Hyperstabilization of Tetrameric Bacillus sp. TB-90 Urate Oxidase by

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Hyperstabilization of Tetrameric Bacillus sp. TB-90 Urate Oxidase by Introducing Disulfide Bonds through Structural Plasticity Takao Hibi, Asami Kume, Akie Kawamura, Takafumi Itoh, Harumi Fukada, and Yoshiaki Nishiya Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.5b01119 • Publication Date (Web): 06 Jan 2016 Downloaded from http://pubs.acs.org on January 15, 2016

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Biochemistry

Hyperstabilization of Tetrameric Bacillus sp. TB-90 Urate Oxidase by Introducing Disulfide Bonds through Structural Plasticity

∞

Takao Hibi,*,† Asami Kume,†,|| Akie Kawamura,†, Takafumi Itoh,†

Harumi Fukada,‡ and Yoshiaki Nishiya§,¶ †

From the Department of Bioscience, Fukui Prefectural University, Fukui 910-1195, Japan;

‡

Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan; §

Tsuruga Institute for Biotechnology, Toyobo Co. Ltd., Tsuruga, Fukui 914-0047, Japan;

KEYWORDS urate oxidase, disulfide bridge, calorimetry, hyperstabilization, oligomeric protein, structural plasticity, protein engineering, protein assembly, salt bridge, flexible loop

ACS Paragon Plus Environment

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ABSTRACT

Bacillus sp. TB-90 urate oxidase (BTUO) is one of the most thermostable homotetrameric enzymes. We previously reported [Hibi et al. (1994) Biochemistry 53, 3879-3888] that specific binding of a sulfate anion induced the thermostabilization of the enzyme, because the bound sulfate formed a salt bridge with two Arg298s, which stabilized the packing between two βbarrel dimers. To extensively characterize the sulfate-binding site, Arg298 was substituted with cysteine by site-directed mutagenesis. This substitution markedly increased the protein melting temperature by about 20 °C compared with the wild type enzyme, which was canceled by reduction with dithiothreitol. Calorimetric analysis of the thermal denaturation suggested that the hyperstabilization resulted from suppressing the dissociation of the tetramer into the two homodimers. The crystal structure of R298C at 2.05 Å resolution revealed a distinct disulfide bond formation between the symmetrically related subunits via Cys298, although the Cβ distance between Arg298s of the wild-type enzyme (5.4 Å apart) was too large to predict stable formation of an engineered disulfide crosslink. The disulfide bonding was associated with local disordering of the interface loop II (residues 277 to 300), which suggested that the structural plasticity of the loop allowed hyperstabilization by disulfide formation. Another conformational change in the Cterminal region led to intersubunit hydrogen bonding between Arg7 and Asp312, which probably promoted the mutant thermostability. Knowledge of the disulfide linkage of flexible loops at the subunit interface will help to develop new strategies for enhancing the thermostabilization of multimeric proteins.


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Introduction of a disulfide bond to a protein has been used as a practical method to increase protein stability. The apparent melting temperatures of mutant proteins with an engineered disulfide bond have been reported to increase by 5–17 °C.1-8 The location of the disulfide bond in a protein is significant for the successful stabilization of protein structure, and several approaches to find the optimal site, such as computer-assisted methods of protein structural analysis, have been tested.9-11 Dani et al.9 analyzed and described engineered disulfide bonds. According to the authors, stabilizing mutations were most often found in regions of medium to high mobility and were more likely to be near the protein surface. It was noted that most mutants successfully thermostabilized through the introduction of a disulfide bond were small proteins with molecular masses of less than 30 kDa. One reason why the thermostabilization of larger proteins is difficult may be that their large hydrophobic cores more significantly contribute to protein stability. However, the strategy of stabilizing the multimerization of protein subunits has been successful. Bjørk et al.12 replaced the Thr187-Thr187 intersubunit hydrogen bond of Chloroflexus aurantiacus malate dehydrogenase with an engineered disulfide bridge. This replacement allowed an increase of 15°C in the apparent melting temperature of the tetrameric enzyme. Gokhale et al.13 introduced two disulfide bridges across the dimer interface of thymidylate synthase, which improved the temperature optimum from 40 °C to 55 °C. They reported that intersubunit crosslinks could impart appreciable thermostability in multimeric enzymes and implied that appreciable conformational flexibility was needed at the interfaces. Bacillus sp. TB-90 urate oxidase (BTUO, 331 amino acid residues, molecular mass: 37,863.8 Da × 4),14,15 is one of the most thermostable urate oxidases, and its optimum temperature for activity is 45–50 °C (as described in US patent number US 4 987 076). A marked increase (over 10 °C) in its thermal stability is induced by high concentrations (0.8–1.2 M) of sodium sulfate.16


