚2, c ¼ 156.7 A˚ and one molecule per
asymmetric unit Both cells are related by a rotation of 30°
around c Only reflections corresponding to the subcell
(k ¼ h ± 3n) show significant intensities, with reflections
from the larger cell being remarkably weaker This
crystallo-graphic problem also affected monomeric, wild-type eIGPS
and has been described previously [25] The structure
presented here corresponds to that of the subcell and
therefore represents an averaged model
The HKL suite of programs [26] was used in data
processing and reduction The data set consisted of 17 306
unique reflections with a multiplicity of 8.8%, an overall
Rmerge of 4.1% and a completeness of 93.7% (the outer
resolution shell, 2.15–2.10 A˚, had values of 25.0% Rmerge,
multiplicity 2.6% and 78% completeness) Structure
solu-tion was carried out by the molecular replacement technique
(AMoRe [27]), using the eIGPS domain from the
bifunc-tional enzyme [1] as a search model For refinement,
reflection data were divided into a working set and a test set
(1057 reflections) usingFREERFLAG Refinement was carried
out using theCNSsoftware [28] and included bulk solvent
correction, overall anisotropic B-factor scaling and
restrained, individual, isotropic B factor refinement The
structure has been refined to a crystallographic R-factor of
24.1% (Rfree31.9%) The model includes protein residues
1–259, the CdRP compound and 187 solvent atoms No
obvious interpretable electron density can be observed for
residues 1 and 2, so these were included as models
The coordinates and structure factors have been
depo-sited at the PDB with accession code 1218477 (1JCM)
R E S U L T S A N D D I S C U S S I O N
Design of disulfide bonds
Engineering of a new disulfide bond into eIGPS must take
into account the presence of three parental cysteines within
the strands b1(C54), b3(C113) and b4(C134) of the (ba)8
barrel (Fig 1) At first sight, any of these native cysteines might be used as disulfide-bonding partners All three residues are, however, solvent-inaccessible: the ASA values, calculated with AREAIMOL [28a], are 1, 2, and 0A2, respectively Although they are not conserved [1], and therefore not directly essential for catalysis, C54 and C113 are nevertheless adjacent to two catalytically essential residues, namely E53 and K114 [5] Initially, 12 potential residue pairs with an appropriate geometry for disulfide formation were identified, using theMODIP program [29] Ten of these pairs were rejected, however, using the following criteria for exclusion: (a) pairs of residues leading
to short-range disulfides, that is, separated by less than 25 positions in the sequence; (b) positions adjacent to catalytic residues; and (c) new cysteines leading to geometrically favourable disulfides [29] with one of the three parental cysteines [30] We avoided using the parental cysteines as partners for new disulfides, because the orientation of E53 and K114 might be altered by the introduction of an adjacent disulfide bridge, thus impairing catalysis
One of the preferred, new disulfide bonds requires the double substitution T3C/R189C, and fixes the N-terminus
to the barrel core of this variant, designated eIGPS(3–189) The disulfide bond is accessible (ASA values of T3 and R189 are 54 and 68 A˚2, respectively) and fortuitously mimics one of the extra salt bridges in both sIGPS [7] and tIGPS [3], which are missing in eIGPS [1] However, the replacement R189C in eIGPS disrupts the parental short-range salt bridge of R189 to E169 on helix a5.
The other selected disulfide variant involves the double substitution I64C/M240C (ASA values of I64 and M240 are
27 and 0 A˚2, respectively), and is designated eIGPS(64– 240) M240 is an invariant but solvent-inaccessible residue that anchors the short helix a8¢ to the core of the protein [1] This proposed disulfide crosslinks the loops b1a1 and b8a8, which are widely separated in sequence but adjacent in space (Fig 1), thus clamping the barrel between the N- and C-terminal modules ba1 and ba8 This disulfide bond is topologically analogous to the strongly stabilizing disulfide bond introduced between helices a1 and a8 of the mono-meric (ba)8-barrel protein yPRAI from yeast [13], and fortuitously mimics the stabilizing salt bridge E73-R241 in tIGPS [3]
Production and purification of disulfide-bonded proteins The eIGPS variants (3–189) and (64–240) were produced by growing transformants of E coli strain BL21 (DE3), as
Fig 1 Stereo representation of indoleglycerol phosphate synthase from E coli The bound phosphate ion indicates the location of the active site The Ca positions of native cysteines (54, 113 and 134) and of the planned disulfide bonds (3–189) and (64–240) are shown 15, position of the single tryptophan residue.
