INTRODUCTION

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Adenylate kinase (AK,¹ ATP:AMP phosphotransferase, EC 2.7.4.3) is a ubiquitous enzyme which contributes to homeostasis of adenine nucleotides in living cells (1). Three classes of AKs, differing in size, subcellular localization, and substrate specificity were identified in vertebrates, AK₁ in the cytosol, AK₂ in the mitochondrial intermembrane space, and AK₃ (called also GTP:AMP phosphotransferase) in the mitochondrial matrix. Only one form of AK was identified in bacteria. Mitochondrial adenylate kinases (AK₂, AK₃) and the vast majority of bacterial adenylate kinases belong to the class of long forms. They differ from AK₁ and some bacterial AKs, the short variants, by a 28-residue long insertion organized into a small domain (2) called LID well exposed to the solvent (Fig. 1A) and undergoing a large movement during catalysis (3, 4).

View larger version (54K): [in this window] [in a new window]   Fig. 1.   Pictorial representation of the three-dimensional structure of AKe and of the LID domain after substitution by 4 cysteines (A) and alignment of partial amino acid sequences of the LID domain in long isoforms of adenylate kinase (B). The model was obtained using the atomic coordinates of AKe (32). The 4 Cys side chains were substituted in the corresponding positions His126, Ser129, Asp146, and Thr149 without searching for side chain optimization after substitution. The figure was drawn using the MOLSCRIPT software (33). The residues critical for coordination of zinc atom in Gram-positive bacteria and their counterparts in Gram-negative organisms are boxed.

AKs from Gram-positive bacteria contain a structural zinc atom (5-7), a property which is due to the presence of 3 or 4 cysteine residues in the LID domain. Sequence alignment of AKs from Gram-positive and Gram-negative organisms, devoid of metal, showed that in the latter species the Cys residues are substituted with four other highly conserved amino acids, His, Ser, Asp, and Thr (Fig. 1B). This conservation suggests that these particular residues have some essential function, but different in the enzyme from the Gram-negative bacteria and eukaryotes. A noticeable exception is AK from the Gram-negative bacterium Paracoccus denitrificans. This enzyme not only conserves the Cys-containing sequence found in AK from Gram-positive species but binds zinc or iron (8).

In this study, we substituted His¹²⁶, Ser¹²⁹, Asp¹⁴⁶, and Thr¹⁴⁹ in Escherichia coli adenylate kinase with cysteine residues. Our aim was to know whether a motif composed of 3 or 4 Cys residues generates a metal-binding site in AK or whether other structural factors contribute to the specificity (zinc versus iron or any other metal) or to the strength of the protein/metal interaction. On the other hand, we wanted to know the relevance of the metal binding for catalysis or stability of AK. A number of variants of AK_e containing one to four cysteine residues were thus obtained. In agreement with previous studies on zinc-binding AKs, we found that the 3 and 4 cysteine modified forms of AK_e acquired zinc binding properties. Moreover, the 4 cysteine-containing AK_e exhibited an increased stability against thermal denaturation as compared with the wild-type form, with full conservation of its catalytic properties.

	EXPERIMENTAL PROCEDURES

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Materials-- Adenine nucleotides, coupling enzymes, T4 DNA ligase, T4 polynucleotide kinase, and restriction enzymes were from Boehringer Mannheim. T4 DNA polymerase was from Biolabs. T7 DNA polymerase and Deaza sequencing mixes kit were from Amersham Pharmacia Biotech. Blue Sepharose (Cibacron Blue 3 G-1 Sepharose CL-6B) was from Pharmacia LKB Biotechnologies Inc. TPCK-treated trypsin, soybean trypsin inhibitor, PMPS, 4-(2-pyridylazo)resorcinol (PAR), and DTNB were purchased from Sigma.

Bacterial Strains, Plasmids, and DNA Manipulations-- The E. coli NM554, CJ236, and BL21(DE3) strains were used for cloning experiments, site-directed mutagenesis, and recombinant protein overproduction, respectively (9, 10). Plasmid pDIA17 harboring the lacI gene provides additional transcriptional control, under nonpermissive conditions. Plasmid pEAK91 carries the E. coli adk gene (11) and was kindly provided by A. Wittinghofer (Max Planck Institut für Molekulare Physiologie, Dortmund). Plasmid pPV1003 is a pET22b derivative carrying the adk gene subcloned from pEAK91.