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Calorimetric measurements and size-exclusion chromatographic analyses have suggested that sulfate-induced thermal stabilization is related to the binding of a sulfate anion that suppresses the dissociation of BTUO tetramers into two homodimers. To determine the sulfate-binding site, the crystal structure was solved at 1.75 Å resolution. The crystal structure of the tetramer molecule exhibits four identical subunits, enclosing a tightly closed tunnel, with each subunit composed of two tandem tunneling-fold motifs17, similar to other known urate oxidases.18-21 The bound sulfate anion was found at the subunit interface of the symmetrically related subunits and formed a salt bridge with two Arg298s in the flexible interface loop II16 that is involved in subunit assembly. Site-directed mutagenesis of Arg298 to glutamate (R298E) was used to extensively characterize the sulfate-binding site at the subunit interface. The network of charged hydrogen bonds via the bound sulfate is suggested to contribute significantly to the thermal stabilization of both subunit dimers and the tetrameric assembly of BTUO. If the sulfate-induced thermostabilization mechanism is entirely efficient, the thermostability of the enzyme could be improved by the introduction of a crosslink between the subunits. Based on this assumption we have studied the effects of the formation of a covalent link at the subunit interface. Although one of the simplest ways to introduce such an intersubunit link is a rational design of disulfide bonds, the results obtained using tools for disulfide bond design10, 11 indicated that there was an energetic disadvantage to the formation of an intersubunit disulfide bridge by the substitution of Arg298 or another residue in the interface loop II with cysteine. Therefore, our experimental approach was to chemically modify the cysteine residues using a sulfhydryl crosslinker to produce a rigid bond. Unexpectedly, the BTUO mutant R298C exhibited dramatic thermostabilization without using any crosslinker, implying the formation of intersubunit


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disulfide bridges. Here, we provide evidence of formation of an intersubunit disulfide bridge, and suggest how the single residue substitution in the loop can greatly stabilize the enzyme.

EXPERIMENTAL METHODS Materials Recombinant BTUO and its mutants were overexpressed in Escherichia coli strain DH5 alpha and purified as described previously.15 The purity and homogeneity of the enzyme were assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and dynamic light scattering analyses. In order to spectrophotometrically determine the concentration of BTUO, we obtained the molar extinction coefficient at 280 nm of (1.39 ± 0.01) × 105 M-1cm-1 using the Edelhoch method.22,

23

The protein concentrations were also determined by the Bradford

method,24 using bovine serum albumin as a standard.

Site-directed mutagenesis and enzyme assays Site-specific mutagenesis was carried out using a QuickChange site-directed mutagenesis kit (Stratagene) or a KOD-Plus Mutagenesis kit (Toyobo). Mutations were verified using a Dye Terminator Cycle Sequencing FS Ready Reaction kit and an ABI PRISM 377 sequencer (PerkinElmer). Urate oxidase was assayed by following the disappearance of uric acid, detected by a decrease in absorbance at 291 nm in the presence of the enzyme. The assay mixture contained 0.05 mL of enzyme solution (50 mM borate buffer, pH 8.5, containing 50 mM potassium chloride) and 120 µM uric acid in a final volume of 2.0 mL. The unit of activity was defined as the amount of enzyme that catalyzed the transformation of 1 µmol of substrate per minute at


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37 °C and pH 8.5. The extinction coefficient for uric acid was assumed to be 1.22 × 104 M-1 cm1 25

.

Calorimetric measurements Calorimetric measurements were performed by differential microcalorimetry using a nano DSC differential scanning microcalorimeter (Calorimetry Sciences Corp.) as previously described.16 The experiments were carried out between 20 °C and 90 °C at a scan rate of 1 K/min. The protein solutions were dialyzed against a 50 mM borate buffer, pH 8.5, and the dialysis buffer was used as a reference.

Protein crystallization and data collection In order to obtain crystals, the 13 C-terminal residues (MFSDEPDHKGALK) were deleted from the wild type BTUO. Crystals of the R298C mutant enzyme were grown by the hangingdrop vapor-diffusion technique as previously described.16 A protein solution (5 µL) was mixed with an equal volume of reservoir solution containing 16% (w/v) polyethylene glycol 8000, 100 mM Tris-HCl pH 8.0, 0.08 M Li2SO4, and 1 mM 9-methyluric acid. The crystal obtained was soaked in the reservoir solution containing 20% polyethylene glycol 400 for less than a minute and was flash-cooled in a 100 K dry nitrogen stream and then exposed to a 1 Å X-ray beam. A data set of the R298C single crystal was collected to 2.05 Å resolution using an ADSC Quantum 315 CCD camera and synchrotron radiation on beam line BL-5A (Photon Factory, Tsukuba, Japan). Individual frames consisted of a 0.5° oscillation angle and were measured for 10 seconds. The crystal belonged to the orthorhombic space group of P21212 with unit cell dimensions of a = 131.86, b = 142.58, c = 70.65 Å. Intensity data were processed, merged, and


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scaled with HKL2000.26 Data collection statistics are given in Table 1. A 99.8% complete data set from the crystal was processed to 2.05 Å, with an overall Rmerge of 12.1%, having a total number of 410,560 reflections containing 83,842 unique reflections. Assuming four subunits (molecular mass 36354.1 × 4) per asymmetric unit, the Matthews coefficient was calculated to be 2.20 Å3 Da-1, corresponding to a solvent content of 43.7%.