Trang 5described in Materials and methods As generally a larger
fraction of soluble protein is expressed during cell growth at
temperature lower than at 37°C [31], the cells were grown at
22°C for 64 h As estimated by SDS/PAGE, 30% of the
variant (3–189) remained soluble under these conditions It
was purified from the soluble fraction of the cell
homogen-ate in the absence of dithiothreitol, to promote spontaneous
formation of the disulfide bond by auto-oxidation
Chro-matography, first on an anion exchange resin, then on
hydroxylapatite and finally on a size-exclusion gel, resulted
in preparations that were at least 95% pure, as estimated by
SDS/PAGE under reducing conditions (in the presence of
2-mercaptoethanol; Fig 2B), and with an overall yield of
45% (Table 1)
When the (3–189) protein was analyzed by SDS/PAGE
under nonreducing conditions, two bands of about equal
strength were observed (Fig 2A) Because the spontaneous
formation of the disulfide bond was apparently incomplete,
total oxidation was achieved by incubating the protein with
an excess of Ellman’s reagent (Nbs2[32]), leading to a single,
but faster migrating band (Fig 2C) SDS micelles decorated
with disulfide-bonded proteins have a smaller hydrodynamic
volume than those decorated with the corresponding dithiol
forms [33], and therefore migrate more rapidly
In contrast, during culture at 22°C of the cells producing the (64–240) variant, most of the protein partitioned into the insoluble fraction of the cell homogenate Attempts to purify this variant by first solubilizing the precipitate in guanidinium chloride in the presence or absence of dithiothreitol, and then dialyzing against phosphate buffer [5], failed to yield significant amounts of soluble material Apparently, parallel substitution of residues 64 and 240 by cysteines prevents the correct folding of the enzyme The available IGPS structures [1,7] suggest that replacement of the long and hydrophobic side chain of the buried and invariable residue M240 by the short, polar side-chain of cysteine may disrupt the abundant hydrophobic interactions
at the C-terminus and hinder the correct folding of the protein Studies with this variant were therefore not pursued further
Crystal structure of the oxidized variant eIGPS(3–189)
In order to assess the extent to which the (3–189) disulfide bond had actually formed in the partially oxidized variant eIGPS(3–189) (see Figure 2A), the crystal structure of this protein was solved by X-ray crystallography to 2.1-A˚ resolution Unfortunately, the 4-A˚ resolution of the previ-ous crystal structure of the monofunctional eIGPS [25] obstructs comparison to both the structure of the eIGPS domain of the bifunctional enzyme and the structure of the variant reported here Hence, throughout this work, the structure of the eIGPS domain from the bifunctional enzyme [1] will be used as reference The Ca trace of partially oxidized eIGPS(3–198) very closely superimposes
on that of the eIGPS domain (the overall rmsd for Ca atoms
is 0.51 A˚), with residues 45–47 and 206–209 involved in crystal packing showing the largest differences
Figure 3 shows the enzyme in a state of partial oxidation,
as confirmed by SDS/PAGE from dissolved crystals (Fig 2A) Indeed, double conformations can be observed for the side-chain of the C3 residue, with one rotamer as part of the disulfide bridge to C189, and a second rotamer corresponding to the -CH2SH side chain of the reduced form Overall, the structure does not reveal any substantial differences compared to the wild-type eIGPS in the vicinity
of the substitutions Additional electron density was observed, however, in the active site of the protein It can
be modelled as the CdRP substrate (data not shown), but a detailed description including a comparison to related
Fig 2 Partial oxidative closure of the engineered (3–189) disulfide
bond SDS PAGE in absence of mercaptoethanol Lane A, purified
and spontaneously oxidized, variant (3–189); lane B, as in (A), but with
dithiothreitol in the sample buffer; lane C, as in (A), but with Nbs 2 ;
lanes M, marker proteins with the given M r -values (kDa).
