In contrast with adenylate kinase from
Gram-negative bacteria, the enzyme from Gram-positive organisms harbors
a structural Zn2+ bound to 3 or 4 Cys residues in the
structural motif
Cys-X2-Cys-X16-Cys-X2-Cys/Asp. Site-directed mutagenesis of His126, Ser129,
Asp146, and Thr149 (corresponding to
Cys130, Cys133, Cys150, and
Cys153 in adenylate kinase from Bacillus
stearothermophilus) in Escherichia coli adenylate
kinase was undertaken for determining whether the presence of Cys
residues is the only prerequisite to bind zinc or (possible) other
cations. A number of variants of adenylate kinase from E. coli, containing 1-4 Cys residues were obtained, purified, and
analyzed for metal content, structural integrity, activity, and
thermodynamic stability. All mutants bearing 3 or 4 cysteine residues
acquired zinc binding properties. Moreover, the quadruple mutant
exhibited a remarkably high thermal stability as compared with the
wild-type form with preservation of the kinetic parameters of the
parent enzyme.
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INTRODUCTION |
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).

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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.
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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 AKe 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 AKe acquired zinc binding properties. Moreover, the 4 cysteine-containing
AKe exhibited an increased stability against
thermal denaturation as compared with the wild-type form, with full
conservation of its catalytic properties.
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EXPERIMENTAL PROCEDURES |
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 AKe 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 AKe.
AKeC4 corresponds to the 4 Cys-substituted enzyme, AKeHC3 to HCCC, AKeC3T to CCCT,
AKeC2DT to CCDT,
AKeHSC2 to HSCC, and
AKeHC2T to HCCT.
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RESULTS |
Overproduction and Purification of Cysteine-substituted Variants of
AKe--
To create a 4-Cys-substituted mutant of
AKe, 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).
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Table I
Oligonucleotides used for the construction of AKe
cysteine-substituted mutants
The asterisk (*) indicates the position of the
mismatches.
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The overproduced variants of AKe (about 20% of
the soluble E. coli proteins) exhibited high specific
activity in crude extracts (90-120 units/mg of protein), close to that
of the wild-type AKe, indicating that substitution with cysteine of any of the four targeted residues has no direct
consequences on enzyme activity. Chromatography on blue Sepharose and
Ultrogel AcA54 yielded homogeneous preparations of enzymes. The double Cys-substituted AKe species, HC2T,
and CSDC were inactivated during purification in the absence of
reducing agents. They conserved only 5% (23 units/mg of protein) and
37% (175 units/mg of protein) of wild-type activity.
Metal Binding--
The zinc content of different variants of
AKe, 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
AKe. No iron was observed in
AKeC4 when E. coli was
cultivated in minimal medium supplemented with this metal (data not
shown).

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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.
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Reaction of Wild-type AKe and of Cysteine-substituted
Enzymes with DTNB--
Wild-type AKe 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 AKe 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 kobs
(5.10
3 s
1 for
AKeC4, 7.10
3
s
1 for AKeHC3 and
14.10
3 s
1 for
AKeC3T) were practically independent
on the concentration of thiol reagent. Thus, the first order process
might reflect the dissociation rate of the
AKe-Zn2+ complex.
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(Eq. 1)
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As preincubation of AKeC4 with
ZnCl2 did not affect the kobs (data
not shown), one might assume that difference in affinity for metal is
primarily due to the dissociation rate constant for protein-metal
complex.

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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.
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The reactivity toward DTNB of the double substituted species of
AKe was relevant for the structural changes into the LID domain of the bacterial enzyme. Thus, the HSC2
variant reacted with DTNB under denaturing conditions. This means that Cys146 and Cys149 in this mutant are not
exposed to the solvent and do not form a disulfide bridge as might be
expected. On the other hand, the C2DT variant easily forms
an intramolecular disulfide bridge, as suggested by its lack of
reactivity toward DTNB both under nondenaturing and denaturing
conditions. An even more complicated behavior was found with CSDC and
HC2T forms of AKe, both of which
were inactivated during the purification. Under nondenaturing conditions, 1.3-1.6 mol of SH/mol of enzymes were titrated with DTNB,
indicating that the extra thiol groups were free and accessible. The
observed inactivation of these species of AKe is likely due to structural deformation of the LID domain, which propagates to the CORE of the molecule.
Thermal Stability and Proteolysis by Trypsin of Cys-modified
Mutants of AKe--
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 AKe 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 AKe, whereas
the C2DT mutant was less resistant (half-inactivation at
46 °C).
The thermal stability of the C4 and HC3
variants was further examined by microcalorimetry. The excess heat
capacity curve for the wild-type AKe,
C4, and HC3 mutants is shown in Fig.
4. The Tm values
(63 and 55.7 °C, respectively, instead of 51.8 °C for the
wild-type enzyme) were reproducible within 0.1 °C. Inspection of
Fig. 4 suggests that, at least under the conditions of the calorimetric
experiments, the cooperativity of the denaturation process decreases
significantly in the case of C4 mutant. A detailed analysis
of structural and energetic properties of this variant is described in
a companion study (20).

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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.
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Limited proteolysis was used as a test of conformational changes in
AKe induced by various amino acid substitutions. Inactivation of the bacterial enzyme by TPCK-trypsin followed first
order kinetics (7). The triple and quadruple Cys mutants of
AKe showed similar or slightly higher resistance
against trypsin digestion (t1/2 between 26 and 40 min) as compared with the wild-type enzyme. The other modified variants
of AKe, except the HCDT mutant, exhibited a much
lower resistance to proteolysis (t1/2 < 3 min).
Sequence analysis of the proteolytic fragments indicated that
131R
V132 and
141K
F142 bonds located into the LID domain
became sensible to the attack by trypsin; the 14-kDa fragment
accumulated upon proteolysis corresponds to the segment 1-131 of the
molecule (Fig. 5).

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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).
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Catalytic Properties of Cys-substituted AKe--
Table
II shows the kinetic parameters of
wild-type AKe, compared with two zinc-containing
variants. The Km 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
apoAKeC4 or
apoAKeHC3, confirming that zinc does
not participate in the kinase activity.
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Table II
Comparative kinetic parameters of wild-type (WT) AKe,
AKeC4, and AKeHC3
Km (ADP) and Vmax (ADP) were
determined from plots of 1/v versus 1/ADP2, which
assumes that the two molecules of ADP bind to the enzyme with the same
affinity. The apparent Km for AMP and for ATP was
determined at a single fixed concentration of cosubstrates (1 mM ATP and 0.2 mM AMP). The
Vmax (ATP, AMP) was obtained by extrapolating the
reaction rates for infinite concentrations of ATP and AMP and assuming
that the concentration of one nucleotide substrate does not affect the
apparent Km for the second nucleotide substrate.
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DISCUSSION |
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 AKe was
observed.
The crystal structure of AKe 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 AKe 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).

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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.
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The biochemical characteristics of the others variants of
AKe 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 AKe 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 AKe 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 AKe, 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 AKe.
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.
We thank O. Bârzu, C. T. Craescu,
H. Sakamoto, and C. Schulz for fruitful suggestions and comments, L. Serina for help in single strand DNA preparation, and M. Ferrand for
excellent secretarial assistance.