Structure determination and refinement The structure of the R298C mutant in complex with 9-methyl uric acid has been determined by molecular replacement techniques. The initial phase was solved using the model 3WLV as a search probe. The MR solution was readily obtained and was rebuilt and refined using the program PHENIX.27 Each round of refinement was alternated with a round of manual rebuilding using COOT,28 and the progress of refinement was monitored by tracking decreases in Rcryst and Rfree. After several rounds of refinement, the electron density from the |Fo|-|Fc| map depicted a clear density for 9-methyl uric acid. The four subunit molecules in the asymmetric unit could be superimposed on each other in the range between 0.11 and 0.39 Å of a root mean square deviation (rmsd) for the Cα atoms, so that the refined overall structure was similar from monomer to monomer except at some crystal contact interfaces. Statistics for the refinement are also given in Table 1. The final coordinates were deposited in the Protein Data Bank as entry 5AYJ.

Table 1. Data collection and refinement statistics. Data collection statistics Space group

P21212


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a = 131.86, b = 142.58,

Unit cell parameters Å

c = 70.65 Resolution range Å

50 - 2.05 (2.09 - 2.05)

Total reflections

410,560

No. of unique reflections

83,842

Completeness %

99.8 (99.9)

Rmerge(=ΣhΣi|Ih,i - | / ΣhΣi|Ih,i|) 0.121 (0.715) Rpim(=Σhkl(1/n-1)1/2Σi|Ih,i - |

0.059 (0.351)

/ ΣhΣi|Ih,i|) CC1/2

0.561

I/σ

20.3 (3.0)

Redundancy

4.9 (4.8)

Wilson B factor Å2

34.0

Values in parentheses are for the outer shell. Refinement statistics PDB entry

5AYJ

Refinement resolution Å

32.27-2.05

No. of reflections (work/free)

79,525/4,245 9,297 (A 2303, B 2350,

No. of protein atoms C 2361, D 2283) No. of ligand atoms

125

No. of water molecules

429

Missing residues

A 1-6, 129-132, 287-290, 311-324, B 1-7, 287-290, 315-324, C 1-7, 287-289, 315-324, D 1-6, 129-134, 287-290, 311-324


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R

work

/R

0.169 / 0.221

free

rmsd Bond length Å

0.007

Bond angle °

0.839 2

Mean B factors Å Main chain atoms

26.37 (A 28.88, B 24.19, C 24.07, D 28.48)

Side chain atoms

28.33 (A 30.66, B 26.29, C 26.12, D 30.39)

Ligand atoms

40.73

Water atoms

33.61

Ramachandran plot Favored region %

98.1

Allowed region %

1.9

RESULTS Hyperstabilization of R298C mutant by formation of a disulfide bridge The substitution of Arg298 with a cysteine residue resulted in a mutant enzyme (R298C) having a kcat value of 1.2 ± 0.1 s-1 and a Km value of 54 ± 9 mM, nearly equal to those of the R298E mutant;16 this was not affected even in the presence of high concentrations of sulfate salt. The irreversible loss of enzyme activity after 30 min incubations at various temperatures was determined. In Figure 1, the irreversible inactivation plots of the R298C mutant show a marked increase in its thermal stability. To approximate the temperature at which the enzyme is half denatured (T1/2), the residual activity data were fit to a Boltzman model as described previously.16 The T1/2 value of R298C was 15 °C higher than that of the wild type enzyme. It


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decreased by approximately 4 °C with the addition of 10 mM of glutathione. When the reaction mixtures were treated with 10 mM of dithiothreitol, the thermal stabilization effect was completely canceled. The effects of the reductants implied that the thermal stabilization resulted from the formation of a disulfide bridge.

FIGURE 1. Plots of the irreversible inactivation of BTUO after 30 min incubations at various temperatures are shown; ( mM glutathione, and (

) wild type BTUO, (

) mutant R298C, (

) R298C treated with 10

) R298C treated with 10 mM dithiothreitol. Error bars represent the

standard errors between different experiments (n = 3). Residual activity data at each temperature were fit to a Boltzmann model using Origin (Origin Lab Co. Ltd.), and the values of wild type BTUO, mutant R298C, R298C treated with 10 mM glutathione, and R298C treated with 10 mM dithiothreitol are represented as black, red, orange, and blue solid lines, respectively.


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Differential Scanning Calorimetry (DSC) of R298C/C305S double mutant DSC experiments were conducted for further investigation of the thermal stabilization mechanism of the mutant enzyme. The BTUO subunit has a single cysteine residue C305, and the formation of other disulfide bonds might disturb the calorimetric measurement in the unfolding process of the R298C mutant by heat. To avoid interference, we made a mutant C305S and a double mutant R298C/C305S, which were used for the DSC measurements (Figure 2). The DSC curves of 2.8 mg/mL (19 mM) enzyme were obtained at a scanning rate of 1 K/min.


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FIGURE 2. Differential scanning calorimetry thermograms of BTUO mutant were measured (A) in the absence of Na2SO4 and in the presence of (B) 0.1 M Na2SO4 and (C) 1.0 M Na2SO4 as a salt. The sample solution contains 2.7 mg/mL protein in 50 mM sodium borate (pH 8.5).