Fig 3 2F obs -F calc /a calc electron density map
showing the disulfide bond contoured at 1.0 r.
The newly introduced cysteine residues C3 and
C189 are labelled (black dots, Sulfur atoms).
Trang 6complex structures will be reported elsewhere Perhaps this
unexpected feature is responsible for the observed
incom-plete autoxidation of eIGPS(3–189) shown in Fig 2A The
following measurements were conducted with the completely
oxidized form (Fig 2C)
Conformational analysis in solution
In order to further analyze possible structural differences
between the reduced and oxidized forms of eIGPS(3–189),
designated red(3–189) and ox(3–189), and the wild-type
eIGPS, we measured far-UV CD spectra in phosphate
buffer (Fig 4) All forms displayed identical spectra within
error limits Therefore, it can be concluded that secondary
structural elements are not perturbed by the double
replacement in both the dithiol and disulfide forms
Near-UV CD measurements were performed to further analyze
the tertiary structure of eIGPS and its variants The spectra
(Fig 5) revealed that eIGPS, red(3–189) and ox(3–189)
have the same minima and maxima, indicating that they have a similar chiral environment for W15, which is the only tryptophan of the eIGPS domain, and partially accessible to solvent (ASA ¼ 48 A˚2) Furthermore, fluorescence mea-surements in phosphate buffer, excited at 295 nm, were employed to monitor polarity changes in the environment of W15 upon disulfide formation (data not shown) The spectra of eIGPS, red(3–189) and ox(3–189) were identical, implying that the indole moiety of W15 is similarly exposed
to solvent in all three cases The spectra were also characterized by identical fluorescence emission maxima at
348 nm, supporting the conclusion that the indole moiety of W15 is exposed to solvent Thus, no significant structural differences local to helix a0 seem to occur in both red(3–189) and ox(3–189), with respect to the wild-type In summary, our near-UV and far-UV CD as well as fluorescence measurements confirm that neither the introduction of the (3–189) disulfide bridge nor specific experimental conditions affect the structure of the eIGPS domain
Thermostability eIGPS can be reversibly unfolded by GdmCl in both Tris [34] and phosphate [35] buffers Red(3–189) displays the same properties (data not shown) However, the unfolding
of ox(3–189) by GdmCl in the absence of dithiothreitol was irreversible, presumably due to thiol-disulfide scrambling [30] Therefore, the relative stability of the three forms could only be estimated by irreversible thermal inactivation (Fig 6) [14] The results show that the maximal velocities (Vmax ¼ kcat· [E0]) of the three forms decay irreversibly and exponentially at 50°C In contrast to eIGPS, ox(3–189)
is stabilized 13-fold, whereas red(3–189) is destabilized fivefold, most likely due to the loss of the salt bridge E167-R189 In other words, ox(3–189) is stabilized 65-fold over the dithiol form, which is the correct reference for estimating
Fig 4 Far-UV CD spectra at 25 °C s, parental eIGPS; h, reduced
(dithiol); n, oxidized (disulfide) forms of the variant eIGPS (3–189).
Protein concentrations were between 10 and 21 l M ; d ¼ 0.1 cm.
Buffer: 0,05 M Na-phosphate, pH 7.5, 1 m M EDTA (in case of red(3–
189) with 1 m M dithiothreitol).
Fig 5 Near-UV CD spectra at 25 °C s, parental eIGPS; h, reduced
(dithiol); n, oxidized (disulfide) forms of the variant eIGPS (3–189).
Protein concentrations were between 10 and 21 l M ; d ¼ 5 cm Buffer
as in Fig 4.