Site-directed Mutagenesis, DNA Sequence Analysis, and Growth Conditions-- Site-directed mutagenesis was carried out according to Kunkel et al. (12). A 91 bases long primer (see Table I) containing 7 mismatched bases allowed several simultaneous substitutions in the adk gene. Mutant plamids from 48 randomly selected clones were further analyzed. A panel of single, double, triple, and quadruple mutants was obtained. Some additional variants, not resulting from this procedure, were created individually with appropriate primers (see Table I). Absence of any other mutation in the adk gene was checked on all plasmids. Overproduction of various AK forms was performed by growing strain BL21(DE3)/pDIA17 containing pVP1003 derivatives in LB medium (13) supplemented with 100 mg/liter ampicillin and 30 mg/liter chloramphenicol. Overproduction was carried out by adding 1 mM isopropyl--D-thiogalactoside when the culture reached an absorbance at 600 nm of 1.0. Bacteria were harvested by centrifugation 3 h after induction.

Purification of AK_e and Activity Assays-- The adenylate kinase overproduced in E. coli was purified as described previously (14). When required, purified proteins were dialyzed against 50 mM ammonium bicarbonate, then lyophilized. Enzyme activity was determined at 30 °C using the spectrophotometric assay (15). Measurements were made at 334 nm (0.5 ml final volume) using an Eppendorf ECOM 6122 photometer. One unit of enzyme activity corresponds to 1 µmol of the product formed in 1 min at 30 °C and pH 7.4 (in the direction of ATP formation). Protein concentration was determined according to Bradford (16), using a Bio-Rad kit. SDS-polyacrylamide gel electrophoresis was performed as described by Laemmli (17).

Zinc Content-- The metal in various forms of AK was quantitated colorimetrically, using the metal-binding dye PAR as described previously (5, 7) and by atomic absorption spectrophotometry, using a graphite furnace instrument. The protein samples and the zinc standard solutions were diluted with water purified to 18.2 megohms/cm resistivity. In all cases, the background levels of zinc were insignificant.

Differential Scanning Calorimetry-- The thermal stability of different proteins was studied by differential scanning calorimetry using an ultrasensitive Microcal MC-2D instrument at a scanning rate of approximately 50 °C/h. Proteins in 50 mM Tris-HCl buffer (pH 7.4) were in the range of 1-1.5 mg/ml. Differential scanning calorimetry data were analyzed by the software provided by Microcal Inc., Northampton, MA.

Nomenclature-- The mutants were named according to the position of key residues in the motif ¹²⁶His-X₂-Ser-X₁₆-Asp-X₂-Thr¹⁴⁹. Thus, the HSDT variant is the wild-type AK_e. AK_eC₄ corresponds to the 4 Cys-substituted enzyme, AK_eHC₃ to HCCC, AK_eC₃T to CCCT, AK_eC₂DT to CCDT, AK_eHSC₂ to HSCC, and AK_eHC₂T to HCCT.

	RESULTS

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Overproduction and Purification of Cysteine-substituted Variants of AK_e-- To create a 4-Cys-substituted mutant of AK_e, a single nondegenerate 91-base oligonucleotide, was designed spanning the adk gene region corresponding to the LID domain in the protein (Table I). The 7 mismatched bases in the oligonucleotide allow simultaneous substitutions of His¹²⁶, Ser¹²⁹, Asp¹⁴⁶, and Thr¹⁴⁹ codons with cysteines. Out of 48 randomly selected clones, two-thirds carried one or several substitutions with cysteine residue(s) and one-third harbored the expected quadruple modification. Considering the length of the oligonucleotide and the relatively low yield of its synthesis, the mutagenesis reaction was fairly effective and produced in one step a panel of single, double, and triple mutants displaying different positions of substitution. Missing species were constructed with appropriate primers (Table I).

Metal Binding-- The zinc content of different variants of AK_e, was quantified either by atomic absorption spectrophotometry or with the metal-binding dye PAR. The enzymes were first reacted with PMPS (18, 19), the formation of the PMPS-sulfhydryl chromophore being followed at 250 nm. Linear incorporation of PMPS into the proteins was observed up to 3.4 ± 0.3 equiv./mole of C₄ mutant, 2.3 ± 0.1 equiv./mole of HC₃ and C₃T mutants (Fig. 2A). The released Zn²⁺ (0.73-0.82 mol of zinc/mol of protein) was determined spectrophotometrically with PAR (Fig. 2B). Atomic absorption spectrophotometry confirmed that the quadruple and the triple Cys mutations conferred to the protein the ability to bind the metal (0.8 ± 0.1 mol of zinc/mol of protein). Less than 0.03 mol of zinc/mol of protein was found in the wild-type AK_e. No iron was observed in AK_eC₄ when E. coli was cultivated in minimal medium supplemented with this metal (data not shown).