The profile of the DSC thermogram of C305S (Figure 2) was composed of two distinct transition peaks; the first peak A and the second peak B demonstrated that at least two unfolding steps exist in the process of thermal unfolding. In the absence of Na2SO4 (Figure 2A), the melting point temperatures of peak A and peak B, Tp,A and Tp,B were determined to be 72.7 °C and 82.4 °C, respectively, by fitting the data to the sequential two-state model described previously:16

N4⇄ 2U2⇄ 2D2

(1).

In the same way as the wild type enzyme, the Tp,A and Tp,B values slightly decreased to 68.4 °C and 79.9 °C, respectively, in the presence of 0.1 M Na2SO4 (Figure 2B) and then Tp,A in the presence of 1.0 M Na2SO4 noticeably increased to 78.5 °C, near to Tp,B (= 83.8 °C) (Figure 2C). In comparison with the wild type and C305S BTUO, the DSC transition curve of the R298C/C305S double mutant changed dramatically (Figure 2). The DSC profile of the double mutant showed an apparent single transition peak, similar to the wild type enzyme in the presence of high concentration (0.8 to 1.2 M) of sulfate salt.16 The analysis of the DSC curves in terms of an approximate model of two-state denaturation with self-dissociation29 was conducted (Figure 3). The statistics of best fit were obtained, assuming that the denaturation process could be considered to take place in three sequential steps as follows:


12

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N4⇄ U4⇄ 2D2

(2),

where N4 is the native form of BTUO tetramer, U4 is the partially unfolded tetramer, and D2 is the final denatured state of the dimer. These analyses revealed that the R298C substitution significantly increased Tp,A and Tp,B up to 86.8 °C and 90.4 °C, respectively (Figure 3). Moreover, the transition peaks of the double mutant R298C/C305S did not change with or without Na2SO4, although those of the wild type enzyme and the mutant C305S were sensitive to the addition of the sodium salt (Figure 2). These results suggest that the formation of the intersubunit disulfide bonds strengthened the interaction between the β-barrel dimers and delayed the initiation of the first unfolding process.

FIGURE 3. DSC scans of R298C/C305S in the absence of Na2SO4. The data points are shown as open circles. The fits of the data to the sequential two-state model of equation (2) are indicated by solid lines. The base line is drawn as a dotted line.


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Structural analysis of the R298C mutant by X-ray crystallography The crystal structure of R298C was determined in complex with the competitive inhibitor 9methyl uric acid at 2.05 Å resolution. In the refinement process, a clear electron density for a left-handed disulfide bridge at the subunit interface was depicted from the σA omit map (Figure 4). Table 2 shows the disulfide bond geometry in the R298C mutant. The disulfide bond formation shortened the distance between the Cβ atoms of Cys298s to 4.54 Å, which is 0.9 Å shorter than that of Arg298s of the wild-type enzyme. The average Cβ-Sγ-Sγ angle of 126.4° was very large, and the averaged χ2 torsion angle (Cα-Cβ-Sγ-Sγ) was 5.5° (eclipse), so that a steric hindrance arose between the Cα-Cβ and the Sγ-Sγ bonds and then the disulfide strain energy was estimated to be relatively high (20.5-21.2 kJ/mol, Figure S1) according to Katz et al.30 and Schmidt et al.31 These conformational distortions contribute to keep almost the same Cα-Cα distance of residue 298s (R298C mutant, 4.2 Å and wild-type enzyme, 4.3 Å, respectively).


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A

B

FIGURE 4. The structures of BTUO R289C in complex with 9-methyl uric acid. (A) Side and top views of a schematized model of the BTUO tetramer. Magnification shows the engineered disulfide bonds as ball and stick models. A β-barrel dimer is surrounded by a dashed line. Figures were prepared using PyMOL.32 (B) σA-weighted |Fo|-|Fc| omit electron density maps for Cys298 shown at the 4.5 σ contour level. The left and right panels represent the ball and stick


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models of an intersubunit disulfide bond which were rotated by 90° to each other. Figures were prepared using PyMOL.32

Table 2. Geometry of the left-handed disulfide bridge in mutant R298C. geometry parameter

distance (Å)

angle (°)

torsion angle (°)

Cα-Cα

Cβ-Cβ

Sγ-Sγ

Cα-Cβ-Sγ

Cβ-Sγ-Sγ

χ1

χ2

χ3

R298C average

4.20

4.57

2.04

112.6

126.4

-71.1 gauche

5.5 eclipse

-78.2 gauche

reported value

5.88± 0.49a)

2.02b)

114b)

105b)

-60, 60, -170c) gauche

-60, 180c) gauche

-85.8± 8.6b) gauche

a) The value was obtained as an average of 39 left-handed disulfide bridges.30 b) The value was averaged across geometry parameters of left-handed disulfide bridges in 351 examples.33 c) The values were the torsion angles at the peaks in the histogram concerning χ1 or χ2 distribution.33

