An Integrated Structural and Computational Study of the ...

JOURNAL OF BACTERIOLOGY, July 2003, p. 4285–4289 0021-9193/03/$08.00⫹0 DOI: 10.1128/JB.185.14.4285–4289.2003 Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Vol. 185, No. 14

An Integrated Structural and Computational Study of the Thermostability of Two Thioredoxin Mutants from Alicyclobacillus acidocaldarius Simonetta Bartolucci,1 Giuseppina De Simone,2 Stefania Galdiero,1 Roberto Improta,2 Valeria Menchise,2 Carlo Pedone,2 Emilia Pedone,2 and Michele Saviano2* Dipartimento di Chimica Biologica, University of Naples “Federico II,”1 and Istituto di Biostrutture e Bioimmagini-CNR,2 80134 Naples, Italy Received 21 February 2003/Accepted 3 April 2003

We report a crystallographic and computational analysis of two mutant forms of the Alicyclobacillus acidocaldarius thioredoxin (BacTrx) done in order to evaluate the contribution of two specific amino acids to the thermostability of BacTrx. Our results suggest that the thermostability of BacTrx may be modulated by mutations affecting the overall electrostatic energy of the protein. method at 22°C. Two microliters of a 16-mg/ml protein solution was mixed with 2 ␮l of reservoir solution. K18G crystals grew in 0.1 M HEPES (pH 8)–25% polyethylene glycol 8000– 0.2 M calcium acetate. R82E crystals grew from a solution containing 0.1 M cacodylate (pH 6.5)–25% polyethylene glycol 8000–0.1 M zinc acetate. ˚ resolution for both K18G and Diffraction data at 1.90 A R82E were collected at the EMBL Beamline X11 of the Deutsches Elektronen-Synchrotron, Hamburg, Germany, at 100K. For data collection, the crystals were cryoprotected by addition of 10% glycerol to the reservoir solution and flash-frozen in a cold nitrogen gas stream. The data sets were processed with the HKL crystallographic data reduction package (Denzo/ Scalepack) (30). The crystal parameters and data collection statistics are reported in Table 1. Molecular replacement and refinement. The K18G and R82E structures were solved by molecular replacement (AMoRe) (27). For K18G, the X-ray structure of the Trx from E. coli (Protein Data Bank code 2trx) (22) was used as the molecular replacement search model. The refined structure of K18G was used as the starting model for the crystal structure resolution of R82E. The refinement and model building of K18G and R82E were performed by using the program CNS (7) and the program O (21), respectively. The statistics for refinement are summarized in Table 1. Poor electron density was observed for the loop regions Gln15-Gly18 in K18G and Gln15-Asp17 in R82E. In both structures, the electron density in the C-terminal region (residues Ala101-Gln105) is weak albeit compatible with an ␣-helical conformation. The coordinates and structure factors determined in this study have been deposited in the Protein Data Bank (accession codes 1NSW and 1NW2). Overall structure. The three-dimensional structures of K18G and R82E are very similar to those of thioredoxins from other sources (18, 22), consisting of a central core made up of a five-stranded mixed ␤-sheet surrounded by four ␣-helices (Fig. 1). Both mutants are oxidized with a disulfide bridge forming a 14-member ring involving residues 29 to 32.

In the past few decades, many studies have aimed at unraveling the relationship between protein structure and thermostability. Sequence and structural comparisons between functionally homologous proteins from mesophilic and thermophilic organisms have pointed out that high thermal stability is usually related to multiple factors (8, 11, 16, 17, 19, 20, 25, 37, 39, 41, 42, 45–48). Recently, a series of findings have suggested that thermostability can be also achieved by single ad hoc mutations, possibly allowing the formation of ion pairs or ion networks (26, 44) and/or optimization of long-range coulombic interactions on the protein surface (15, 23, 36, 40). A striking example of this phenomenon is given by previous studies of two mutant forms, Lys18Gly and Arg82Glu, of the thermophilic Alicyclobacillus acidocaldarius thioredoxin (BacTrx) (5, 28) and of a Glu85Arg mutant of the mesophilic Escherichia coli thioredoxin (Trx) (22). Molecular dynamic simulations (33–35) allowed the identification of two surface residues able to modulate the thermostability of both proteins. In fact, replacement of Lys18 or Arg82 of BacTrx with the corresponding residues of Trx (Gly21 and Glu85, respectively) led to a dramatic decrease in thermostability (34). Alternatively, the reverse replacement of Glu85 with one Arg residue made Trx remarkably more thermostable (35). Here we report a crystallographic analysis of the Lys18Gly (K18G) and Arg82Glu (R82E) mutant forms of BacTrx that was done in order to ascertain the structural consequences of these two mutations. Moreover, on the basis of the experimental structures, we performed computational studies on K18G, R82E, and the Glu85Arg (E85R) mutant form of Trx, both at ab initio quantum mechanical and molecular mechanic levels, to evaluate both the energetic contribution of a hydrogen bond highlighted by the X-ray experiments and the effect of the mutations on the overall electrostatic energy of the proteins. Crystallization and data collection. Crystals of the K18G and R82E mutant forms were grown by the hanging-drop * Corresponding author. Mailing address: Istituto di Biostrutture e Bioimmagini-CNR, Via Mezzocannone 6, 80134 Naples, Italy. Phone: 39-081-2536651. Fax: 39-081-5514305. E-mail: saviano@chemistry .unina.it. 4285

