INTRODUCTION
Carboxypeptidasefamily proteins catalyze the removal of Cterminal aminoacid residues from pro teins and peptides. Carboxypeptidase T (CPT) from Thermoactinоmyces vulgaris belongs to metallocarbox ypeptidases containing the catalytic zinc ion in the active site. Carboxypeptidase T has 30 and 27% iden tical aminoacid residues with pancreatic metallocar boxypeptidases A (CPA) and B (CPB) and is their dis tant homologue [1]. The threedimensional structure of wildtype carboxypeptidase determined at 2.3 Å resolution [2] is very similar to the structures of CPA and CPB. Although CPA, CPB, and CPT have similar threedimensional structures, these enzymes substan tially differ in the substrate specificity. CPA cleaves off bulky Cterminal hydrophobic residues, CPB removes positively charged residues, and CPT has a mixed specificity and cleaves both types of substrates but at lower rates compared to CPA and CPB. Due to these properties, CPT is the most convenient enzyme for investigations of the structural basis of the specificity.
In a number of studies it was shown that the sub strate specificity of enzymes can be influenced not only by the aminoacid residues directly interacting with the substrate, but also distant aminoacid resi dues [3, 4]. The kinetic study of the substrate hydroly sis by carboxypeptidase T showed that the binding of calcium ions to the enzyme leads to substantial
changes in the kinetic parameters of hydrophobic and charged substrates [5]. The Xray diffraction study of the threedimensional structure of wildtype CPT showed that the CPT molecule, unlike the CPA and CPB molecules, contains four bound calcium ions [2]. In this study we produced recombinant CPT with the aim of revealing possible differences in the three dimensional structure of the enzyme with occupied and free calciumbinding sites. Crystals of recombi nant CPT were grown in the absence of calcium ions. The structure of calciumfree CPT was determined at 1.69 Å resolution. Threedimensional structures of calciumcontaining and calciumfree CPT were com pared. Based on the analysis of the conformational changes, the possible mechanisms of the influence of metal ions on the kinetic parameters of the enzymatic reaction are considered.
MATERIALS AND METHODS
Isolation and purification of CPT. Recombinant CPT was produced by cloning the CPT gene into E. coli followed by renaturation from inclusion bodies and purification by affinity chromatogra phy on paminobenzylsuccinic acid coupled to acti vated Sepharose [6].
Crystal growth of CPT. Crystals of CPT were grown by the counterdiffusion method in a capillary inserted
STRUCTURE OF MACROMOLECULAR COMPOUNDS
ThreeDimensional Structure of Recombinant Carboxypeptidase T
from
Thermoactinomyces vulgaris without Calcium Ions
V. Kh. Akparova, V. I. Timofeevb, and I. P. Kuranovab
a Scientific Center of Russian Federation Research Institute for Genetics and Selection of Industrial Microorganisms, Pervyi Dorozhnyi proezd 1, Moscow, 113545 Russia
email: valery@akparov.ru
b Shubnikov Institute of Crystallography, Russian Academy of Sciences, Leninskii pr. 59, Moscow, 119333 Russia
email: inna@ns.crys.ras.ru Received March 9, 2011
Abstract—Crystals of recombinant carboxypeptidase T (CPT) from Thermoactinomyces vulgaris were grown
in a capillary by the counterdiffusion method in the absence of calcium ions. The threedimensional structure of CPT was solved at 1.69Å resolution using the Xray diffraction data collected from the crystals of the enzyme on the SPring8 synchrotron radiation facility and was then refined to Rfact = 16.903% and Rfree = 18.165%. The coordinates of the refined model were deposited in the Protein Data Bank (PDB ID: 3QNV). A comparison of this structure with the structure of wildtype CPT containing bound calcium ions, which was determined earlier, revealed a number of conformational changes both in the calciumbinding sites and the enzyme active site. Based on the results of this comparison, the possible factors responsible for the difference in the catalytic activity of the two forms of the enzyme are considered.
DOI: 10.1134/S106377451104002X
into a gel tube according to the procedure described in [7]. A protein solution (10 µL) composed of the pro tein with a concentration of 10 mg/mL in a 50 mM MES/NaOH buffer, pH 6.0, containing 0.25 M NaCl was placed in a 0.5mm capillary. The free end of the capillary was hermetically sealed with plasticine, and then the opposite end of the capillary was inserted into a silicone tube filled with a 1% agarose gel containing 0.04% NaN3. The end of the tube was cut with a blade
to a length of 10 mm. The capillary with the tube was placed into a vessel with a screwedon cap containing a reservoir solution (1 mL) composed of 1.8 M (NH4)2SO4 in a 50 mM MES/NaOH buffer, pH 6.0, and 3% MPD. Photos of CPT crystals in the capillary are shown in Fig. 1.