The whole main-chain trace of the R298C mutant was in good agreement with that of the wild type enzyme. Superimposition of the R298C structure onto that of the wild type enzyme (PDB entry 3WLV) using GASH revealed a rmsd of 0.28 Å for 1187 Cα atoms overall. In spite of the high degree of structural main chain similarity, some significant differences were locally observed: the loop region 129 to 132 in subunits A and D, and the region 287 to 290 in the interface loop II were missing (Table 1), while the N-terminal Arg7 in subunits A and D, and the C-terminal region 311 to 314 in the subunits B and C, missing in the wild type enzyme, were constructed. The guanidino group of Arg7 formed bidentate hydrogen bonds with the carboxy group of Asp 312 in the neighbor subunit that was the mate of the β-barrel dimer (Figure 5),


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which may have contributed to the stabilization of the dimer via electrostatic interactions (Table 3).

FIGURE 5. The structure of the intersubunit salt bridge between Arg7 and Asp312. The Nterminal residues 6 to 8 of subunit A and the C-terminal residues 311 to 315 of subunit B are drawn as stick models. Other parts of subunit A (green) and subunit B (orange) are depicted as cartoon models and estimated hydrogen bonds are drawn as dashed lines; numbers show the contact distances between the nitrogen atoms of Arg7 in subunit A and the oxygen atoms of Asp312 in subunit B.

To investigate the effect on the subunit assemblies due to the disulfide formation, the buried interface surface areas of the wild type and R298C enzymes were analyzed by structure-based computational methods using the PISA server.34 The total buried interface areas for the wild type and R298C enzymes were 22554 and 21266 Å2, respectively. The buried surface areas of the dimer-dimer interfaces (subunit A to D, subunit B to C) of the R298C mutant were 5178 Å2, corresponding to 24.3% of the total buried interface area. This buried surface area of the mutant


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is comparable to that of the wild type enzyme (5563 Å2, 24.7%), as well as to the averaged buried surface area of the subunit-subunit interfaces (subunit A to B, subunit C to D) in the βbarrel dimer of the R298C mutant which were 2569 Å2 (12.1%) for R298C, and 2593 Å2 (11.5%) for the wild type enzyme. Accordingly, there is no significant increase in the buried interface area between the wild-type and R298C enzymes. The structure around the interface loop II of subunit C is shown in Figure 6, which is placed at the interface between subunits A and B. As described previously,16 the role of the loop was suggested to be a hinge between subunits A and B, participating in the open and closing motion of the active site cleft at the subunit interface. In the R298C mutant structure, the electron densities around residues Pro287–Glu290, and the middle of the loop, were completely lost. Moreover, comparison with the B-factor profile (Figure 7) showed that the B-factors of the residues 272 to 283 and 291 to 300 of the R298C mutant specifically increased compared to those of the wild type enzyme, implying that the loop becomes more flexible after the formation of the disulfide bond.


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FIGURE 6. The structures of residues 280 to 300 in the interface loop II. (a) A ball-stick model of the interface loop II of the R298C. (b) Comparison between the loop structures of R298C (colored) and wild type (gray) BTUO.


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FIGURE 7. Scaled B-factor plots of R298C (solid) and wild type (dashed) BTUO. The B-factors of R298C were scaled using the ratio of Wilson's B-factors between the mutant and wild type enzyme. Upper schematic diagram of the BTUO secondary structure, with α-helix depicted as cylinders, β-strand as block arrows, and others as lines; which were assigned using DSSP.35 Black pins graphically mapped on the sequence label show the location of active-site residues. Thick lines represent the regions (residues 100 to 104, 129 to 132, 272 to 283, 287 to 289, and 291 to 300) in the R298C polypeptide that have B-factors significantly higher than those of wild type enzyme.

Table 3. Predicted hydrogen bond pairs around interface loop II16. In order to specify each atom of hydrogen bond donors and acceptors, the chain name, residue name, residue number, and atom name are represented. R298C

Wild-type

interface loop II … bond length (Å) … bonding interface loop II … bond length (Å) … bonding partner partner C:ASP 280 OD2 … 3.21 … A:ARG 298 NE C:ASP 280 OD2 … 3.10 … A:ARG 298 NH2 C:ARG 298 NE