Fig 6 The disulfide bond of ox(3–189) stabilizes the fold of eIGPS kinetically Thermal inactivation at 50 °C is an irreversible, exponen-tial process eIGPS, reduced (dithiol) and oxidized (disulfide) variants
of eIGPS(3–189) were incubated at concentrations of 10 l M protein
in 0.1 M potassium phosphate pH 7.5, 1 m M EDTA eIGPS and red(3–189) also contained 1 m M dithiothreitol Enzyme activity was determined in samples drawn at the indicated times and quenched on ice Half-lives: ox(3–189), 49 min; eIGPS, 3.7 min; red(3–189), 0.75 min.
Trang 7the effect of closing this disulfide bridge These results imply
that the engineered disulfide bond crosslinks parts of the
structure that probably separate in the parental protein
before the rate-determining step of its irreversible unfolding
is attained [15] Thus, the disulfide-linked variant apparently
unfolds via a transition state that is different from to that of
the wild-type eIGPS Variation of the phosphate
concen-tration between 5 and 100 mM revealed that the kinetic
stabilities of eIGPS and its variants increase with increasing
phosphate concentration (data not shown) These
observa-tions support the idea that phosphate may serve as an
additionally stabilizing electrostatic clamp within the active
site Note that phosphate specifically interacts with K55
(loop b1a1), G216 (loop b7a7) (not shown in Fig 1) and the
helix dipole of helix a8¢ [1], i.e protein segments that are far
apart in the protein sequence but adjacent in space
These considerations could also explain why the second
disulfide variant (64–240) does not fold properly, even in
the presence of phosphate M240 is an invariant,
solvent-inaccessible residue (ASA ¼ 0 A˚2) that anchors the short
helix a8¢ to the core of the protein [1] Merz et al [36] have
shown that disruptive substitutions near the closure
between the ba1 and ba8 modules of the (ba)8-barrel of
sIGPS are generally destabilizing Replacing M240 of
eIGPS with cysteine may therefore destabilize the
hydro-phobic interface between the terminal modules ba1and
ba8, as well as helix a8¢, thus decreasing the protein’s
affinity for phosphate
Reactivity of cysteines as a measure for protein flexibility
The accessibilities of the three buried cysteines (C54, C113
and C134) of eIGPS and the dithiol and disulfide forms of
the (3–189) variant can be assessed by measuring the
kinetics of their irreversible reaction with Nbs2[32] [37]
ESH þ DTNB ÿ!k ESTNB þ TNB ð1Þ
This reaction, which can be followed by the increase of
absorption of TNB at 440 nm, becomes pseudo-first order,
i.e exponential, when Nbs2is in excess:
ÿd½ESH
dt ¼ d½TNB
dt ¼ kobs½ES ð2Þ where kobs ¼ k[DTNB] is the observed first-order rate
constant Both the total number of reactive cysteine
sulfhydryls as well as their average rate constant k as
expressed by the observed half-lives t1=2 ¼ ln 2
k½DTNB are presented in Table 2 The measurements were performed
in 50 mM phosphate buffer, when the active site is 97% saturated with phosphate [38]
As determined by SDS/PAGE under nonreducing condi-tions (as described in Fig 2), no intermolecular bridges were formed during the oxidation process, as there was no evidence for either aggregation or cross-linking The possi-bility that a further intramolecular disulfide bridge had formed between C113 and C134 (cf Fig 1) was also excluded by measurements of the forms in 0.5% SDS, which unfolds the proteins immediately and allows Nbs2to react with all free thiols that were not accessible in the folded state No decrease of the maximally expected number of free sulfhydryl groups was found
Only one of three native cysteines of eIGPS reacted with Nbs2, albeit slowly As the three cysteines are basically solvent inaccessible, as judged from the calculated accessible surface areas (ASA values are: C45¼ 1 A˚2; C113¼ 2 A˚2; C134 ¼ 0 A˚2), we cannot identify the reactive cysteine of eIGPS from structural considerations In ox(3–189), how-ever, in which the two newly introduced cysteines form a disulfide bond, the reactive cysteine is almost completely protected
In contrast, at least two cysteines of red(3–189) react rapidly As the positions of T3 and R189 of eIGPS are partially accessible to solvent (ASA values are: T3¼ 54 A˚2; R189 ¼ 68 A˚2), it is likely that C3 and C189 of red(3–189) are the two reactive cysteines They are converted to the corresponding disulfide via a mixed disulfide intermediate [E(SH)STNB], which does not accumulate
EðSHÞ2 þ DTNB ! EðSHÞSTNB þ TNB ð3Þ
The cysteine group in excess of C3 and C189 that reacts
to 40% completion in red(3–189) is likely the same as that which reacts in eIGPS to 90% The particularly slow reaction of this parental cysteine in ox(3–198) to only 10% completion must be due to the decreased structural fluctuations of this form of the variant hindering the access
of DTNB by comparison to eIGPS
Catalytic constants
As the active site is located in a depression at the C-terminal end of the b-barrel, between the structured segments that carry the newly introduced pairs of cysteines (see Fig 1), enzyme activity is a sensitive monitor for detecting changes
in both the structure and flexibility of the three enzymes Steady-state kinetic measurements were conducted in Tris
Table 2 Reaction of protein sulfhydryl groups with Nbs 2 The protecting effect of the introduced disulfide bridge.