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TABLE 1. Data collection and refinement statistics Parameters

Crystal parameters Space group a (Å) b (Å) c (Å) ␤ (°) Z Data collection Resolution (Å) Total reflections Unique reflections Completeness (%) Overall Outermost data shell Rsymc Overall Outermost data shell Mean I/s(I) overall Overall Outermost data shell T (K) Refinement statistics Resolution range (Å) No. of reflections (all) No. of reflections (test) R factor (%)a Rfree (%)b Root mean square deviation from ideal geometry Bond lengths (Å) Bond angles (°) No. of protein atoms No. of nonprotein atoms Avg B factor (Å2), all atoms a b c

K18G

R82E

P21 46.73 73.89 54.91 103.79 8

P21 79.61 60.21 82.95 93.30 16

1.90 120,205 26,803

1.90 234,705 52,158

93.6 95.6

84.1 85.7

3.0 11.5

5.1 26.2

25.5 7.09 100

20.1 4.3 100

20.0–1.90 26,803 1,350 20.5 24.9

20.0–1.90 52,158 5,263 21.2 25.5

0.010 1.64 3224 242 24.86

0.009 1.56 6480 722 26.10

R factor ⫽ ⌺|Fo ⫺ Fc|/⌺Fo. Rfree was calculated with 5% of data withheld from refinement. Rsym ⫽ ⌺|Ic ⫺ ⬍I⬎|/⌺Ii.

The four independent molecules occurring in the asymmetric unit of K18G form two dimers, related by a twofold axis, consisting of the pairs A-B and C-D. The eight independent molecules occurring in the asymmetric unit of R82E form two

tetramers, ABCD and EFGH, which present an approximate 222 symmetry. The two tetramers are related by a twofold axis. In both structures, the only differences between the individual monomers are observed in the N- and C-terminal regions (residues 1 to 4 and 101 to 105), as well as in the region Gln15Gly/Lys18. Except for these regions, the backbones of all of the molecules in each structure are highly similar and the differences can be related to differences in packing. For this reason, one single monomer was arbitrarily chosen for the following discussion and comparisons unless otherwise stated. Comparison of K18G and R82E with BacTrx. The overall fold of K18G and R82E is very similar to that of the nuclear magnetic resonance (NMR) structure of BacTrx (28) (Fig. 1). Superposition of the C␣ atoms of BacTrx onto K18G and R82E yielded root mean square deviations, averaged over all ˚ , respectively. The of the 20 NMR models, of 1.38 and 1.37 A only noticeable differences among the three structures involve very flexible regions, located in the N- and C-terminal segments, in loop 15 to 18 between ␣1 and ␤2 and in loop His46Val50 between ␤2 and ␤3. The Gln15-Asp17 region is very flexible in all three enzymes, while there are important differences in the residue at position 18 (Fig. 2A). In fact, in R82E, there is a hydrogen bond between the Lys18 side chain (Nz) and the carbonyl oxygen of Ala47. The corresponding residue in K18G (Gly18) is disordered and very poorly defined in the electron density maps. Lastly, in the NMR structure of BacTrx, the side chain of Lys18 establishes electrostatic interactions with Asp17 and/or Asp48. In the region corresponding to mutation 82, the three proteins superimpose well (Fig. 2B). In the three structures, residue 82 is exposed to the solvent and whereas in BacTrx it is hydrogen bonded to the carbonyl oxygen of Pro83, in K18G and R82E, it is not involved in any significant intramolecular interaction. Computational results. Since an H bond between a charged residue and the backbone is reported to have a favorable effect on protein thermostability (24, 43), we verified whether the loss of the hydrogen bond between Lys18Nz and Ala47O plays

FIG. 1. Stereo view showing superposition of the C␣ trace of BacTrx (red), K18G (blue), and R82E (green).