… 3.26 … A:ASP 280 OD2

C:ARG 298 NH2 … 3.04 … A:ASP 280 OD2 C:LYS 292 N

… 3.01 … B:THR 21 OG1

C:LYS 292 N

… 3.02 …

B:THR 21 OG1

C:LYS 292 O

… 3.18 … B:THR 21 N

C:LYS 292 O

… 3.22 …

B:THR 21 N

C:TYR 294 N

… 2.84 … B:TYR 19 O

C:TYR 294 N

… 2.78 …

B:TYR 19 O

C:TYR 294 O

… 2.90 … B:TYR 19 N

C:TYR 294 O

… 2.87 …

B:TYR 19 N

C:THR 295 OG1 … 2.72 …

B: PHE 17 O

C:THR 295 OG1 … 2.63 … B:PHE 17 O


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Biochemistry

C:GLU 296 OE2 … 2.53 … B:TYR 19 OH

C:THR 295 OG1 … 3.16 …

B: GLN 81 NE2

C:GLU 296 OE2 … 2.37 …

B:TYR 19 OH

Role of the C-terminal disordered region in thermal stability The C-terminal 17 to 22 residues of BTUO were disordered as revealed by the crystal structures (PDB entries, 1J2G, 3WLV, and 5AYJ). Recently, Feng et al.21 determined the crystal structure of urate oxidase from Bacillus fastidious (BFUO), which has 57% sequence identity with BTUO. It was reported that the C-terminal region of BFUO formed an ordered structure containing an α-helix (Q305-A313) and a random coil (S314-L322), and the decrease of its thermal stability at pH 7.4 was caused by the mutations that disrupted the interactions between Arg3 and Asp307. To investigate the relationship of the C-terminal residues with thermal stabilization, we examined the wild type and R298C BTUO with serial truncations of the disordered C-terminal residues. Each mutant used in this study is represented by the number of truncated C-terminal residues (for example, WT∆C13 means that the C-terminal residues 320 to 332 of the wild type enzyme are deleted.). Figure 8 shows the irreversible loss of activity of the truncated mutants during 30 min incubations at various temperatures, and Table 4 summarizes the T1/2 values of the mutants determined by the irreversible inactivation plots.


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FIGURE 8. Plots of irreversible inactivation of C-terminal truncated mutants after 30 min incubations at various temperatures; ( (

) R298C∆C8, (

) R298C∆C9, (

) wild-type BTUO, ( ) mutant R298C, ( ) R298C∆C6, ) R298C∆C10, and (

) R298C∆C10 treated with 10 mM

dithiothreitol. Error bars of the wild type and R298C BTUO are represented. Residual activity data at each temperature were fit as described in the legend of Figure 1.

Table 4. T1/2 values of C-terminus truncated mutants. enzyme

C-terminal sequence

T1/2 (°C)

Wild-type (WT)

…316NILMFSDEPDHKGALK

68

WT∆C8

…316NILMFSDE

66

WT∆C13

…316NIL

66

R298C

…316NILMFSDEPDHKGALK

83

R298C∆C6

…316NILMFSDEPD

80


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R298C∆C8

…316NILMFSDE

81

R298C∆C9

…316NILMFSD

81

R298C∆C10

…316NILMFS

73

R298C∆C10+10 mM DTT

…316NILMFS

66

R298C∆C8-D323A/E324A

…316NILMFSAA

83

R298C∆C8-D323N/E324Q

…316NILMFSNQ

85

R298C∆C8-D323L/E324L

…316NILMFSLL

83

R298C∆C8-D323V/E324V

…316NILMFSVV

83

A significant decrease of 11 °C was observed by deleting 10 disordered C-terminal residues (Asp323–Lys332), however the changes in the T1/2 of the mutants from R298C to R298C∆C9 were negligible. When the R298C∆C10 was treated with 10 mM dithiothreitol, its T1/2 value further decreased to the same level as that of the wild type enzyme. To study the individual contributions of the C-terminal Asp323 and Glu324 to the thermal stabilization of R298C∆C8, the thermal stability of four double mutants of Asp323 and Glu324 (D323A/E324A, D323N/E324Q, D323L/E324L, and D323V/E324V) were analyzed. None of the four mutants demonstrated any significant changes in T1/2, suggesting that the thermal stabilization of the R298C mutants depended more upon the length of the C-terminal disordered region than the electrostatic or hydrophobic interactions through the specific side chains of the C-terminal residues. The truncation of the randomly coiled C-terminal region might make the unfolded state thermodynamically unfavorable due to the loss of conformational entropy. Further work is needed to elucidate the mechanism.


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DISCUSSION Cystine is hydrophobic, and thus, most naturally occurring disulfide bonds are buried in the protein. It has generally been thought that the introduction of an engineered disulfide bond into the protein hydrophobic core better maintains the biophysical properties of a target protein. Although Cys298 in the BTUO mutant R298C had a relative accessible surface area of 34 %, our studies demonstrated that the substitution of the surface residue enabled spontaneous formation of an intersubunit disulfide bond and significant stabilization of the enzyme. In our DSC experiments, the Tp,A and Tp,B values of the double mutant R298C/C305S were 18 °C and 11 °C higher than those of the mutant C305S in the presence of 0.1 M Na2SO4. The analyses of the DSC curves demonstrated that the pronounced increase in the first peak temperature Tp,A was primarily caused by the disulfide bond linkages between the two homodimers, which prevented the tetramer from dissociating in the first step of denaturation. The thermal stability of pyroglutamyl peptidase from Bacillus amyloliquefaciens was noticeably increased by the introduction of an intersubunit disulfide bond within the subunit interface, the site of which was predicted by the structural comparison with the thermophilic enzyme from Thermococcus litoralis36. Such a higher oligomerization state is frequently observed in extremophilic organisms, and is presumably favorable for stability.37 Whereas our previous studies16 have shown that high concentrations of sodium sulfate also suppressed the dissociation of BTUO tetramers, the effect of the sulfate anion was limited to an increase in Tp,A, and the second peak temperature Tp,B remained largely unaffected. The introduction of the disulfide bond at Cys298 increased both Tp,A and Tp,B, which implied that the surface-exposed disulfide bond stabilized not only intersubunit packing of the two β-barrel dimers, but also the native structure of each subunit.