Variants
Free sulfhydryl groups per protein chaina Totalb Accessible Protectedc t d
1=2 (min)
a Buffer: 0.05 M potassium phosphate buffer, pH 7.5, 1 m M EDTA; T ¼ 25 °C b Evaluated by conducting the reaction at 25 °C in 0.05 M
Tris buffer, pH 7.5, 0.5% SDS.cRounded, integral numbers.dHalf-life evaluated from exponential progress curves recorded at 440 nm Nbs concentration ¼ 1 m
Trang 8buffer and in the absence of dithiothreitol Measurements in
phosphate buffer are not feasible because phosphate is a
competitive inhibitor (Ki ¼ 2.8 mM [38]) The Michaelis
constants (KCdRPM ) of the two forms of both ox(3–189) and
red(3–189) are only 15% smaller than that of eIGPS
(Table 3) The turnover numbers, however, are decreased to
45% in red(3–189) and to 10% in ox(3–189) As the poor
activity of the thermostable IGPS from S solfataricus at
low temperature is due to the rate-limiting release of the
product IGP [36], it is likely that ox(3–189) is ‘constipated’
[36] by the rigidified structure This finding suggests that
covalent crosslinking the helix a0 to the loop b6a6 is
responsible for the retarded release of product in ox(3–189),
and is supported by the decreased reactivity with Nbs2of the
single most reactive cysteine in ox(3–189), in contrast to
eIGPS (Table 2)
C O N C L U S I O N
We have demonstrated that a mesophilic (b/a)8-barrel
enzyme from the tryptophan biosynthesis pathway, namely
indoleglycerol phosphate synthase from E coli, can be
stabilized against irreversible thermal denaturation by the
introduction of a new disulfide bridge The new disulfide
crosslink of eIGPS(3–189) fastens the N-terminal extension
to the catalytic face of the (ba)8-barrel fold, thus rigidifying
it and changing the pathway of unfolding Despite obeying
the structural criteria of good disulfide geometry, as well as
sufficient distance from both catalytic residues and parental
cysteines, the variant eIGPS(64–240) failed to fold to the
native structure, even in the reduced state We conclude that
another important criterion is to avoid replacing
solvent-inaccessible, hydrophobic residues by cysteines Although
surface disulfide bridges have not generally been selected
during the evolution of thermophilic proteins [39], perhaps
because of the chemically reducing environment of the
cytoplasm, correctly designed disulfide bridges, which form
only after release from the cytoplasm, are of specific interest
for biotechnological applications
A C K N O W L E D G E M E N T S
The authors thank Drs Thorsten Kno¨chel and Ralf Thoma for advice,
Dr Reinhard Sterner for critical discussion and Gu¨nter Pappenberger
for designing the stereo figure This work was supported by grant Nr.
31–45855.95 of the Swiss National Science Foundation (to K K.).
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