Xray diffraction data collection and processing for CPT crystals. The Xray diffraction data were collected from crystals at 100 K to 1.50 Å resolution at the BL41XU beamline of the SPring8 synchrotron radia tion facility (Japan) using a MX225HE CCD detector. Before the Xray diffraction experiment, the crystals were flashfrozen with the use of a reservoir solution containing an additional 20% glycerol as the cryopro tectant solution. The Xray diffraction data were mea sured by the rotation method from one crystal at a crystaltodetector distance of 185 mm. The angles of oscillation and rotation were 0.3° and 180°, respec tively. The wavelength was 0.8 Å. The experimental Xray intensities were processed using the HKL2000 program package [8]. The crystals belong to sp. gr. R6322. The unitcell parameters are a = b = 158.002 Å, c = 103.913 Å, α = β = 90°, and γ = 120°. The Xray datacollection statistics are given in Table 1. The sol vent content in the unit cell is 76.01%. The Matthews coefficient [9] is 5.12 Å3/Da. There is one enzyme monomer per asymmetric unit.
Structure solution and refinement. The structure of CPT was solved by the molecularreplacement method with the use of the Phaser program [10] and the coordinates of this enzyme (which were deter mined at a lower resolution) as the starting model (PDB ID 1OBR). The structure refinement was car ried out using the Refmac program [11]. The manual rebuilding was performed with the use of the Coot interactive graphics program [12] based on electron density maps calculated with the coefficients (2|Fo|– |Fc|) and (|Fo|–|Fc|). Water molecules, the zinc ion, sulfate ions, and glycerol molecules were also located in electrondensity maps using the Coot program. The structure was refined at 1.69 Å resolution to Rcryst = 16.903% and Rfree = 18.165%. The coordinates of the refined model were deposited in the Protein Data Bank (PDB ID 3QNV). The refinement statistics are summarized in Table 2.
RESULTS AND DISCUSSION
The threedimensional structure of recombinant CPT was determined with the use of crystals of the
enzyme grown in the absence of calcium ions. Metal ions were found in none of four calciumbinding sites. The calciumfree form of the enzyme was used for a comparison with the wildtype enzyme containing four bound calcium ions [2].
As was shown in [13], the thermal stability of the enzyme is substantially enhanced in the presence of calcium ions. Earlier, A.M. Grishin et al. studied the kinetics of the hydrolysis of various substrates by
Fig. 1. Crystals of recombinant CPT grown in a capillary by the counterdiffusion method.
Table 1. Xray datacollection statistics for crystals of CPT
Sp. gr. P6322
Unitcell parameters, Å, deg a = b = 158.002, c = 103.913,
α = β = 90, γ = 120
Resolution range, Å 30.00–1.50 (1.51–1.5)*
Number of reflections 7 810 976
Number of reflections per asymmetric unit 964318 Redundancy 8.1 (3.6) Crystaltodetector distance, mm 185.00
Oscillation angle, deg 0.3
Rotation angle, deg 180
Completeness of the data set, %
98.4 (95.8) I/σ 14.209 (2.318) Rsym(I), % 9.3 (41.6)
l (wavelength), Å 0.8
recombinant CPT and found that calcium ions have an effect on the parameters of the enzymatic reaction [5]. The sigmoidal character of the dependence on the calcium ion concentration shows that the kinetic parameters are influenced by the structurally bound metal ions. The enzyme containing bound calcium ions hydrolyzes hydrophobic substrates and charged substrates 1.5 times faster and 1.7–2.2 times slower than the calciumfree enzyme. With the aim of eluci dating possible factors responsible for changes in the activity depending on the presence of calcium ions, we compared the threedimensional structures of the cal ciumcontaining and calciumfree forms of the enzyme.
The model of recombinant CPT refined at 1.69 Å resolution was compared with the structure of the wildtype enzyme determined earlier. For this pur
pose, the structures were superimposed based on Cα atoms with the use of the Lsqkab program from the CPP4 program suite [14]. The polypeptidechain fold and the arrangement of the functionally important regions of the CPT molecule are presented in Fig. 2, where the zinc ion and its environment in the catalytic site, as well as the selected aminoacid residues of the substratebinding site, are shown. The calcium ions are represented as smaller filled spheres. The main dif ferences in the secondary structure of recombinant and wildtype (1OBR) CPT—additional helical regions at the N terminus of the molecule (residues 3– 7) and in the active site (residues 144–148) in the cal ciumfree structure—are shown in black and indi cated by arrows. A detailed pattern of the changes in the secondary structure is presented in Fig. 3.
The main differences between the model of recom binant CPT and the structure of the wildtype enzyme determined earlier are the absence of calcium ions in the calciumbinding sites, changes in the conforma tion of aminoacid residues, and changes in the posi tion and number of solvent molecules involved in the coordination sphere (Figs. 4a, 4b). Of the four cal ciumbinding sites in CPT, three sites are located in the loop 51–61 as a cluster at a distance of approxi mately 30 Å from the zinc ion in the active site, and one calcium ion is located in the Nterminal region of the molecule.