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The distance between the Cβ atoms of Cys298s became shorter by 0.9 Å in comparison with those of Arg298s of the wild-type enzyme, though the crystal structure of R298C was highly similar to that of the wild type BTUO as described earlier. This shortening of the Cβ−Cβ distance caused abnormal distortions in the disulfide bond geometry, Cβ-Sγ-Sγ angle, and χ2 torsion angle. The disulfide strain energy is estimated to be 20.5-21.2 kJ/mol using the Disulfide Bond Dihedral Angle Energy Server.30 Therefore, some energetic gain other than that from forming the disulfide bond is required for the spontaneous formation of the intersubunit disulfide bonds and the stabilization of the native subunit structure. The enthalpic loss due to the distortions may be partially compensated for by the increased conformational entropy of the disordered state of the interface loop II (residues 287 to 290). Moreover, a missing part of the Cterminal region of the wild type enzyme was ordered in the R298C structure, and then an intersubunit bidentate hydrogen bond formed between Arg7 and Asp312. Recently, electrostatic interactions on the protein surface were shown to be more important for stability than previously suggested.37 The polar interaction between Arg7 and Asp312 probably makes the intersubunit packing tighter and contributes to the thermal stabilization of the β-barrel dimer structures. In general, disulfide bonds engineered into proteins do not always enhance stability and the prediction of appropriate loci for disulfide crosslinking is still difficult. This is because unfavorable contacts are formed in the surrounding residues around disulfide bonds or existing favorable interactions are lost within the folded protein. An intersubunit residue in the flexible surface loop is a potential target for the introduction of a disulfide linkage, to avoid the strained formation, to minimize unfavorable contacts with the inner residues, and to maximize the entropic effect. Our studies also demonstrate the possibility of using an intersubunit salt bridge as a probe to predict the loci for disulfide crosslinking.


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AUTHOR INFORMATION *Corresponding Author Department of Bioscience, Fukui Prefectural University, Fukui 910-1195, Japan; Telephone: +81 (776) 61-6000; Fax: +81 (776) 61-6015; E-mail: [email protected]

Present Addresses ǁ

A.K.: Daito Kasei Kogyo Co. Ltd., 3-7-4 Technoport, Kawashiri, Fukui 910-3136, Japan;

⊥A.K.:

¶

Kobayashi Kako Co. Ltd., Yachi, Awara, Fukui 919-0603, Japan;

Y.N.: Department of Life Science, Setsunan University, 17-8 Ikedanaka-machi, Neyagawa,

Osaka 572-8508, Japan

Author Contributions T.H. was the Principal Investigator and conducted X-ray crystallography and enzyme kinetics. A.K.|| conducted purification, kinetic analysis of the enzyme, and crystallization screening. A.K.⊥ conducted purification and kinetic analysis of the enzyme. H.F. conducted calorimetry. T.I. conducted X-ray crystallography. Y.N.¶ conducted genetic engineering and enzyme purification.

ACKNOWLEDGMENT


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The authors thank the staff at the beamline BL-5A of the Photon Factory (Tsukuba, Japan) for providing data collection facilities and support (Proposal No. 2011G674). The synchrotron radiation experiments were also performed at the BL26B1 of SPring-8 with the approval of the Japan Synchrotron Radiation Research Institute (JASRI) (Proposal No. 2011A1882). We thank W. MacDonald (Fukui Prefectural University) for generous advice and support.

Supporting Information Available FIGURE S1. The geometry parameters of the disulfide bonds of R298C and the histogram of disulfide strain energy. The pink bars represent the frequency distribution of disulfide strain energy calculated for a set of 16,554 disulfide bond structures from Protein Data Bank. The blue bars in the histogram represent the strain energies of the disulfide bonds of BTUO R298C. The results

were

obtained

using

Disulfide

Bond

Dihedral

Angle

Energy

Server

(http://services.mbi.ucla.edu/disulfide/).

ABBREVIATIONS BTUO, urate oxidase from Bacillus sp. TB-90; OHCU, 2-oxo-4-hydroxy-4-carboxy-5ureidoimidazoline; E. coli, Escherichia coli; AFUO, urate oxidase from Aspergillus flavus; AGUO, urate oxidase from Arthrobacter globiformis; CCD, charged coupled device; PDB, protein data bank; rmsd, root mean square deviation; DSC differential scanning calorimetry; SEC, size exclusion chromatography.

REFERENCES


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[21] Feng, J., Wang, L., Liu, H., Yang, X., Liu, L., Xie, Y., Liu, M., Zhao, Y., Li, X., Wang, D., Zhan, C. G., and Liao, F. (2015) Crystal structure of Bacillus fastidious uricase reveals an unexpected folding of the C-terminus residues crucial for thermostability under physiological conditions, Applied microbiology and biotechnology 99, 7973-7986. [22] Edelhoch, H. (1967) Spectroscopic

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[37] Eijsink, V. G., Bjork, A., Gaseidnes, S., Sirevag, R., Synstad, B., van den Burg, B., and Vriend, G. (2004) Rational engineering of enzyme stability, Journal of biotechnology 113, 105120.