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FIG. 2. Stereo views showing the superposition in the region close to residue 18 of BacTrx (red), K18G (blue), and R82E (green) (A) and the superposition in the region close to residue 82 of BacTrx (red), K18G (blue), and R82E (green) (B). Hydrogen bonds are shown.

a relevant role in destabilizing the K18G mutant with respect to the wild type. In particular, we evaluated the energetic stabilization resulting from this specific hydrogen bond, performing quantum mechanical calculations on a model system composed of dipeptide analogues of Lys18 and Ala47. Starting from the crystallographic structure of R82E, the geometry of the hydrogen atoms and of the lysine side chain was optimized by means of HF/6-31G(d) calculations, freezing all of the remaining geometrical parameters to their crystallographic values. Finally, accurate single-point PBEO/6-311⫹G(d,p) calculations (1) were performed both on the adduct and on the insulated molecules, taking into account solvent effects by means of CPCM/HF/6-31G(d) calculations (2–4). All of the quantum mechanical calculations were performed with the Gaussian 98 package (13) by using standard 6-31G(d) and 6-311⫹G(d,p) basis sets (12). After calculation of the basis set superposition error (6), the adopted DFT/PCM (density functional theory/ polarizable continuum model) computational strategy, that already gave reliable estimates of inter- and intramolecular interaction energies in several biological systems (14, 29), provided a final value of 1.55 kcal/mol for the hydrogen bond strength in an aqueous solution. However, this interaction should lead to a comparable entropy loss (10) and thus, it should play only a minor role in determining the thermostability of BacTrx in vivo, as confirmed by NMR experiments performed on BacTrx in an aqueous solution at room temperature (28) that did not indicate a Lys18Nz-Ala47O H bond. We then evaluated the effects of the Lys18Gly and Arg82Glu mutations in BacTrx on the overall interresidue electrostatic energy of the protein. The electrostatic energy of the native protein and of the two mutant forms was computed in accordance with the Coulomb law (with the AMBER6 package [9], the 1,994 atomic charges, and a protein dielectric constant of 80) on the basis of models built starting from the experimental structure of R82E with the opportune side chain mutation and without alteration of the backbone structures. We used stan-

dard pH 7 titration states for all of the amino acid side chains, checking that the histidine protonation state did not significantly affect our results. The same procedure was repeated also starting from the crystal structure of K18G and the NMR structure of BacTrx, thus demonstrating that our results were not biased by the choice of the starting model. The electrostatic interaction energy decreased in the order native ⬎ K18G ⬎ R82E and suitably matched the relative thermostability trend from calorimetric studies (Table 2) (34). Electrostatic calculations on the Lys18Gly/Arg82Glu BacTrx double mutant (K18G/R82E) (last column of Table 2), built in accordance with the procedure outlined above, predicted that this species is less stable than K18G and R82E, in agreement with the experimental data (34). The decomposition of the total electrostatic energy in the individual amino acid contributions made it possible to determine which interactions were responsible for the larger stabilization of the native protein. Interestingly, if only the charged residues closest to amino acid 82 were considered, Glu would be more favored than Arg at position 82, since that position is

TABLE 2. Relative interresidue electrostatic energy of the three BacTrx mutants and the Trx mutant with respect the native proteinsa Relative interresidue electrostatic energy (kcal/mol)

Starting structure

Wild type K18G R82E Trx

R82E

K18G

K18G/R82E

2.65 2.60 2.93

2.26 1.86 2.56

5.38 4.94 5.90

E85R ⫺2.04

a Interresidue electrostatic energies of wild-type proteins: ⫺91.9975 (wild type); ⫺93.3476 (K18G); ⫺95.0848 kcal/mol (R82E); Trx, ⫺103.1202 kcal/mol.

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FIG. 3. CPK representation of BacTrx. Acid residues are shown in red, basic residues are shown in blue, and Lys18 and Arg82 are shown in cyan.

surrounded by two positively charged residues (Lys79 and Lys85) and is not very distant from Lys18 (Fig. 3). However, nonspecific nonlocal interactions stabilized Arg over Glu. Lys18 exhibits two strong interactions with the side chains of Asp48 and Asp17. In the NMR bundle of structures obtained for BacTrx (28), Lys18 indeed forms a salt bridge with either the former or the latter of these two aspartic acid residues. However, analysis of our results suggests that the stabilizing effect of Lys18 is due not to these specific interactions but to the maximization of all of the favorable long-range interactions between Lys18 and negatively charged spots present within the protein, especially with the Glu40-Glu44 and C-terminal stretches of the protein (Fig. 3). In fact, the electrostatic stabilization coming from the Lys18-Asp48 interaction is reduced by a corresponding increase in repulsion with the adjacent Lys49 residue. Also, the Lys18-Asp17 interaction should not have any significant effect on the thermostability of BacTrx. It is likely to be present also in the unfolded state, since its presence seems to depend only on the proximity of adjacent residues and not on any specific structure-dependent interaction (31). The same consideration also holds true for the hydrogen bond formed, according to the NMR experiments, by Arg82 and the carbonyl group of Pro83. The importance of surface electrostatics is fully confirmed by analogous calculations performed on Trx and on its E85R mutant (see Table 2). Indeed, this mutation leads to a relevant increase in overall electrostatic interaction energy, in agree-

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