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Titration of the mutated forms of AKe with PMPS. A, AKe (25 nmol in 0.6 ml of 10 mM Tris-HCl, pH 8.0) was treated with PMPS (1 mM solution in the same buffer) to give the indicated molar ratios of PMPS to enzyme. The absorbance at 250 nm was measured relative to the control value at the beginning of the titration. B, titration of each variant (3.5 µM in the same buffer) was also performed in the presence of PAR, where the absorbance developed at 500 nm reflects formation of a zinc-dye complex (m = 6.6 × 104). , AKeC4; , AKeHC3; and , AKeC3T variants.

Reaction of Wild-type AK_e and of Cysteine-substituted Enzymes with DTNB-- Wild-type AK_e contains a buried cysteine residue in position 77. It reacted with DTNB only in the presence of urea over 2 M (15). The same was true for AK from Bacillus subtilis and AK from Bacillus stearothermophilus, although they contain, besides the conserved Cys⁷⁷, 3 and, respectively, 4 other Cys residues in the LID domain (5, 7). It was, therefore, surprising to find that Zn²⁺-chelating AK_e variants reacted with DTNB under native conditions (Fig. 3). The kinetics of the reaction with DTNB of these mutants was fitted to a single exponential equation. Over 0.5 mM DTNB, the values of k_obs (5.10³ s¹ for AK_eC₄, 7.10³ s¹ for AK_eHC₃ and 14.10³ s¹ for AK_eC₃T) were practically independent on the concentration of thiol reagent. Thus, the first order process might reflect the dissociation rate of the AK_e-Zn²⁺ complex.

(Eq. 1)

As preincubation of AK_eC₄ with ZnCl₂ did not affect the k_obs (data not shown), one might assume that difference in affinity for metal is primarily due to the dissociation rate constant for protein-metal complex.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Reaction of AKe with DTNB under native conditions. AKe (in 10 mM Tris-HCl, pH 8.0) was treated with 20 µl of 10 mM DTNB, then the absorbance increase was read at 412 nm. The ratio of thiols reacted to moles of AKe was calculated using a molecular mass of 23.5 kDa. Symbols are the same as those used in Fig. 2, to which wild-type AKe () and AKsub () were added.

Thermal Stability and Proteolysis by Trypsin of Cys-modified Mutants of AK_e-- In preliminary experiments, different proteins were heated for 10 min at various temperatures between 40 and 80 °C, after which the residual enzyme activity was determined. The wild-type AK_e and the C₃T, CSDT, HCDT, and HSDC mutants were half-inactivated at temperature between 51 and 54 °C; the C₄ and HC₃ variants exhibited a higher thermal stability (half-inactivation at 65 and 58 °C, respectively) than the wild-type AK_e, whereas the C₂DT mutant was less resistant (half-inactivation at 46 °C).

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Differential scanning calorimetric traces for wild-type AKe (A), HC3 (B) and C4 (C) mutants. The curves were obtained by smoothing raw calorimetric data and substracting from them the base lines using a cubic interpolation procedure.

View larger version (65K): [in this window] [in a new window]   Fig. 5.   Proteolysis by TPCK-trypsin of the wild-type and of the mutated forms of AKe. AKe at 1 mg/mL in 50 mM Tris-HCl, pH 7.4, was incubated at 30 °C with TPCK-trypsin (2 µg/mL). At different time intervals, 10-µl aliquots were withdrawn, boiled with electrophoresis buffer, and analyzed by SDS-polyacrylamide gel electrophoresis (12.5% gel) and Coomassie Blue staining. Lanes 1 and 2, wild-type AKe (0 and 25 min); lanes 3 and 4, HCDT mutant (0 and 25 min); lanes 5 and 6, C4 mutant (0 and 25 min); and lanes 7 and 8, CSDT mutant (0 and 3 min). Lane 9, standard proteins, from top to bottom, phosphorylase a (94,000), bovine serum albumin (66,200), ovalbumin (43,000), carbonic anhydrase (30,000), soybean trypsin inhibitor (21,100), and lysozyme (14,400).