In the absence of calcium ions in the binding site 1, the mainchain oxygen of Glu57 is in the opposite orientation, the side chain of Asp56 is rotated by approximately 90°, and the position of one water mol ecule substantially changes. In the binding site 2, sub stantial changes are observed in the positions of Asp 51, Glu57, and Glu59 and a new water molecule (HOH 562) appears. In the binding site 3, a water mol ecule is located at a distance of 0.76 Å from the site occupied by the calcium ion in the structure of the wildtype enzyme, whereas the water site that is involved in the coordination environment of the cal cium ion in the wildtype enzyme remains unoccupied (Fig. 4a). The formation of a turn of the helix at the N terminus of the protein molecule after the removal of the calcium ion from the binding site 4 is accompanied by a slight shift in the mainchain oxygen of Ser7 and the oxygen atom OE2 of Glu14 (Fig. 4b).
The removal of the bound calcium ions leads to the destabilization of the threedimensional structure of CPT. This is manifested, in particular, in a decrease in thermal stability of the molecule, as was shown in [13]. The higher lability of the aminoacid residues of the calciumbinding sites after the removal of the calcium ions is also confirmed by a comparison of the ratios of the average B factor for the aminoacid residues of the calciumbinding sites to the average B factors in the calciumcontaining and calciumfree structures. This ratio is 0.684 and 0.593 in the calciumcontaining and calciumfree structures, respectively, which is indica tive of an increase in the average B factor of the resi Table 2. Refinement statistics for CPT
Resolution, Å 10.00–1.69 (1.73–1.69)*
Number of reflections in the re finement
79903 (5668)
Number (5%) of reflections in the test set
4183 (287)
Completeness of the data set, % 99.12 (98.01)
Number of refined residues in the protein molecule
323
Number of refined atoms in the protein molecule
2712
Number of water molecules 265
Number of sulfate ions 4
Number of glycerol molecules 4
Rcryst, % 16.903 (26.7)
Rfree,% 18.165 (27.7)
rmsd bond lengths, Å 0.006 rmsd bond angles, deg 1.006
Average temperature factor (Å2) for:
mainchain atoms 11.802
sidechain atoms 12.111
water 26.564
Ramachandran statistics
in most favored regions, % 88.7
in allowed regions, % 11.4
in disallowed regions, % 0.0
Gln250 Ala251 Asp260 Arg147 Thr262 Ca1 Ca2 Ca3 His204 Glu72 Arg129 His69 Ca4 Asn146
Fig. 2. Threedimensional structure of CPT. Calcium ions are represented as four spheres of the same size. The zinc ion in the active site is shown as a larger sphere. The aminoacid residues coordinating zinc and selected aminoacid residues of the sub stratebinding site are shown. The main differences in the secondary structure of recombinant and wildtype (PDB ID 1OBR) CPT are shown in black and indicated by arrows.
1 3QNV: Sequence: 1OBR: 71 3QNV: Sequence: 1OBR: 141 3QNV: Sequence: 1OBR: 211 3QNV: Sequence: 1OBR: 281 3QNV: Sequence: 1OBR:
dues involved in the closest environment of the cal cium ions compared to the average B factor in the cal ciumfree structure.
In addition to the changes in the calciumbinding sites of the enzyme, the absence of calcium ions leads to certain conformation shifts in the activesite cavity (Fig. 5). In the structure of recombinant calciumfree CPT, three additional water molecules (HOH369, HOH499, and HOH568) are located in the region of the active site. The molecule 568 appears in the vicin ity of the oxygen atom OE1 of Gln250, which is accompanied by a shift of the side chain of this residue (the oxygen atom OE1 is shifted by 2.89 Å). The mol ecule 369 is found at a hydrogenbond distance from this residue. Yet another new water molecule is found at a distance of 2.68 Å from the mainchain oxygen atom of Ala251, at a distance of 3.2 Å from the main chain oxygen atom of Tyr255, and at a distance of 2.56 Å from the oxygen atom O3 of glycerol 329 (which, apparently, appeared in the active site as a result of soaking of the crystal in the cryoprotectant solution). It should be noted that the glycerol mole cule has low occupancy and a high B factor. The appearance of the abovementioned water molecule is accompanied by a shift of the mainchain oxygen atom of Ala251 by 0.48Å.