BRIEFS Structural Plasticity Allows Hyperstabilization of Uricase via Disulfide Linkage.

SYNOPSIS Symmetrically-related arginine298s of Bacillus sp. TB-90 urate oxidase form intersubunit salt bridges via sulfate anions. Cysteine substitution of arginine298 induces conformational changes to form a disulfide bridge, which leads to markedly increased thermostability.


33

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For Table of Contents Use Only Hyperstabilization of Tetrameric Bacillus sp. TB-90 Urate Oxidase by Introducing Disulfide Bonds through Structural Plasticity

Takao Hibi, Asami Kume, Akie Kawamura, Takafumi Itoh, Harumi Fukada, and Yoshiaki Nishiya


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FIGURE 1. Plots of the irreversible inactivation of BTUO after 30 min incubations at various temperatures are shown; (white) wild type BTUO, (red) mutant R298C, (orange) R298C treated with 10 mM glutathione, and (blue) R298C treated with 10 mM dithiothreitol. Error bars represent the standard errors between different experiments (n = 3). Residual activity data at each temperature were fit to a Boltzmann model using Origin (Origin Lab Co. Ltd.), and the values of wild type BTUO, mutant R298C, R298C treated with 10 mM glutathione, and R298C treated with 10 mM dithiothreitol are represented as black, red, orange, and blue solid lines, respectively. 85x68mm (300 x 300 DPI)


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

FIGURE 2. Differential scanning calorimetry thermograms of BTUO mutant were measured (A) in the absence of Na2SO4 and in the presence of (B) 0.1 M Na2SO4 and (C) 1.0 M Na2SO4 as a salt. The sample solution contains 2.7 mg/mL protein in 50 mM sodium borate (pH 8.5). 357x617mm (72 x 72 DPI)


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Biochemistry

FIGURE 3. DSC scans of R298C/C305S in the absence of Na2SO4. The data points are shown as open circles. The fits of the data to the sequential two-state model of equation (2) are indicated by solid lines. The base line is drawn as a dotted line. 85x114mm (300 x 300 DPI)


Biochemistry

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FIGURE 4. The structures of BTUO R289C in complex with 9-methyl uric acid. (A) Side and top views of a schematized model of the BTUO tetramer. Magnification shows the engineered disulfide bonds as ball and stick models. A β-barrel dimer is surrounded by a dashed line. Figures were prepared using PyMOL.32 (B) σA-weighted |Fo|-|Fc| omit electron density maps for Cys298 shown at the 4.5 σ contour level. The left and right panels represent the ball and stick models of an intersubunit disulfide bond which were rotated by 90° to each other. Figures were prepared using PyMOL.32 170x209mm (300 x 300 DPI)


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Biochemistry

FIGURE 5. The structure of the intersubunit salt bridge between Arg7 and Asp312. The N-terminal residues 6 to 8 of subunit A and the C-terminal residues 311 to 315 of subunit B are drawn as stick models. Other parts of subunit A (green) and subunit B (orange) are depicted as cartoon models and estimated hydrogen bonds are drawn as dashed lines; numbers show the contact distances between the nitrogen atoms of Arg7 in subunit A and the oxygen atoms of Asp312 in subunit B. 85x73mm (300 x 300 DPI)


Biochemistry

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FIGURE 6. The structures of residues 280 to 300 in the interface loop II. (a) A ball-stick model of the interface loop II of the R298C. (b) Comparison between the loop structures of R298C (colored) and wild type (gray) BTUO. 170x124mm (300 x 300 DPI)


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FIGURE 7. Scaled B-factor plots of R298C (solid) and wild type (dashed) BTUO. The B-factors of R298C were scaled using the ratio of Wilson's B-factors between the mutant and wild type enzyme. Upper schematic diagram of the BTUO secondary structure, with α-helix depicted as cylinders, β-strand as block arrows, and others as lines; which were assigned using DSSP.35 Black pins graphically mapped on the sequence label show the location of active-site residues. Thick lines represent the regions (residues 100 to 104, 129 to 132, 272 to 283, 287 to 289, and 291 to 300) in the R298C polypeptide that have B-factors significantly higher than those of wild type enzyme. 86x44mm (300 x 300 DPI)


Biochemistry

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FIGURE 8. Plots of irreversible inactivation of C-terminal truncated mutants after 30 min incubations at various temperatures; (white) wild-type BTUO, (red) mutant R298C, (green) R298C∆C6, (purple) R298C∆C8, (yellow) R298C∆C9, (blue) R298C∆C10, and (black) R298C∆C10 treated with 10 mM dithiothreitol. Error bars of the wild type and R298C BTUO are represented. Residual activity data at each temperature were fit as described in the legend of Figure 1. 313x223mm (72 x 72 DPI)


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Hyperstabilization of Tetrameric Bacillus sp. TB-90 Urate Oxidase by

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