Catalytic Properties of Cys-substituted AK_e-- Table II shows the kinetic parameters of wild-type AK_e, compared with two zinc-containing variants. The K_m for nucleotide substrates was similar for the three variants of bacterial enzyme, and excess of AMP (above 0.3 mM) inhibited the activity of all forms at a similar extent. It should be mentioned that removal of metal ion did not affect the phosphorylating activity of apoAK_eC₄ or apoAK_eHC₃, confirming that zinc does not participate in the kinase activity.

	DISCUSSION

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Zinc in proteins is responsible for a wide range of functions (21-24). The design of zinc binding sites generates proteins with new interesting properties (25-28). The fact that zinc is a structural component of AKs from Gram-positive bacteria (6) prompted us to create a similar metal site in the enzyme from Gram-negative species. As shown here, the presence of three or four cysteine residues in the consensus sequence ¹²⁶Cys-X₂-Cys-X₁₆-Cys-X₂-Cys¹⁴⁹ led to a zinc binding site in E. coli AK. Moreover, a significant increase in thermostability of the C₄ variant as compared with the wild-type AK_e was observed.

The crystal structure of AK_e shows that the LID domain forms a single distorted antiparallel -sheet, two turns and one loop structure (29). The -sheet is stabilized by hydrogen backbone interactions and attractive forces between few side chains inside the -sheet. The four amino acids (His¹²⁶, Ser¹²⁹, Asp¹⁴⁶, and Thr¹⁴⁹) replaced by cysteine residues in the mutagenized protein belong to this hydrogen binding network (Fig. 6). Three additional amino acid side chains (Arg¹³¹, Glu¹⁵¹, and Tyr¹³³) stabilize the network of hydrogen bonds by connecting the -sheet segments in a sandwich-like structure. In the LID domain of adenylate kinase from Gram-positive bacteria, Zn²⁺ which is held by cysteine residues seems to substitute efficiently the hydrogen bond network (30). The crystal structure of adenylate kinase from B. stearothermophilus (entry 1.Zip in the Protein Data Bank) confirms this observation. Metal chelation not only preserves the above mentioned network but also enhanced the thermal stability of the protein. As the catalytic properties of the C₄ and C₃ containing variants of AK_e are conserved, the zinc-chelating LID domain of the protein conserves also intact the ability to rotate and to move like a solid block on the ATP-binding pocket. In other words, the overall conformation of this domain remains intact, in agreement with circular dichroism and NMR structural analysis (20).

View larger version (21K): [in this window] [in a new window]   Fig. 6.   Amino acid side chains forming the hydrogen bonding network into the LID domain of AKe. Gray ovals are the positions for the cysteine replacement.

The biochemical characteristics of the others variants of AK_e might be also viewed in the light of hydrogen bond network located into the LID domain. The double mutants with vicinal thiols (C₂DT and HSC₂) conserve over 65% of the activity of the wild-type enzyme. On the contrary the two mutants, where each Cys residue is located on one side of the sandwich-like structure (HC₂T and CSDC), are greatly affected in their activity. The loss of activity was independent on disulfide bridge formation as in the latter cases the SH groups are free.

Among the Cys-monosubstituted variants, the most conservative substitution concerns Ser¹²⁹. The S129C mutant exhibited similar structural and catalytic properties with the wild-type enzyme and with another AK_e mutant (S129F) previously described by Haase et al. (31). This last mutation, however, is conditionally lethal, and the bacteria do not survive at 42 °C. It was concluded that AK_e might be involved in other essential cellular functions, independent of phosphotranferase activity, such as phospholipid synthesis. This attractive hypothesis still awaits for experimental proofs. All other single Cys variants (except HCDT form) of the AK_e, although active and with similar thermal stability as the wild-type enzyme, exhibited a considerably lower resistance against trypsin digestion. In other words, despite the fact that the single amino acid substitutions were "conservative" in terms of hydrogen bond formation, some subtle conformational changes into the LID domain occur, yielding proteins with higher susceptibility to proteolytic digestion.

In conclusion, this study highlighted the importance of some key residues into the LID domain of the AK_e. Quadruple and triple Cys mutations stabilized the protein by chelation with zinc. Double mutants are the most exposed to conformational changes leading to inactivation, irrespective of the presence or absence of disulfide bridges. All single Cys mutants are active but only one (HCDT) conserves the stability of the wild-type protein.