To elucidate the possible functional role of water molecules found in the active site, the structure of cal ciumfree CPT was superimposed with the structure of CPB containing the inhibitor covalently bound to the zinc ion in the active site (PDB ID 1ZG9). The structures were superimposed based on the Cα atoms of 17 homologous aminoacid residues of the active site with the use of the Lsqkab program from the CPP4 program suite [14]. The position of the inhibitor
(a) (b) Asn101 Ca3 Ca4 Ca2 Ca1 Glu61 Glu59 Glu104 Asp56 Glu57 Asp51 Ser50 Ser7 Tyr9 Glu14
Fig. 4. Conformational changes in the calciumbinding sites (a) 1, 2, 3, and (b) 4 of wildtype (pale gray) and recombinant (black) CPT. The calcium ions and water molecules are shown as large and small spheres, respectively. The calcium ions and the ligands in their coordination environment are connected by dashed lines.
Tyr255 499 GOL329 Ala251 568 369 Gln250
Fig. 5. Conformational changes in the activesite cavity of wildtype (pale gray) and recombinant (black) CPT. The water molecules located in the active site of recombinant CPT are shown as filled spheres; GOL329 is the glycerol molecule 329.
placed in the active site of CPT is shown in Fig. 6. It appeared that the water molecule 499 is present in the vicinity of the guanidine group of the inhibitor (Fig. 7) in the primary specificity pocket of CPT [6, 15]. All these water molecules are in the primary specificity site of the enzyme, and one of these molecules (499) can directly interact with the substrate.
According to [16], the appearance of additional water molecules and additional hydrogen bonds in the activesite pocket can affect the strength of the binding of the substrate in the active site, which would be reflected in the rate of hydrolysis. The results of this study provide an explanation for the influence of cal cium ions on the enzyme catalysis (this fact was found earlier) and show that calcium ions can be considered distal specificity determinants of the enzyme, which was hypothesized in [5].
ACKNOWLEDGMENTS
This study was supported by the Russian Founda tion for Basic Research (project no. 100401541a)
and the Central ScientificResearch Institute of Mechanical Engineering of the Russian Federal Space Agency (Roscosmos).
REFERENCES
1. S. V. Smulevitch, A. L. Osterman, O. V. Galperina, et al., FEBS Lett. 291, 75 (1991).
2. A. Teplyakov, K. Polyakov, G. Obmolova, et al., Eur. J. Biochem. 208, 281 (1992).
3. J. J. Perona and C. S. Craik, Protein Sci. 4, 337 (1995). 4. B. Jelinek, J. Antal, I. Venekei, and L. Gráf, Protein
Eng. Design Selection 17, 127 (2004).
5. A. M. Grishin, V. Kh. Akparov, and G. G. Chestukhina, Biokhimiya 73, 1422 (2008).
6. V. Kh. Akparov, A. M. Grishin, M. P. Yusupova, et al., Biokhimiya 72, 515 (2007).
7. H. Tanaka, K. Inaka, Sh. Sugiyama, et al., J. Synchro tron Rad. 11, 45 (2004).
8. Z. Otwinowski and W. Minor, Methods in Enzymology. 276, 307 (1997). Arg129 Asn146 354 499 380 Arg147 Asp260 Thr257 Ala251 Tyr255 Gln250
Fig. 6. Arrangement of the inhibitor 5guanidino2mer captomethylpentanoic acid (shown in black) placed in the active site of CPT as a result of the superposition of the structures of recombinant CPT and CPB containing the ligand (PDB ID: 3ZG9). The water molecules located in the activesite cavity in the vicinity of the inhibitor are rep resented as filled spheres.
499
Ala251
Gln250
568
369
Fig. 7. Conformational shifts of the aminoacid residues Gln250 and Ala251 of the substratebinding sites in wildtype (pale gray) and recombinant (black) CPT. The position of the inhibitor (shown in black) placed in the active site of CPT as a result of the superposition of the structures of recombinant CPT and CPB containing the ligand (PDB ID: 3ZG9) is shown in the upper right corner in the vicinity of the zinc ion (a gray sphere). The water molecules are represented as filled spheres.
9. B. W. Matthews, J. Mol. Biol. 33, 491 (1968).
10. A. J. McCoy, R. W. GrosseKunstleve, L. C. Storoni, et al., Acta Crystallogr. D 61, 458 (2005).
11. G. N. Murshudov, A. A. Vagin, and E. J. Dodson, Acta Crystallogr. 53, 240 (1997).
12. P. Emsley and K. Cowtan, Acta Crystallogr. D 60, 2126 (2004).
13. A. L. Osterman, V. M. Stepanov, G. N. Rudenskaya, et al., Biokhimiya 49, 292 (1984).
14. COLLABORATIVE COMPUTATIONAL PROJECT, NUMBER 4, Acta Crystallogr. D 50, 760 (1994). 15. V. Kh. Akparov, A. M. Grishin, V. I. Timofeev, and
I. P. Kuranova, Kristallografiya 55, 851 (2010) [Crys tallogr. Rep. 55, 802 (2010)].
16. A. R. Fersht, J. P. Shi, and J. KnillJones, Nature 314 (6008), 235 (1985).
